Maria O’Donovan

Acceptability and accuracy of a non-endoscopic screening test for Barrett's oesophagus in primary care: cohort study

Objectives To determine the accuracy and acceptability to patients of non-endoscopic screening for Barrett’s oesophagus, using an ingestible oesophageal sampling device (Cytosponge) coupled with immunocytochemisty for trefoil factor 3. Design Prospective cohort study. Setting 12 UK general practices, with gastroscopies carried out in one hospital endoscopy unit. Participants 504 of 2696 eligible patients (18.7%) aged 50 to 70 years with a previous prescription for an acid suppressant (H2 receptor antagonist or proton pump inhibitor) for more than three months in the past five years. Main outcome measures Sensitivity and specificity estimates for detecting Barrett’s oesophagus compared with gastroscopy as the ideal method, and patient anxiety (short form Spielberger state trait anxiety inventory, impact of events scale) and acceptability (visual analogue scale) of the test. Results 501 of 504 (99%) participants (median age 62, male to female ratio 1:1.2) successfully swallowed the Cytosponge. No serious adverse events occurred. In total, 3.0% (15/501) had an endoscopic diagnosis of Barrett’s oesophagus (≥1 cm circumferential length, median circumferential and maximal length of 2 cm and 5 cm, respectively) with intestinal metaplasia. Compared with gastroscopy the sensitivity and specificity of the test was 73.3% (95% confidence interval 44.9% to 92.2%) and 93.8% (91.3% to 95.8%) for 1 cm or more circumferential length and 90.0% (55.5% to 99.7%) and 93.5% (90.9% to 95.5%) for clinically relevant segments of 2 cm or more. Most participants (355/496, 82%, 95% confidence interval 78.9% to 85.1%) reported low levels of anxiety before the test, and scores remained within normal limits at follow-up. Less than 4.5% (2.8% to 6.1%) of participants reported psychological distress a week after the procedure. Conclusions The performance of the Cytosponge test was promising and the procedure was well tolerated. These data bring screening for Barrett’s oesophagus into the realm of possibility. Further evaluation is recommended.

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.

Accuracy, Safety, and Tolerability of Tissue Collection by Cytosponge vs Endoscopy for Evaluation of Eosinophilic Esophagitis

BACKGROUND & AIMS Management of eosinophilic esophagitis (EoE) requires repeated endoscopic collection of mucosal samples to assess disease activity and response to therapy. An easier and less expensive means of monitoring of EoE is required. We compared the accuracy, safety, and tolerability of sample collection via Cytosponge (an ingestible gelatin capsule comprising compressed mesh attached to a string) with those of endoscopy for assessment of EoE. METHODS Esophageal tissues were collected from 20 patients with EoE (all with dysphagia, 15 with stricture, 13 with active EoE) via Cytosponge and then by endoscopy. Number of eosinophils/high-power field and levels of eosinophil-derived neurotoxin were determined; hematoxylin-eosin staining was performed. We compared the adequacy, diagnostic accuracy, safety, and patient preference for sample collection via Cytosponge vs endoscopy procedures. RESULTS All 20 samples collected by Cytosponge were adequate for analysis. By using a cutoff value of 15 eosinophils/high power field, analysis of samples collected by Cytosponge identified 11 of the 13 individuals with active EoE (83%); additional features such as abscesses were also identified. Numbers of eosinophils in samples collected by Cytosponge correlated with those in samples collected by endoscopy (r = 0.50, P = .025). Analysis of tissues collected by Cytosponge identified 4 of the 7 patients without active EoE (57% specificity), as well as 3 cases of active EoE not identified by analysis of endoscopy samples. Including information on level of eosinophil-derived neurotoxin did not increase the accuracy of diagnosis. No complications occurred during the Cytosponge procedure, which was preferred by all patients, compared with endoscopy. CONCLUSIONS In a feasibility study, the Cytosponge is a safe and well-tolerated method for collecting near mucosal specimens. Analysis of numbers of eosinophils/high-power field identified patients with active EoE with 83% sensitivity. Larger studies are needed to establish the efficacy and safety of this method of esophageal tissue collection. ClinicalTrials.gov number: NCT01585103.

Gastro-esophageal reflux disease symptoms and demographic factors as a pre-screening tool for Barrett's esophagus.

