Complex clonal mosaicism within microdissected intestinal metaplastic glands without concurrent gastric cancer.

BACKGROUND: The transformation of healthy gastric tissue into intestinal metaplasia (IM) is thought to be a critical premalignant step in the development of intestinal-type gastric adenocarcinoma (GA). How such premalignancies contribute to the development of GA is, however, poorly understood. METHODS: In this study, the extent and clonal complexity in IM tissue from patients without gastric cancer were analysed by measuring variations of multiple microsatellite (MS) markers. RESULTS: Even though these tissues are generally regarded as clinically benign, we found extensive MS length heterogeneity between and within individual IM glands, indicating that complex genome diversity is already pervasive in these tissues. Based on a clonal relationship analysis, we found that there exist multiple clones within individual IM glands and that MS alterations can accumulate in these clones. Moreover, we found spatially distant IM glands with the same MS phenotype, suggesting that these MS alterations were progressed by gland fission. CONCLUSIONS: These results provide evidence that genetic instability is an early event, present within metaplastic tissues of otherwise non-cancer patients, and such frequent genetic alterations can be part of the pathophysiological rationale for the requirement of this phase during gastric carcinogenesis.

Exploring the Wnt pathway-associated LncRNAs and genes involved in pancreatic carcinogenesis driven by Tp53 mutation.

PURPOSE: Study the contribution of long non-coding RNAs (lncRNAs) to progression of pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma (PDAC). METHODS: We explored lncRNAs profilings in PanIN cell line (SH-PAN) isolated from Pdx-1-Cre; LSL-Kras (G12D/+) mice and PDAC cell line (DT-PCa) isolated from Pdx-1-Cre; LSL- Kras (G12D/+) ; LSL- Tp53 (R172H/+) mice by lncRNAs microarray, and detected expression of lncRNAs and genes in PDAC by Real-time PCR, Western blot, ChIP and immunohistochemistry. RESULTS: Eight lncRNAs and five protein-coding genes, associated with Wnt pathway, were identified with more than five-fold changes between DT-PCa cells and SH-PAN cells. Of them, lincRNA1611 and Ppp3ca were validated significantly high expression in DT-PCa cells and in 22 of 26 fresh resected human PDAC tissues, compared to SH-PAN cells and normal pancreatic tissues, respectively. Moreover, Tp53 mutation status displayed a positive correlation with lincRNA1611 or Ppp3ca level. Immunohistochemical staining for Ppp3ca was weak or lack in 91 of 107 normal pancreatic tissues, 24 of 29 PanIN-I and 13 of 16 PanIN-II tissues, however, was strong in 10 of 27 PanIN-III and 62 of 107 PDAC tissues post operation. CONCLUSIONS: LincRNA1611 and Ppp3ca were high expression in PDAC and may serve as new potential targets for intervention of the disease.

Reduction of TIP30 in esophageal squamous cell carcinoma cells involves promoter methylation and microRNA-10b.

TIP30 is a putative tumor suppressor that can promote apoptosis and inhibit angiogenesis. However, the role of TIP30 in esophageal squamous cell carcinoma (ESCC) biology has not been investigated. Immunohistochemistry was used to investigate the expression of TIP30 in 70 ESCC. Hypermethylation of TIP30 was evaluated by the methylation specific PCR (MSP) method in ESCC (tumor and paired adjacent non-tumor tissues). Lost expression of TIP30 was observed in 50 of 70 (71.4%) ESCC. 61.4% (43 of 70) of primary tumors analyzed displayed TIP30 hypermethylation, indicating that this aberrant characteristic is common in ESCC. Moreover, a statistically significant inverse association was found between TIP30 methylation status and expression of the TIP30 protein in tumor tissues (p=0.001). We also found that microRNA-10b (miR-10b) targets a homologous DNA region in the 3'untranslated region of the TIP30 gene and represses its expression at the transcriptional level. Reporter assay with 3'UTR of TIP30 cloned downstream of the luciferase gene showed reduced luciferase activity in the presence of miR-10b, providing strong evidence that miR-10b is a direct regulator of TIP30. These results suggest that TIP30 expression is regulated by promoter methylation and miR-10b in ESCC.

Expression and regulatory function of miRNA-34a in targeting survivin in gastric cancer cells.

