The apelin­apelin receptor signaling pathway in fibroblasts is involved in tumor growth via p53 expression of cancer cells.

Cancer­associated fibroblasts (CAFs) are pivotal in tumor progression. TP53­deficiency in cancer cells is associated with robust stromal activation. The apelin­apelin receptor (APJ) system has been implicated in suppressing fibroblast­to­myofibroblast transition in non­neoplastic organ fibrosis. The present study aimed to elucidate the oncogenic role of the apelin­APJ system in tumor fibroblasts. APJ expression and the effect of APJ suppression in fibroblasts were investigated for p53 status in cancer cells using human cell lines (TP53­wild colon cancer, HCT116, and Caco­2; TP53­mutant colon cancer, SW480, and DLD­1; and colon fibroblasts, CCD­18Co), resected human tissue samples of colorectal cancers, and immune­deficient nude mouse xenograft models. The role of exosomes collected by ultracentrifugation were also analyzed as mediators of p53 expression in cancer cells and APJ expression in fibroblasts. APJ expression in fibroblasts co­cultured with p53­suppressed colon cancer cells (HCT116sh p53 cells) was significantly lower than in control colon cancer cells (HCT116sh control cells). APJ­suppressed fibroblasts treated with an antagonist or small interfering RNA showed myofibroblast­like properties, including increased proliferation and migratory abilities, via accelerated phosphorylation of Sma­ and Mad­related protein 2/3 (Smad2/3). In addition, xenografts of HCT116 cells with APJ­suppressed fibroblasts showed accelerated tumor growth. By contrast, apelin suppressed the upregulation of phosphorylated Smad2/3 in fibroblasts. MicroRNA 5703 enriched in exosomes derived from HCT116sh p53 cells inhibited APJ expression, and inhibition of miR­5703 diminished APJ suppression in fibroblasts caused by cancer cells. APJ suppression from a specific microRNA in cancer cell­derived exosomes induced CAF­like properties in fibroblasts. Thus, the APJ system in fibroblasts in the tumor microenvironment may be a promising therapeutic target.

A novel role for Helicobacter pylori cytotoxin-associated gene A in negative regulation of autophagy in human gastric cells.

BACKGROUND: Autophagy plays an important role in carcinogenesis and tumor progression in many cancers, including gastric cancer. Cytotoxin-associated gene A (CagA) is a well-known virulent factor in Helicobacter pylori (H. pylori) infection that plays a critical role in gastric inflammation and gastric cancer development. However, its role in autophagy during these processes remains unclear. Therefore, we aimed to clarify the role of CagA in autophagy in CagA-related inflammation. METHODS: We evaluated the autophagic index of AGS cells infected with wild-type cagA-positive H. pylori (Hp-WT) and cagA-knockout H. pylori (Hp-ΔcagA) and rat gastric mucosal (RGM1) cells transfected with CagA genes. To identify the mechanisms underlying the down regulation of autophagy in AGS cells infected with H. pylori, we evaluated protein and mRNA expression levels of autophagy core proteins using western blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR). To determine whether autophagy induced the expression of the pro-inflammatory mediator, cyclooxygenase-2 (COX-2), we evaluated COX-2 expression in AGS cells treated with an autophagy inducer and inhibitor and infected with H. pylori. In addition, we evaluated whether COX-2 protein expression in AGS cells influenced beclin-1 (BECN1) expression with si-RNA transfection when infected with H. pylori. RESULTS: Autophagic flux assay using chloroquine showed that autophagy in AGS cells was significantly suppressed after H. pylori infection. The autophagic index of AGS cells infected with Hp-WT was decreased significantly when compared with that in AGS cells infected with Hp-ΔcagA. The autophagic index of RGM1 cells transfected with CagA was lower, suggesting that CagA inhibits autophagy. In addition, BECN1 expression levels in AGS cells infected with Hp-WT were reduced compared to those in AGS cells infected with Hp-ΔcagA. Furthermore, COX-2 expression in AGS cells infected with H. pylori was controlled in an autophagy-dependent manner. When AGS cells were transfected with small interfering RNA specific for BECN1 and infected with Hp-WT and Hp-ΔcagA, COX-2 was upregulated significantly in cells infected with Hp-ΔcagA. CONCLUSIONS: In conclusion, the H. pylori CagA protein negatively regulated autophagy by downregulating BECN1. CagA-induced autophagy inhibition may be a causative factor in promoting pro-inflammatory mediator production in human gastric epithelial cells.

Suppression of autophagy promotes fibroblast activation in p53-deficient colorectal cancer cells.