Background Barrett’s esophagus (BE) occurs as consequence of reflux and is a risk factor for esophageal adenocarcinoma. The current “gold-standard” for diagnosing BE is endoscopy which remains prohibitively expensive and impractical as a population screening tool. We aimed to develop a pre-screening tool to aid decision making for diagnostic referrals. Methodology/Principal Findings A prospective (training) cohort of 1603 patients attending for endoscopy was used for identification of risk factors to develop a risk prediction model. Factors associated with BE in the univariate analysis were selected to develop prediction models that were validated in an independent, external cohort of 477 non-BE patients referred for endoscopy with symptoms of reflux or dyspepsia. Two prediction models were developed separately for columnar lined epithelium (CLE) of any length and using a stricter definition of intestinal metaplasia (IM) with segments ≥2 cm with areas under the ROC curves (AUC) of 0.72 (95%CI: 0.67–0.77) and 0.81 (95%CI: 0.76–0.86), respectively. The two prediction models included demographics (age, sex), symptoms (heartburn, acid reflux, chest pain, abdominal pain) and medication for “stomach” symptoms. These two models were validated in the independent cohort with AUCs of 0.61 (95%CI: 0.54–0.68) and 0.64 (95%CI: 0.52–0.77) for CLE and IM≥2 cm, respectively. Conclusions We have identified and validated two prediction models for CLE and IM≥2 cm. Both models have fair prediction accuracies and can select out around 20% of individuals unlikely to benefit from investigation for Barrett’s esophagus. Such prediction models have the potential to generate useful cost-savings for BE screening among the symptomatic population.

Amplification of TRIM44: Pairing a Prognostic Target With Potential Therapeutic Strategy

Background Many prognostic biomarkers have been proposed recently. However, there is a lack of therapeutic strategies exploiting novel prognostic biomarkers. We aimed to propose therapeutic options in patients with overexpression of TRIM44, a recently identified prognostic gene. Methods Genomic and transcriptomic data of epithelial cancers (n = 1932), breast cancers (BCs; n = 1980) and esophago-gastric cancers (EGCs; n = 163) were used to identify genomic aberrations driving TRIM44 overexpression. The driver gene status of TRIM44 was determined using a small interfering RNA (siRNA) screen of the 11p13 amplicon. Integrative analysis was applied across multiple datasets to identify pathway activation and potential therapeutic strategies. Validation of the in silico findings were performed using in vitro assays, xenografts, and patient samples (n = 160). Results TRIM44 overexpression results from genomic amplification in 16.1% of epithelial cancers, including 8.1% of EGCs and 6.1% of BCs. This was confirmed using fluorescent in situ hybridization. The siRNA screen confirmed TRIM44 to be a driver of the amplicon. In silico analysis revealed an association between TRIM44 and mTOR signalling, supported by a decrease in mTOR signalling after siRNA knockdown of TRIM44 in cell lines and colocalization of TRIM44 and p-mTOR in patient samples. In vitro inhibition studies using an mTOR inhibitor (everolimus) decreased cell viability in two TRIM44-amplified cells lines by 88% and 70% compared with 35% in the control cell line. These findings were recapitulated in xenograft models. Conclusions Genomic amplification drives TRIM44 overexpression in EGCs and BCs. Targeting the mTOR pathway provides a potential therapeutic option for TRIM44-amplified tumors.

Endoscopic TriModal imaging and biomarkers for neoplasia conjoined: a feasibility study in Barrett's esophagus.

In Barretts esophagus (BE), the normal squamous lining of the esophagus is replaced by specialized columnar epithelium. Endoscopic surveillance with autofluorescence imaging (AFI) and molecular biomarkers have been studied separately to detect early neoplasia (EN) in BE. The combination of advanced-imaging modalities and biomarkers has not been investigated; AFI may help detecting biomarkers as a risk-stratification tool. We retrospectively evaluated a cohort of patients undergoing endoscopy for EN in BE with AFI and correlated five biomarkers (HPP1, RUNX3, p16, cyclin A, and p53) in tissue samples with AFI and dysplasia status. Fifty-eight samples from a previous prospective study were selected: 15 true-positive (TP: AFI-positive, EN), 21 false-positive (FP: AFI-positive, no EN), 12 true-negative (TN1; AFI-negative, no EN in sample), 10 true-negative (TN2: AFI-negative, no EN in esophagus). Methylation-specific RT-PCR was performed for HPP1, RUNX3, p16, and immunohistochemistry for cyclin A, p53. P < 0.05 was considered statistically significant. Bonferroni correction was used for multiple comparisons. P16, cyclin A, p53 correlated with dysplasia (P < 0.01, P = 0.003, P < 0.001, respectively). Increased p16 methylation was observed between TP versus TN2 (P = 0.003) and TN1 versus TN2 (P = 0.04) subgroups, suggesting a field defect. Only p53 correlated with AFI-status (P = 0.003). After exclusion of EN samples, significance was lost. Although correlation with dysplasia status was confirmed for p16, cyclin A and p53, underlining the importance of these biomarkers as an early event in neoplastic progression, none of the investigated biomarkers correlated with AFI status. A larger prospective study is needed to assess the combination of AFI and a larger panel of biomarkers to improve risk stratification in BE.