We aimed to investigate the expression of microRNA-34a (miR-34a) in human gastric cancer cells and to evaluate the effects of miR-34a, acting via its gene survivin, on gastric cancer cell HGC-27 to provide potential new strategies for treating gastric cancer. In vitro cultures of the human gastric cancer cell lines MGC80-3, HGC-27, NCI-N87, and SGC-7901 and the normal human gastric epithelial cell line GES-1 were established. The expression of miR-34a in each gastric cancer cell line and GES-1 normal human gastric epithelial cell line was detected using quantitative real-time polymerase chain reaction (qRT-PCR). After the HGC-27 cells were transfected with a miR-34a mimic for 48 h, the changes in the expression levels of miR-34a were detected using qRT-PCR. The effect of miR-34a on HGC-27 cell viability was measured using a tetrazolium-based colorimetric [-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazoliumromide (MTT)] assay. Flow cytometry was used to analyze the effects of miR-34a on HGC-27 cell proliferation. Annexin V/propidium iodide double staining and flow cytometry were used to analyze the effects of miR-34a on HGC-27 cell apoptosis. A Transwell invasion chamber was used to detect the effects of miR-34a on HGC-27 cell invasion. Finally, western blotting was used to analyze the effects of miR-34a on survivin protein expression. The qRT-PCR test determined that miR-34a expression in gastric cancer cells was significantly reduced compared to the normal gastric epithelial cell line GES-1 (p < 0.01). Compared to the control group, cellular miR-34a expression levels were significantly increased in HGC-27 human gastric carcinoma cells after transfection with a miR-34a mimic for 48 h (p < 0.01). The MTT assay demonstrated that after overexpressing miR-34a in HGC-27 cells, cellular viability was significantly reduced (p < 0.05). Flow cytometry analysis determined that upon miR-34a overexpression, the proliferation index decreased significantly (p < 0.05), and cellular apoptosis was significantly increased (p < 0.01). The Transwell invasion chamber assay illustrated that after increasing the expression of miR-34a, the number of cells passing through the Transwell chamber was significantly reduced (p < 0.01). Based on western blotting, compared with the control group, survivin protein expression levels were significantly decreased in the HGC-27 cells transfected with the miR-34a mimic for 48 h (p < 0.01). In conclusion, the expression level of miR-34a was downregulated in human gastric cancer cell lines. miR-34a can negatively regulate survivin protein expression and inhibit gastric cancer cell proliferation and invasion. Therapeutically enhancing miR-34a expression or silencing the survivin gene may benefit patients with gastric cancer.

Double-balloon enteroscopy in the evaluation of small bowel disorders in pediatric patients.

BACKGROUND: The aim of the present study was to investigate the indications, clinical utility, feasibility and safety of double-balloon enteroscopy (DBE) in the diagnosis and management of small intestinal diseases in pediatric patients. METHODS: This is a retrospective analysis of pediatric patients younger than 18 years referred to Shanghai Rui Jin Hospital from May 2003 to June 2008 for investigation of suspected small bowel disorders. Demographic, clinical, procedural and outcome data were collected for analysis. RESULTS: A total of 35 DBE were carried out in 30 children with a mean age of 13 years. Indications for DBE were obscure gastrointestinal (GI) bleeding (n = 22), chronic abdominal pain (n = 4), chronic diarrhea (n = 3), and incomplete small bowel obstruction (n = 1). DBE evidenced pathological findings in 29 patients (96.7%). DBE altered management in 90% of patients with positive findings. Follow up was obtained on all patients with a mean (range) of 40 months (14-75 months). The procedure was successful in all patients and there were no serious complications related to sedation. CONCLUSION: DBE is feasible and safe and has a high diagnostic yield and therapeutic impact on the diagnosis and management of small bowel disorders in selected pediatric patients.

Epigenetic repression of microRNA-129-2 leads to overexpression of SOX4 in gastric cancer.

High levels of SOX4 expression have been found in a variety of human cancers, such as lung, brain and breast cancers. However, the expression of SOX4 in gastric tissues remains unknown. The SOX4 expression was detected using immunohistochemical staining and semi-quantitative RT-PCR, and our results showed that SOX4 was up-regulated in gastric cancer compared to benign gastric tissues. To further elucidate the molecular mechanisms underlying up-regulation of SOX4 in gastric cancers, we analyzed the expression of microRNA-129-2 (miR-129-2) gene, the epigenetic repression of which leads to overexpression of SOX4 in endometrial cancer. We found that up-regulation of SOX4 was inversely associated with the epigenetic silencing of miR-129-2 in gastric cancer, and restoration of miR-129-2 down-regulated SOX4 expression. We also found that inactivation of SOX4 by siRNA and restoration of miR-129-2 induced apoptosis in gastric cancer cells.

Clinical characteristics of small bowel tumors diagnosed by double-balloon endoscopy: Experience from a Chinese tertiary hospital.

