Shelby D. Melton

Acid and Bile Salt–Induced CDX2 Expression Differs in Esophageal Squamous Cells From Patients With and Without Barrett's Esophagus

BACKGROUND & AIMS It is not clear why only a minority of patients with gastroesophageal reflux disease (GERD) develop Barretts esophagus. We hypothesized that differences among individuals in molecular pathways activated when esophageal squamous epithelium is exposed to reflux underlie the development of Barretts metaplasia. METHODS We used esophageal squamous cell lines from patients who had GERD with Barretts esophagus (normal esophageal squamous [NES]-B3T and NES-B10T) and without Barretts esophagus (NES-G2T and NES-G4T) to study effects of acid and bile salts on expression of the CDX2 gene. Bay 11-705, Ad5 inhibitor kappaB(IkappaB)alpha-SR, and site-directed mutagenesis were used to explore effects of nuclear factor-kappaB (NF-kappaB) inhibition on CDX2 promoter activity; DNA binding of the NF-kappaB subunits p50 and p65 was assessed by chromatin immune-precipitation. RESULTS Acid and bile salts increased CDX2 messenger RNA (mRNA), protein, and promoter activity in NES-B3T and NES-B10T cells, but not in NES-G2T or NES-G4T cells. Inhibition of NF-kappaB abolished the increase in CDX2 promoter activity. Increased CDX2 promoter activity was associated with nuclear translocation of p50, which bound to the promoter. We found CDX2 mRNA in 7 of 10 esophageal squamous biopsy specimens from patients with Barretts esophagus, but in only 1 of 10 such specimens from patients who had GERD without Barretts esophagus. CONCLUSIONS Acid and bile salts induce CDX2 mRNA and protein expression in esophageal squamous cells from patients with Barretts esophagus, but not from GERD patients without Barretts esophagus. We speculate that these differences in acid- and bile salt-induced activation of molecular pathways may underlie the development of Barretts metaplasia.

Frequent occurrence of gastritis and duodenitis in patients with inflammatory bowel disease.

Background: The purpose was to study the epidemiology of Helicobacter‐negative gastritis among a large group of patients with inflammatory bowel disease (IBD) and healthy controls. Methods: From a computerized database of surgical pathology reports we selected 5493 patients who underwent colonoscopy and upper gastrointestinal endoscopy with biopsy results from both procedures. The presence of gastritis and duodenitis was compared among 550 case subjects with IBD and 4943 healthy control subjects. The results are expressed as prevalence rates, as well as age‐ and sex‐adjusted odds ratios with 95% confidence intervals. Results: Any type gastritis was found in 13% of controls and 25% of IBD patients (Crohns disease [CD] 33%, ulcerative colitis [UC] 19%). Duodenitis was found in 1% of controls and 13% of IBD patients (CD 26%, UC 3%). In subjects younger than 18 years the prevalence of gastritis and duodenitis were 53% and 40% in CD, respectively, and 38% and 0% in UC, respectively. Similar prevalence rates were found in men and women. The odds ratio for Helicobacter‐negative chronic active gastritis associated with CD was 11.7 (7.5–18.0) and with UC 2.8 (1.4–5.0). The corresponding values for focally enhanced gastritis were 40.1 (15.5–114.9) in CD and 0 in UC. Conclusions: Helicobacter‐negative gastritis and duodenitis occur significantly more often in patients with IBD than healthy controls. Such upper gastrointestinal inflammation appears to be particularly common in CD and younger patients. (Inflamm Bowel Dis 2011;)

Malignant transformation of non-neoplastic Barrett's epithelial cells through well-defined genetic manipulations.

Background Human Barretts cancer cell lines have numerous, poorly-characterized genetic abnormalities and, consequently, those lines have limited utility as models for studying the early molecular events in carcinogenesis. Cell lines with well-defined genetic lesions that recapitulate various stages of neoplastic progression in Barretts esophagus would be most useful for such studies. Methodology/Principal Findings To develop such model cell lines, we started with telomerase-immortalized, non-neoplastic Barretts epithelial (BAR-T) cells, which are spontaneously deficient in p16, and proceeded to knock down p53 using RNAi, to activate Ras by introducing oncogenic H-RasG12V, or both. BAR-T cells infected with either p53 RNAi or oncogenic H-RasG12V alone maintained cell-to-cell contact inhibition and did not exhibit anchorage-independent growth in soft agar. In contrast, the combination of p53 RNAi knockdown with expression of oncogenic H-RasG12V transformed the p16-deficient BAR-T cells, as evidenced by their loss of contact inhibition, by their formation of colonies in soft agar, and by their generation of tumors in immunodeficient mice. Conclusions/Significance Through these experiments, we have generated a number of transformed and non-transformed cell lines with well-characterized genetic abnormalities recapitulating various stages of carcinogenesis in Barretts esophagus. These lines should be useful models for the study of carcinogenesis in Barretts esophagus, and for testing the efficacy of chemopreventive and chemotherapeutic agents.

Biomarkers and molecular diagnosis of gastrointestinal and pancreatic neoplasms.