Autofluorescence-Directed Confocal Endomicroscopy in Combination With a Three-Biomarker Panel Can Inform Management Decisions in Barrett’s Esophagus

OBJECTIVES:Barrett’s esophagus (BE) surveillance with white-light endoscopy and quadrantic biopsies (Seattle protocol) is resource intensive and limited by sampling error. Previous work suggests that autofluorescence imaging (AFI) in combination with a molecular panel might reduce the number of biopsies, but this was not sufficiently sensitive for low-grade dysplasia, now a point for endoscopic intervention. Here we used AFI to direct narrow-field imaging tools for real-time optical assessment of dysplasia and biopsies for a biomarker panel. We compared the new diagnostic algorithm with the current standard.METHODS:A total of 55 patients with BE were recruited at a single tertiary referral center. Patients underwent high-resolution endoscopy followed by AFI. AFI-targeted areas (n=194) were examined in turn by narrow-band imaging with magnification (NBIz) and probe-based confocal laser endomicroscopy (pCLE). Biopsies were taken from AFI-targeted areas and tested using an established molecular panel comprising aneuploidy plus cyclin A and p53 immunohistochemistry.RESULTS:In the per-patient analysis the overall sensitivity and specificity of AFI-targeted pCLE were 100% and 53.6% for high-grade dysplasia/intramucosal cancer and 96.4% and 74.1% for any grade of dysplasia, respectively. NBIz had equal specificity for dysplasia detection (74.1%), but significantly lower sensitivity (57.1%) than pCLE. The time required to perform AFI-targeted pCLE was shorter that that taken by the Seattle protocol (P=0.0004). We found enrichment of molecular abnormalities in areas with optical dysplasia by pCLE (P<0.001), regardless of histologic dysplasia. The addition of the 3-biomarker panel reduced the false positive rate of pCLE by 50%, leading to sensitivity and specificity for any grade of dysplasia of 89.2% and 88.9%, respectively.CONCLUSIONS:The combination of pCLE on AFI-targeted areas and a 3-biomarker panel identifies patients with dysplasia.

Methylation panel is a diagnostic biomarker for Barrett’s oesophagus in endoscopic biopsies and non-endoscopic cytology specimens

Objective Barrett’s oesophagus is a premalignant condition that occurs in the context of gastro-oesophageal reflux. However, most Barrett’s cases are undiagnosed because of reliance on endoscopy. We have developed a non-endoscopic tool: the Cytosponge, which when combined with trefoil factor 3 immunohistochemistry, can diagnose Barrett’s oesophagus. We investigated whether a quantitative methylation test that is not reliant on histopathological analysis could be used to diagnose Barrett’s oesophagus. Design Differentially methylated genes between Barrett’s and normal squamous oesophageal biopsies were identified from whole methylome data and confirmed using MethyLight PCR in biopsy samples of squamous oesophagus, gastric cardia and Barrett’s oesophagus. Selected genes were then tested on Cytosponge BEST2 trial samples comprising a pilot cohort (n=20 cases, n=10 controls) and a validation cohort (n=149 cases, n=129 controls). Results Eighteen genes were differentially methylated in patients with Barrett’soesophagus compared with squamous controls. Hypermethylation of TFPI2, TWIST1, ZNF345 and ZNF569 was confirmed in Barrett’s biopsies compared with biopsies from squamous oesophagus and gastric cardia (p<0.05). When tested in Cytosponge samples, these four genes were hypermethylated in patients with Barrett’s oesophagus compared with patients with reflux symptoms (p<0.001). The optimum biomarker to diagnose Barrett’s oesophagus was TFPI2 with a sensitivity and specificity of 82.2% and 95.7%, respectively. Conclusion TFPI2, TWIST1, ZNF345 and ZNF569methylation have promise as diagnostic biomarkers for Barrett’s oesophagus when used in combination with a simple and cost effective non-endoscopic cell collection device.