BACKGROUND/AIMS: To determine the characteristics of small bowel tumors (SBTs) in patients underwent double balloon endoscopy (DBE) and to compare the clinical value of DBE with other diagnostic tools. MATERIALS AND METHODS: A retrospective study was conducted in patients underwent DBE procedures from March 2008 to April 2017.The demographic, clinical and pathological characteristics of patients with SBTs were recorded, while the diagnosis of SBTs was achieved either by DBE biopsy or surgical specimens. RESULTS: One thousand one hundred and two patients (761 males, range 3-85 years) were enrolled in this study, with 1140 procedures completed in total. 99/1102 patients (9.0%) had SBTs, including benign polyps (20, 20.2%), gastrointestinal stromal tumors (GISTs) (24, 24.2%), lymphomas (13, 13.1%), adenocarcinoma (39, 39.4%), and neuroendocrine tumors (3, 3.0%). The most common clinical symptom for benign polyps was obscure gastrointestinal bleeding (OGIB) (75.0%). But among patients with malignant SBTs, the main indication for DBE was chronic abdominal pain (43.8%), followed by OGIB (36.3%), vomit (10.0%), abnormal images (6.3%) and diarrhea (3.8%) (P<0.001). Moreover, SBTs were primarily located in the jejunum alone (40/99, 40.4%). DBE had better sensitivity (89.2%), specificity (95.2%), positive predictive value (PPV) (90.0%), and negative predictive value (NPV) (94.8%) than other tools for suspected SBTs. CONCLUSION: Small bowel tumor is mainly located in jejunum and with OGIB and abdominal pain as major complaints. DBE is a reliable method for the diagnosis of SBTs compared with other diagnostic tools.

Magnetically Guided Capsule Endoscopy in Pediatric Patients with Abdominal Pain.

BACKGROUND AND AIMS: Magnetically guided capsule endoscopy (MGCE) offers a noninvasive method of evaluating both the gastric cavity and small intestine; however, few studies have evaluated MGCE in pediatric patients. We investigated the diagnostic efficacy of MGCE in pediatric patients with abdominal pain. PATIENTS AND METHODS: We enrolled 48 patients with abdominal pain aged 6-18 years. All patients underwent MGCE to evaluate the gastric cavity and small intestine. RESULTS: The cleanliness of the gastric cardia, fundus, body, angle, antrum, and pylorus was assessed satisfactorily in 100%, 85.4%, 89.6%, 100%, 97.9%, and 100% of patients, respectively. The subjective percentage visualization of the gastric cardia, fundus, body, angle, antrum, and pylorus was 84.8%, 83.8%, 88.5%, 87.7%, 95.2%, and 99.6%, respectively. Eighteen (37.5%) patients had 19 gastrointestinal tract lesions: one esophageal, three in the gastric cavity, and 15 in the small intestine. No adverse events occurred during follow-up. CONCLUSIONS: MGCE is safe, convenient, and tolerable for evaluating the gastric cavity and small intestine in pediatric patients. MGCE can effectively diagnose pediatric patients with abdominal pain.

Association of XRCC3 gene rs861539 polymorphism with gastric cancer risk: evidence from a case-control study and a meta-analysis.

The association between the X-ray repair cross-complementing group 3 (XRCC3) gene Thr241Met polymorphism (rs861539) and gastric cancer has been widely evaluated, but a definitive answer is so far lacking. We first conducted a case-control study to assess this association in a large Han Chinese population, and then performed a meta-analysis to further address this issue. Although our case-control association study and the following meta analysis involving 6,520 subjects indicated null association of XRCC3 gene rs861539 polymorphism between gastric cancer patients and controls under both allelic (odds ratio (OR) = 1.02; 95% confidence interval (CI): 0.91-1.14; P = 0.739) and dominant (OR = 0.97; 95% CI: 0.78-1.21; P = 0.803) models. Stratified analysis by ethnicity demonstrated a significant association in Asians. We conclude that the XRCC3 gene rs861539 polymorphism was associated with the risk for gastric cancer in Asian populations.

BRCA2 dysfunction promotes malignant transformation of pancreatic intraepithelial neoplasia.

