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Stromal genes discriminate preinvasive from invasive disease, predict outcome, and highlight inflammatory pathways in digestive cancers

The stromal compartment is increasingly recognized to play a role in cancer. However, its role in the transition from preinvasive to invasive disease is unknown. Most gastrointestinal tumors have clearly defined premalignant stages, and Barrett’s esophagus (BE) is an ideal research model. Supervised clustering of gene expression profiles from microdissected stroma identified a gene signature that could distinguish between BE metaplasia, dysplasia, and esophageal adenocarcinoma (EAC). EAC patients overexpressing any of the five genes (TMEPAI, JMY, TSP1, FAPα, and BCL6) identified from this stromal signature had a significantly poorer outcome. Gene ontology analysis identified a strong inflammatory component in BE disease progression, and key pathways included cytokine–cytokine receptor interactions and TGF-β. Increased protein levels of inflammatory-related genes significantly up-regulated in EAC compared with preinvasive stages were confirmed in the stroma of independent samples, and in vitro assays confirmed functional relevance of these genes. Gene set enrichment analysis of external datasets demonstrated that the stromal signature was also relevant in the preinvasive to invasive transition of the stomach, colon, and pancreas. These data implicate inflammatory pathways in the genesis of gastrointestinal tract cancers, which can affect prognosis.

In vivo and in vitro evidence for transforming growth factor-β1-mediated epithelial to mesenchymal transition in esophageal adenocarcinoma

There is increasing evidence that epithelial to mesenchymal transition (EMT) is involved in cancer progression. Because local invasion and metastasis occurs early in the pathogenesis of esophageal adenocarcinoma, we hypothesized that EMT may be important in this disease. Using immunohistochemistry in a well-characterized set of adenocarcinoma tissues, we showed down-regulation of epithelial markers (E-cadherin and cytokeratin 18) and up-regulation of mesenchymal markers (vimentin and alpha-smooth muscle actin) with concomitant transforming growth factor-beta1 (TGF-beta1) expression at the invasive margin compared with the central tumor. A panel of esophageal cell lines was examined for the ability of TGF-beta1 to induce EMT in vitro. TE7 cells were selected as a model because TGF-beta1 (0-5 ng/mL) treatment induced morphologic and molecular expression changes suggestive of EMT. In TE7 cells, these TGF-beta1-induced changes were reversed by 100 ng/mL of bone morphogenetic protein 7 (BMP7), another member of the TGF-beta1 superfamily. EMT was mediated via canonical TGF-beta1 signaling with concomitant up-regulation of SMAD-interacting protein 1. Alterations in functional variables (aggregation, wounding, motility, and invasion) following TGF-beta1 treatment were consistent with a more invasive phenotype. These functional changes were reversed by BMP7 and SMAD4 RNA interference in vitro. These data suggest that TGF-beta1-mediated EMT may be relevant in esophageal carcinogenesis.

Retinoic acid induced glandular differentiation of the oesophagus

Background: Retinoic acid (RA) is a powerful differentiation agent. Barrett’s oesophagus occurs when duodeno-gastro-oesophageal reflux causes squamous epithelium (SE) tissue to become columnar epithelium tissue by an unknown mechanism. The bile acid lithocholic acid (LCA) competes for the retinoid X receptor retinoid binding site. Hence, RA pathways may be implicated in Barrett’s oesophagus. Methods: RA activity in tissues and cell lines treated with all-trans retinoic acid (ATRA) with or without LCA was assessed using a reporter. Expression of p21 was determined by real-time PCR in Barrett’s oesophagus cell lines with or without LCA. SE and Barrett’s oesophagus biopsy specimens were exposed to 100 μM of ATRA or 20 mM of a RA inhibitor, citral, in organ culture for >72 h. Characteristics of treated specimens, compared with untreated controls, were analysed by immunohistochemical analysis (cytokeratins (CKs), vimentin) and RT-PCR (CKs). Confocal microscopy assessed temporal changes in co-localisation of CK8/18 and vimentin. Cell proliferation was assessed by bromo-deoxyuridine incorporation and immunohistochemical analysis for Ki67 and p21. Results: RA biosynthesis was increased in Barrett’s oesophagus compared with SE (p<0.001). LCA and ATRA caused a synergistic increase in RA signalling as shown by increased p21 (p<0.01). Morphological and molecular analysis of SE exposed to ATRA showed columnar differentiation independent of proliferation. Metaplasia could be induced from the stromal compartment alone and vimentin expression co-localised with CK8/18 at 24 h, which separated into CK8/18-positive glands and vimentin-positive stroma by 48 h. Citral-treated Barrett’s oesophagus led to phenotypic and immunohistochemical characteristics of SE, which was independent of proliferation. Conclusion: RA activity is increased in Barrett’s oesophagus and is induced by LCA. Under conditions of altered RA activity and an intact stroma, the oesophageal phenotype can be altered independent of proliferation.

An unusual cause of anaemia

A 64-year-old gentleman presented to the emergency department with an episode of angioneurotic oedema ascribed to Valsartan and was noted to have iron deficiency anaemia. He had undergone cardiac transplantation in 1999 and subsequently developed several skin cancers (squamous cell carcinomas and a melanoma removed by the plastic surgeons). Two years prior to this presentation he had been diagnosed with a T3N2 M0 non-small cell lung cancer treated with radiotherapy. The patient was discharged with a plan for outpatient CT study, gastroscopy and colonoscopy but before …

475d A Clinically Applicable Three Gene Signature is Independently Highly Prognostic in Esophageal and Junctional Adenocarcinoma

Introduction The incidence of oesophageal and junctional adenocarcinoma has increased sixfold in the last 30 years and 5-year survival remains Methods Gene expression profiling was performed and the resulting 42 000 gene signatures correlated with clinical features for 91 snap frozen oesophageal and junctional resection specimens. External validation of selected targets was carried out on 371 independent cases using immunohistochemistry to maximise clinical applicability. Results Gene expression profiles were obtained from 75/91 of the samples (82%). 119 genes were significantly associated with survival and 270 genes with the number of involved lymph nodes. Ten of these genes were taken forward to external validation at the protein level. Three genes were prognostic in the external validation data set (TRIM44, SIRT2 and PAPSS2). Patients with dysregulation of none of these three genes had a significantly better outcome (5-year survival 48%) than those with dysregulation of one gene (5-year survival 29%), who in turn did better than those with two or more genes dysregulated (5-year survival 15%) (p=0.004). The three gene signature was independently prognostic in a multivariable model together with the existing clinical TNM staging system (p=0.003). Conclusion We have generated and externally validated an immunohistochemical prognostic gene signature which is independently highly prognostic in an external validation data set. In addition these three genes are possible therapeutic targets raising the possibility of personalised therapy based on a patient9s molecular prognostic signature. Our three gene signature is suitable for application in routine clinical practice and this study may provide a framework for similar future projects.