Comparative study of endoscopic surveillance in hereditary diffuse gastric cancer according to CDH1 mutation status

Background and Aims Hereditary diffuse gastric cancer (HDGC) accounts for 1% of gastric cancer cases. For patients with a germline CDH1 mutation, risk-reducing gastrectomy is recommended. However, for those delaying surgery or for families with no causative mutation identified, regular endoscopy is advised. This study aimed to determine the yield of signet ring cell carcinoma (SRCC) foci in individuals with a CDH1 pathogenic variant compared with those without and how this varies with successive endoscopies. Methods Patients fulfilling HDGC criteria were recruited to a prospective longitudinal cohort study. Endoscopy was performed according to a strict protocol with visual inspection followed by focal lesion and random biopsy sampling to detect foci of SRCC. Survival analysis determined progression to finding of SRCC according to CDH1 mutation status. The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 and 36-item Short Form Health Survey questionnaires assessed quality of life before surveillance and each endoscopy. Results Eighty-five individuals fulfilling HDGC criteria underwent 201 endoscopies; 54 (63.5%) tested positive for CDH1 mutation. SRCC yield was 61.1% in CDH1 mutation carriers compared with 9.7% in noncarriers, and mutation-positive patients had a 10-fold risk of SRCC on endoscopy compared with those with no mutation detected (P < .0005). Yield of SRCC decreased substantially with subsequent endoscopies. Surveillance was associated with improved psychological health. Conclusions SRCC foci are prevalent in CDH1 mutation carriers and can be detected at endoscopy using a standardized, multiple biopsy sampling protocol. Decreasing yield over time suggests that the frequency of endoscopy might be reduced. For patients with no CDH1 pathogenic variant detected, the cost-to-benefit ratio needs to be assessed in view of the low yield.

PTU-154 Sirt2 Modulates the Inflammatory Response in Oesophageal Adenocarcinoma

Introduction Gastro-oesophageal reflux disease is the main risk factor for Barrett’s oesophagus (BE), the precursor lesion to oesophageal adenocarcinoma. In BE, GORD leads to chronic inflammation and to NF-κB pathway activation. SIRT2 is a histone deacetylase involved in deacetylation of key players in the cell, including p65, one subunit of the NF-κB transcription complex. SIRT2 is part of our previously published gene signatures in which loss of SIRT2 confers a poor prognosis and over-expression a good prognosis in keeping with its known role as a tumour suppressor. We hypothesised that exerts its protective effect through recruitment of inflammatory cells to the tumour site via the NF-κB pathway The aim of this study was to assess the inflammatory infiltrate in positive tumoursto assess the relationship betweenthe NF-κB pathway. Methods 76 surgical resection specimen of oesophageal adenocarcinoma were immunostained for SIRT2. An in-depth analysis of the nature of inflammatory cells localised to high SIRT2 areas was done in 5 cases using immune cell markers (CD3, CD4, CD8, CD20, CD56 and CD68). NF- κB and SIRT2 luciferase reporter assays were used with SIRT2 overexpression and TNFα stimulation to study the interplay between the NF- κB pathway and SIRT2. A panel of SIRT2 promoter mutants with mutations of one or two or both NF-κB putative binding sites, identified through an in silico analysis, were also used. Results 32% of the cases were strongly positive for SIRT2 (+3 and +2 on a scale from 0 to +3 where 0 is negative). A higher number of inflammatory cells were identified in SIRT2-positive cases compared to SIRT2 negative cases. In particular, SIRT2 positive cases showed strong staining for CD68 indicating an enrichment in the number of macrophages. SIRT2 overexpression significantly down-regulated NF-κB activity (p = 0.0011). Immunoblotting suggests that this downregulation is probably conferred by the deacetylation of Lysine 310 at the p65 subunit of NF- κB. Luciferase assays with the full-length SIRT2-promoter reporter revealed that the SIRT2 promoter was induced by TNFα stimulation (activates NF- κB pathway). This stimulation resulted in decreased luciferase activity when the NF- κB binding sites mutants were used, suggesting a direct action of NF- κB on SIRT2. Conclusion In oesophageal adenocarcinoma, SIRT2 expression is linked with an increased inflammatory infiltrate, especially macrophages. Luciferase reporter assays suggest that SIRT2 and NF-κB regulate each other. Taken together, downregulation of NF- κB by SIRT2 could be an explanation for the protective effect of SIRT2 overexpression in oesophageal adenocarcinoma. Further work is required to confirm these findings. Disclosure of Interest None Declared