Pancreatic ductal adenocarcinoma (PDAC) is an almost lethal disease. Thus, it is important to better understand its genetic progression from normal cells through precancerous lesions pancreatic intraepithelial neoplasia (PanIN) to invasive pancreatic cancer. Carriers of a germline mutation in BRCA2 have an increased risk of developing PDAC when compared with the general population. The purpose of our study was to examine the role of BRCA2 dysfuction in the progression of PDAC. Here we generated a novel in vitro model of pancreatic carcinogenesis. Cancerous PanIN-BR1 cells were established by stable transduction with lentiviral-mediated BRCA2 RNA interference in PanIN cell isolated from mice with oncogenic KrasG12D. Our data showed that silencing of BRCA2 promoted cell proliferation, migration and invasion in vitro. The tumorigenic potential of PanIN-BR1 were assessed by xenograft tumor formation in BALB/c nude mice. The expression of PCNA , Snail and Slug in the tumor xenografts was detected by immunohistochemistry. The staining for PCNA, Snail and Slug in PanIN-BR1-formed xenograft tissue was significantly more intense than PanIN-formed xenograft tissue. Microarray assay was also performed. Based on gene expression profiling and further validation by real-time PCR and Western Blot, we found that the expression of Cyclinb2, Cyclina2, Twist1, Wisp1 and Cxcr4 revealed a significant increase in the PanIN-BR1 cells, however, the expression of p15, p16 and Wisp2 showed a significant decrease in the PanIN-BR1 cells, compared to the PanIN cells. Collectively, these data reported here demonstrate that BRCA2 may be a promising therapeutic targets for PDAC progression.

The biological features of PanIN initiated from oncogenic Kras mutation in genetically engineered mouse models.

Pancreatic intraepithelial neoplasia (PanIN) is the most common premalignant lesion of the pancreas. Further understanding of the biological behavior and molecular genetic alterations in the stepwise progression of PanINs is necessary toward the development of pancreatic ductal adenocarcinoma (PDAC) interventions. In this study, we analyzed the morphological characteristics, molecular alterations, and biological behavior of pancreatic wild-type and neoplasia tissues, including analysis of PanIN cell line SH-PAN (isolated from Pdx-1-Cre; LSL-KrasG12D/+ mouse) and PDAC cell line DT-PCa (isolated from Pdx1-Cre; LSL-KrasG12D/+; LSL-Tp53R172H/+ mouse. Results show that KrasG12D induces ductal lesion PanINs. Increased expression of EGFR, Her-2/Neu, p-MAPK and ß-Catenin was observed in low-grade PanINs. Tp53 was not expressed in wild-type and low-grade PanINs, however, increased expression was observed in high-grade PanINs. Furthermore, SH-PAN cells did not exhibit any colony formation and showed significantly lower migration and invasion ability compared with DT-PCa cells. Notably, we first found PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) expression was significantly higher in SH-PAN cells than DT-PCa cells, and was high in 96 of 172 peritumoral normal human pancreatic tissues and 20 of 36 human low- or middle-grade PanIN tissues, whereas, was weak or negligible in 12 of 20 human high-grade PanIN tissues and 124 of 172 human PDAC tissues post-operation. The expression of PPP2R2A appears to be correlated with clinical survival. Taken together, Kras(G12D) - driven PanIN showed the tumorigenic ability, however, did not undergo a malignant transformation, and decreased expression of PPP2R2A in PDACs may provided a new target for pancreatic carcinoma intervention.

Diagnostic yield and therapeutic impact of double-balloon enteroscopy in a large cohort of patients with obscure gastrointestinal bleeding.

OBJECTIVES: Double-balloon enteroscopy (DBE) is a novel endoscopic technique developed to investigate small bowel disease. There are limited available data on its impact in the diagnosis and management of obscure gastrointestinal bleeding (OGIB). The aim of this study was to evaluate the diagnostic yield and therapeutic impact of DBE in the management of patients with OGIB. METHODS: This study is a retrospective analysis of patients referred to our hospital from December 2003 to January 2005 for the investigation of overt or occult OGIB who underwent DBE after negative upper endoscopy and colonoscopy. Demographic, clinical, procedural, and outcome data were collected for analysis. RESULTS: One hundred fifty-two patients (73 women and 79 men) were studied, with a mean age of 48.2 yr. Seventeen patients presented with occult OGIB while 135 patients had overt OGIB. A total of 191 DBEs was performed. Antegrade and retrograde approaches were performed in 60 and 53 patients, respectively, and 39 patients had a combination of both routes. DBE demonstrated a potential bleeding site in 115 (75.7%) patients (102 overt, 13 occult). The more common abnormalities detected were small bowel tumors (39.1%) and angioectasia (30.4%). DBE altered management in 83.5% of patients with positive findings. Follow-up was obtained on 119 patients (mean 16 months, range 8-23 months). Of the 95 patients with follow-up and a positive DBE finding, 85 (89%) had no further rebleeding. The procedure was well tolerated with 23 patients (15.1%) experiencing mild self-limited bleeding during the procedure. CONCLUSIONS: DBE appears to have a high diagnostic yield and therapeutic impact in patients with OGIB with previously negative upper endoscopy and colonoscopy.