Xinxue Liu

A genome-wide association study identifies new susceptibility loci for esophageal adenocarcinoma and Barrett's esophagus.

Esophageal adenocarcinoma is a cancer with rising incidence and poor survival. Most such cancers arise in a specialized intestinal metaplastic epithelium, which is diagnostic of Barretts esophagus. In a genome-wide association study, we compared esophageal adenocarcinoma cases (n = 2,390) and individuals with precancerous Barretts esophagus (n = 3,175) with 10,120 controls in 2 phases. For the combined case group, we identified three new associations. The first is at 19p13 (rs10419226: P = 3.6 × 10−10) in CRTC1 (encoding CREB-regulated transcription coactivator), whose aberrant activation has been associated with oncogenic activity. A second is at 9q22 (rs11789015: P = 1.0 × 10−9) in BARX1, which encodes a transcription factor important in esophageal specification. A third is at 3p14 (rs2687201: P = 5.5 × 10−9) near the transcription factor FOXP1, which regulates esophageal development. We also refine a previously reported association with Barretts esophagus near the putative tumor suppressor gene FOXF1 at 16q24 and extend our findings to now include esophageal adenocarcinoma.

Randomized crossover study comparing efficacy of transnasal endoscopy with that of standard endoscopy to detect Barrett's esophagus

BACKGROUND Unsedated transnasal endoscopy (TNE) may be safer and less expensive than standard endoscopy (SE) for detecting Barretts esophagus (BE). Emerging technologies require robust evaluation before routine use. OBJECTIVE To evaluate the sensitivity, specificity, and acceptability of TNE in diagnosing BE compared with those of SE. DESIGN Prospective, randomized, crossover study. SETTING Single, tertiary-care referral center. PATIENTS This study enrolled consecutive patients with BE or those referred for diagnostic assessment. INTERVENTION All patients underwent TNE followed by SE or the reverse. Spielberger State-Trait Anxiety Inventory short-form questionnaires, a visual analogue scale, and a single question addressing preference for endoscopy type were administered. MAIN OUTCOME MEASUREMENTS Diagnostic accuracy and tolerability of TNE were compared with those of SE. RESULTS Of 95 patients randomized, 82 completed the study. We correctly diagnosed 48 of 49 BE cases by TNE for endoscopic findings of columnar lined esophagus compared with the criterion standard, SE, giving a sensitivity and specificity of 0.98 and 1.00, respectively. The BE median length was 3 cm (interquartile range [IQR] 1-5 cm) with SE and 3 cm (IQR 2-4 cm) with TNE, giving high correlations between the two modalities (R(2) = 0.97; P < .001). The sensitivity and specificity for detecting intestinal metaplasia by TNE compared with those by SE was 0.91 and 1.00, respectively. The mean (± standard deviation) post-endoscopy Spielberger State-Trait Anxiety Inventory short-form score for TNE (30.0 ± 1.10 standard error of the mean [SEM]) was lower than that for SE (30.7 ± 1.29 SEM), (P = .054). The visual analogue scale scores were no different (P = .07). The majority of patients (59%) expressed a preference for TNE. LIMITATIONS This is a small study, with limited generalizability, a high prevalence of patients with BE, differential drop-out between the two procedures, and use of sedation. CONCLUSION TNE is an accurate and well-tolerated method for diagnosing BE compared with SE. TNE warrants further evaluation as a screening tool for BE.