Deficiency of p53 in cancer cells activates the transformation of normal tissue fibroblasts into carcinoma-associated fibroblasts; this promotes tumor progression through a variety of mechanisms in the tumor microenvironment. The role of autophagy in carcinoma-associated fibroblasts in tumor progression has not been elucidated. We aimed to clarify the significance of autophagy in fibroblasts, focusing on the TP53 status in co-cultured human colorectal cancer cell lines (TP53-wild-type colon cancer, HCT116; TP53-mutant colon cancer, HT29; fibroblast, CCD-18Co) in vitro. Autophagy in fibroblasts was significantly suppressed in association with ACTA2, CXCL12, TGFß1, VEGFA, FGF2, and PDGFRA mRNA levels, when co-cultured with p53-deficient HCT116sh p53 cells. Exosomes isolated from the culture media of HCT116sh p53 cells significantly suppressed autophagy in fibroblasts via inhibition of ATG2B. Exosomes derived from TP53-mutant HT29 cells also suppressed autophagy in fibroblasts. miR-4534, extracted from the exosomes of HCT116sh p53 cells, suppressed ATG2B in fibroblasts. In conclusion, a loss of p53 function in colon cancer cells promotes the activation of surrounding fibroblasts through the suppression of autophagy. Exosomal miRNAs derived from cancer cells may play a pivotal role in the suppression of autophagy.

Use of a convolutional neural network for classifying microvessels of superficial esophageal squamous cell carcinomas.

BACKGROUND AND AIM: The morphological diagnosis of microvessels on the surface of superficial esophageal squamous cell carcinomas using magnifying endoscopy with narrow-band imaging is widely used in clinical practice. Nevertheless, inconsistency, even among experts, remains a problem. We constructed a convolutional neural network-based computer-aided diagnosis system to classify the microvessels of superficial esophageal squamous cell carcinomas and evaluated its diagnostic performance. METHODS: In this retrospective study, a cropped magnifying endoscopy with narrow-band images from superficial esophageal squamous cell carcinoma lesions was used as the dataset. All images were assessed by three experts, and classified into three classes, Type B1, B2, and B3, based on the Japan Esophagus Society classification. The dataset was divided into training and validation datasets. A convolutional neural network model (ResNeXt-101) was trained and tuned with the training dataset. To evaluate diagnostic accuracy, the validation dataset was assessed by the computer-aided diagnosis system and eight endoscopists. RESULTS: In total, 1777 and 747 cropped images (total, 393 lesions) were included in the training and validation datasets, respectively. The diagnosis system took 20.3 s to evaluate the 747 images in the validation dataset. The microvessel classification accuracy of the computer-aided diagnosis system was 84.2%, which was higher than the average of the eight endoscopists (77.8%, P < 0.001). The area under the receiver operating characteristic curves for diagnosing Type B1, B2, and B3 vessels were 0.969, 0.948, and 0.973, respectively. CONCLUSIONS: The computer-aided diagnosis system showed remarkable performance in the classification of microvessels on superficial esophageal squamous cell carcinomas.

Usefulness of a dedicated mouthpiece for the Valsalva maneuver to visualize the hypopharynx during transoral endoscopy.

Background and study aims Patients with esophageal squamous cell carcinoma (SCC) are at high risk of developing second primary SCCs in the hypopharynx. However, such second primary tumors are difficult to observe because of lumen closure. The Valsalva maneuver using a dedicated mouthpiece is a promising technique to visualize the hypopharynx during transoral endoscopy. In the current study, we investigated the utility of this method. Patients and methods The current study was a randomized, controlled, crossover trial. Patients with esophageal SCC were randomly assigned first to undergo pharyngeal observation using the dedicated mouthpiece followed by observation using a conventional mouthpiece, or vice versa. The primary endpoint was complete visualization of the hypopharynx, which was assessed blindly by three external evaluators. Results A total of 68 pharyngeal examinations were analyzed - 34 with the dedicated mouthpiece and 34 with a conventional mouthpiece. Complete visualization was achieved in 68 % of the examinations (23/34) using the dedicated mouthpiece, whereas none of the examinations using the conventional mouthpiece achieved complete visualization of the hypopharynx. Observation scores of the oropharynx were not significantly different between both types of examination ( P  = 0.50). No serious adverse events (AEs) occurred. Conclusions Endoscopic view of the hypopharynx was markedly improved by the Valsalva maneuver using the dedicated mouthpiece, with no serious AEs. This procedure should be included in the endoscopic examinations for the patients with esophageal SCCs.

Esophageal carcinoma originating in the surface epithelium with immunohistochemically proven esophageal gland duct differentiation: A case report.