Standard protocols for the diagnosis of neoplasms in the gastrointestinal tract are based on histopathologic analysis in combination with clinical information. With the completion of the Human Genome Project in 2003, our understanding of the contribution of genetics to human disease has increased exponentially. This knowledge is gradually being incorporated into clinical decision-making. However, the rate at which molecular biomarkers are validated for use in mainstream clinical applications has lagged far behind that of biomarker discovery. Nevertheless, a number of molecular biomarkers are available for use in the diagnosis and management of gastrointestinal tract neoplasms. This article reviews the most common molecular biomarkers currently available for neoplasms of the luminal gastrointestinal tract and pancreas. In neoplasms of the esophagus, for which no biomarkers are currently used in routine clinical practice, those that have shown the most promise in early clinical validation studies are discussed.

Gastric cardiac polyps: a clinicopathologic study of 330 cases.

As endoscopists have become more skilled in sampling the gastroesophageal junction, pathologists are being increasingly challenged to characterize previously unknown or neglected findings. One such example is the cardiac polyp. Originally described in the radiology literature as the sentinel fold, the first histologic descriptions of polyps at the gastroesophageal junction did not appear until less than a decade ago. Current clinicopathologic information is limited and somewhat conflicting. This study was designed to define the clinical, endoscopic, and histopathologic associations in patients with cardiac polyps. Using an electronic database, we extracted information on all patients who had a distal esophageal or esophagogastric junctional biopsy during a 24-month period. We then reviewed the slides of 330 adult patients diagnosed with a cardiac polyp and used semiquantitative or qualitative scales to score foveolar hyperplasia, inflammation, erosion or ulcers, epithelial type, and metaplasia. As controls we used 120,487 patients who had biopsies from the same anatomic sites during the same period, but were not diagnosed with a cardiac polyp. There were no significant differences among any clinical indications for esophagogastroduodenoscopy between study and control patients. Endoscopically, a polyp or nodule at the gastroesophageal junction was noted in 59.1% of the patients who had a histopathologic diagnosis of cardiac polyp. Histologically, Barrett mucosa, active esophagitis, and Helicobactor pylori gastritis were all significantly less common in patients with a cardiac polyp than in controls. Although relatively infrequent, synchronous hyperplastic polyps elsewhere in the stomach were significantly more common in patients than in controls. In conclusion, this large series suggests that cardiac polyps are rare but histologically distinct lesions. They are benign and are not uniquely associated with esophagitis, Barrett esophagus, gastroesophageal reflux disease, reactive gastropathy, or gastritis, with or without H. pylori.

Microbial Sensing by Intestinal Myeloid Cells Controls Carcinogenesis and Epithelial Differentiation

SUMMARY Physiologic microbe-host interactions in the intestine require the maintenance of the microbiota in a luminal compartment through a complex interplay between epithelial and immune cells. However, the roles of mucosal myeloid cells in this process remain incompletely understood. In this study, we identified that decreased myeloid cell phagocytic activity promotes colon tumorigenesis. We show that this is due to bacterial accumulation in the lamina propria and present evidence that the underlying mechanism is bacterial induction of prostaglandin production by myeloid cells. Moreover, we show that similar events in the normal colonic mucosa lead to reductions in Tuft cells, goblet cells, and the mucus barrier of the colonic epithelium. These alterations are again linked to the induction of prostaglandin production in response to bacterial penetration of the mucosa. Altogether, our work highlights immune cell-epithelial cell interactions triggered by the microbiota that control intestinal immunity, epithelial differentiation, and carcinogenesis.

171 COMMD1 Restrains Tumor Promoting Activities of Myeloid Cells in Colon Cancer

G A A b st ra ct s primary resistance to imatinib. Amongst c-KIT/PDGFR wild type GIST, the most frequent driver mutation is BRAF-V600E, especially in small intestinal GIST. Thus, a mouse model of mutant Braf GIST might allow for development of more effective therapeutic strategies for these less common forms of GIST. Somatic activating mutations in c-Kit induce ICC hyperplasia and GIST in mouse models. However, whether GIST can arise from other cell types is unknown. In this study, we sought to generate a mouse model for Braf-mutant GIST from intestinal smooth muscle. Methods: Mice with a conditionally active (CA) mutant Braf allele, Braf-lox-stop-lox-V600E (Braf-CA), were crossed to mice carrying the tamoxifeninducible smooth muscle driver (Myh11-CreERT2). Adult mice received a single intraperitoneal injection of tamoxifen, ranging from 2.5-25mg/kg. The stomach, small intestine and colon were evaluated by H&E staining and immunohistochemistry. Results: Three weeks after tamoxifen injection, clusters of dysplastic cells with high nuclear-cytoplasmic ratio were observed in the intestinal muscular layer. The phenotype was most striking at the outer longitudinal layer of the greater curvature of stomach, but was also seen in the small intestinal and colonic muscle layer. The dysplastic cells were spindle-shaped and surrounded ganglion cells. CD34 immunoreactivity decorated, the dysplastic cells, a common finding in human GIST. Some of the clusters of dysplastic cells exhibited c-Kit-positivity and decreased a-SMA staining. c-Kit-positive cell clusters were also positive for anoctamin-1 (Ano1), an additional marker for human c-Kit-positive GIST. Conclusions: By conditionally activating mutant Braf in smooth muscle cells, we have developed a new mouse model for Braf-mutant GIST, as determined by characteristic histological features and markers. These results suggest that smooth muscle, rather than ICC, may be the cell-of-origin for Brafmutant GIST.