DNA Methylation as an Adjunct to Histopathology to Detect Prevalent, Inconspicuous Dysplasia and Early-Stage Neoplasia in Barrett's Esophagus

Purpose: Endoscopic surveillance of Barretts esophagus is problematic because dysplasia/early-stage neoplasia is frequently invisible and likely to be missed because of sampling bias. Molecular abnormalities may be more diffuse than dysplasia. The aim was therefore to test whether DNA methylation, especially on imprinted and X-chromosome genes, is able to detect dysplasia/early-stage neoplasia. Experimental design: 27K methylation arrays were used to find genes best able to differentiate between 22 Barretts esophagus and 24 esophageal adenocarcinoma (EAC) samples. These were validated using pyrosequencing on a retrospective cohort (60 Barretts esophagus, 36 dysplastic, and 90 EAC) and then in a prospective multicenter study (98 Barretts esophagus patients, including 28 dysplastic and 9 early EAC) designed to utilize biomarkers to stratify patients according to their prevalent dysplasia/EAC status. Results: Genes (23%) on the array, including 7% of X-linked and 69% of imprinted genes, have shown statistically significant changes in methylation in EAC versus Barretts esophagus (Wilcoxon P < 0.05). 6/7 selected candidate genes were successfully internally (Pearsons P < 0.01) and externally validated (ANOVA P < 0.001). Four genes (SLC22A18, PIGR, GJA12, and RIN2) showed the greatest area under curve (0.988) to distinguish between Barretts esophagus and dysplasia/EAC in the retrospective cohort. This methylation panel was able to stratify patients from the prospective cohort into three risk groups based on the number of genes methylated (low risk: <2 genes, intermediate: 2, and high: >2). Conclusion: Widespread DNA methylation changes were observed in Barretts carcinogenesis including ≈70% of known imprinted genes. A four-gene methylation panel stratified patients with Barretts esophagus into three risk groups with potential clinical utility. Clin Cancer Res; 19(4); 878–88. ©2012 AACR.

Three-Gene Immunohistochemical Panel Adds to Clinical Staging Algorithms to Predict Prognosis for Patients With Esophageal Adenocarcinoma

PURPOSE Esophageal adenocarcinoma (EAC) is a highly aggressive disease with poor long-term survival. Despite growing knowledge of its biology, no molecular biomarkers are currently used in routine clinical practice to determine prognosis or aid clinical decision making. Hence, this study set out to identify and validate a small, clinically applicable immunohistochemistry (IHC) panel for prognostication in patients with EAC. PATIENTS AND METHODS We recently identified eight molecular prognostic biomarkers using two different genomic platforms. IHC scores of these biomarkers from a UK multicenter cohort (N = 374) were used in univariate Cox regression analysis to determine the smallest biomarker panel with the greatest prognostic power with potential therapeutic relevance. This new panel was validated in two independent cohorts of patients with EAC who had undergone curative esophagectomy from the United States and Europe (N = 666). RESULTS Three of the eight previously identified prognostic molecular biomarkers (epidermal growth factor receptor [EGFR], tripartite motif-containing 44 [TRIM44], and sirtuin 2 [SIRT2]) had the strongest correlation with long-term survival in patients with EAC. Applying these three biomarkers as an IHC panel to the validation cohort segregated patients into two different prognostic groups (P < .01). Adjusting for known survival covariates, including clinical staging criteria, the IHC panel remained an independent predictor, with incremental adverse overall survival (OS) for each positive biomarker (hazard ratio, 1.20; 95% CI, 1.03 to 1.40 per biomarker; P = .02). CONCLUSION We identified and validated a clinically applicable IHC biomarker panel, consisting of EGFR, TRIM44, and SIRT2, that is independently associated with OS and provides additional prognostic information to current survival predictors such as stage.

The combination of autofluorescence endoscopy and molecular biomarkers is a novel diagnostic tool for dysplasia in Barrett's oesophagus