A case of esophageal carcinoma exclusively composed of adenocarcinoma simulating an esophageal gland duct in a 61-year-old man is presented. The tumor arose as a slightly elevated lesion in the middle intrathoracic esophagus. It was almost completely overlaid with non-neoplastic stratified squamous epithelial cells. Beneath the overlying surface epithelium, an adenocarcinoma that was bilayered in structure diffusely invaded both the mucosal and submucosal layers. Although the tumor consisted exclusively of adenocarcinomatous cells, a keratinizing squamous cell carcinoma component was focally observed. The invasive carcinoma was focally continuous with the small area of the surface squamous epithelial layer, which was confirmed to be neoplastic by immunohistochemistry. Morphological and immunohistochemical examinations suggested that the adenocarcinomatous component arose from the esophageal surface epithelium and clearly differentiated into an esophageal gland duct. It is important to consider the possibility of this type of adenocarcinoma when diagnosing a ductal or glandular lesion of the esophagus in small biopsy specimens.

Frequency of coexistent carcinoma in sessile serrated adenoma/polyps and traditional serrated adenomas removed by endoscopic resection.

BACKGROUND AND STUDY AIMS: Sessile serrated adenoma/polyps (SSA/Ps) have a different potential than traditional adenomatous polyps for developing into malignant colorectal cancer. However, little is known about the coexistent cancer rate. Here, we evaluate the frequency of carcinoma in serrated polyps removed by endoscopic resection (ER). PATIENTS AND METHODS: This was a retrospective single-center cohort study of consecutive patients with colorectal polyps who underwent ER from March 2003 to October 2014. We determined the frequency of serrated polyps among all resected colorectal polyps and analyzed the clinicopathological findings as well as the frequency and characteristics of coexistent carcinoma in the serrated polyps resected by ER based on pathology reports. RESULTS: A total of 21,048 polyps from 15,326 patients were identified, including 15,984 traditional adenomatous polyps (75.9 %), 621 SSA/Ps (3.0 %), 136 traditional serrated adenomas (TSAs) (0.6 %), 1,121 hyperplastic polyps (5.3 %), and 3,186 polyps of other types (15.1 %). The clinical and endoscopic findings of SSA/Ps revealed a male predominance (68.6 %), with 61.7 % of the polyps located in the proximal colon. Males accounted for 77.2 % of all patients with TSAs, and 77.2 % of these polyps were located in the distal colon. The mean sizes of the SSA/Ps and TSAs were 8.8 and 10.7 mm, respectively. Among the SSA/Ps, 8 (1.3 %) cases had coexistent carcinoma, and 1 (0.7 %) patient with TSA showed coexistent carcinoma. In the patients with SSA/Ps, female sex and a tumor size ≥ 10 mm were predictive factors for coexistent carcinoma. CONCLUSIONS: The frequency of SSA/Ps with carcinoma was lower than that for traditional adenoma. Female sex and tumor size ≥ 10 mm were significant predictive factors for coexistent carcinoma.

Colon adenocarcinoma with dome-like phenotype: characteristic endoscopic ultrasonography (EUS) findings.

An 80-year-old man underwent colonoscopy for proctorrhagia. Conventional white-light imaging showed a superficially flat and elevated lesion that appeared to be a submucosal tumor of the sigmoid colon. Chromoendoscopy with Indigo Carmine showed that the margin of the tumor was covered with normal epithelium but that there was a slight depression on its surface. Magnification endoscopy with Crystal Violet staining revealed the amorphous surface structure of the depressed lesion, but the surrounding mucosa showed a normal pit pattern. Endoscopic ultrasonography demonstrated that a hypoechoic mass was located in the submucosal layer, and a biopsy specimen obtained from the surface of the lesion showed evidence of adenocarcinoma. We then performed sigmoidectomy on the patient. Immunohistochemically, the tumor cells were positive for two mismatch repair proteins (MLH1 and MSH2), but in situ hybridization revealed that the specimen was negative for the Epstein - Barr virus. We finally diagnosed the lesion as adenocarcinoma with a dome-like phenotype of the sigmoid colon.

Endoscopic submucosal dissection for large superficial colorectal tumors using the "clip-flap method".

BACKGROUND AND STUDY AIMS: Endoscopic submucosal dissection (ESD) is technically difficult because of poor visualization and instability in the cutting area. Although mucosal flap formation improves visualization of the cutting area, it is difficult to achieve, especially in colorectal ESD. To facilitate mucosal flap formation, we developed the "clip-flap method" by substituting an endoclip for the mucosal flap until it is formed. PATIENTS AND METHODS: The clip-flap method was applied to 114 of 119 large superficial colorectal tumors being treated by ESD. Therapeutic efficacy and safety were assessed. RESULTS: Mean tumor diameter, resected specimen diameter, and procedure time were 32.5 mm, 38.9 mm, and 82.0 minutes, respectively. The en bloc resection rate was 97.5 %. Intraoperative perforation occurred in one patient who was treated conservatively. A single endoclip was used for 92 lesions and improved visualization of the cutting area. A cross pattern of endoclips was used for 22 lesions and further stabilized the visual field, especially near the lateral side. CONCLUSIONS: The clip-flap method is a simple, safe, and effective option for ESD of large superficial colorectal tumors.