Objective Endoscopic surveillance for Barretts oesophagus (BO) is limited by sampling error and the subjectivity of diagnosing dysplasia. We aimed to compare a biomarker panel on minimal biopsies directed by autofluorescence imaging (AFI) with the standard surveillance protocol to derive an objective tool for dysplasia assessment. Design We performed a cross-sectional prospective study in three tertiary referral centres. Patients with BO underwent high-resolution endoscopy followed by AFI-targeted biopsies. 157 patients completed the biopsy protocol. Aneuploidy/tetraploidy; 9p and 17p loss of heterozygosity; RUNX3, HPP1 and p16 methylation; p53 and cyclin A immunohistochemistry were assessed. Bootstrap resampling was used to select the best diagnostic biomarker panel for high-grade dysplasia (HGD) and early cancer (EC). This panel was validated in an independent cohort of 46 patients. Results Aneuploidy, p53 immunohistochemistry and cyclin A had the strongest association with dysplasia in the per-biopsy analysis and, as a panel, had an area under the receiver operating characteristic curve of 0.97 (95% CI 0.95 to 0.99) for diagnosing HGD/EC. The diagnostic accuracy for HGD/EC of the three-biomarker panel from AFI+ areas was superior to AFI− areas (p<0.001). Compared with the standard protocol, this panel had equal sensitivity for HGD/EC, with a 4.5-fold reduction in the number of biopsies. In an independent cohort of patients, the panel had a sensitivity and specificity for HGD/EC of 100% and 85%, respectively. Conclusions A three-biomarker panel on a small number of AFI-targeted biopsies provides an accurate and objective diagnosis of dysplasia in BO. The clinical implications have to be studied further.

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.

The impact of prophylactic total gastrectomy on health-related quality of life: a prospective cohort study.

Background:The advice to individuals with identified CDH1 mutations is generally to undertake prophylactic total gastrectomy (PTG). This study evaluated the effect of PTG on health-related quality of life (HRQL) in asymptomatic individuals with identified CDH1 mutations at high risk for gastric cancer. Methods:Individuals with hereditary diffuse gastric cancer (HDGC) were recruited to a prospective, multicenter UK study. Questionnaires, including the European Organization for Research and Treatment for Cancer core Quality-of-Life Questionnaire (EORTC QLQ C30); the gastric cancer specific module (EORTC QLQ STO22); and the 36-item short form health survey version 2.0, were completed before and at regular intervals after surgery. Results:Sixty individuals fulfilled HDGC criteria; 38 (63%) had a CDH1 mutation and 32 (53%) underwent PTG. At baseline, there was no significant difference in mental health depending on CDH1 mutation status and treatment preference. Physical functioning reduced in the first month after surgery but recovered to baseline by 12 months. Similarly mental functioning reduced in the first month after surgery but recovered by 3 to 9 months. However, specific symptoms were identified, such as diarrhoea (70%), fatigue (63%), discomfort when eating (81%), reflux (63%), eating restrictions (45%), and body image (44%), which persisted after PTG. Conclusions:Patients contemplating prophylactic gastrectomy can be reassured about the long-term HRQL outcomes, but some residual symptoms require adjustment.

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.

Sa1852 Autofluorescence-Targeted Optical Biopsy Accurately Diagnoses Dysplasia in Barrett's Esophagus and Can Detect the Field of Molecular Change

Introduction Probe-based confocal laser endomicroscopy (pCLE) allows optical biopsies in Barrett’s oesophagus (BO) to predict histological outcome but it is subject to sampling error if performed in a random fashion. We used autofluorescence imaging (AFI) to direct pCLE and added molecular biomarkers to the histopathological diagnosis. The aims of this study were to assess the diagnostic accuracy for dysplasia of AFI-targeted optical biopsies and to investigate the correlation between pCLE patterns and field of molecular change. Methods 46 patients with BO (non-dysplastic BE n = 20, indefinite for dysplasia n = 4, low grade dysplasia n = 10, high grade dysplasia (HGD) or intramucosal cancer (IMC) n = 12) were recruited at a single centre. Patients underwent high-resolution endoscopy followed by AFI and then pCLE was performed on AFI positive (AFI+) areas. Targeted biopsies were taken from AFI+ areas, followed by random biopsies as per Seattle protocol. pCLE sequences were graded according to published criteria. Cyclin A and p53 expression were assessed by immunohistochemistry and aneuploidy by flow-cytometry on AFI-targeted biopsies. Statistical analyses were performed using chi-square test. Results AFI-targeted pCLE correctly classified all the HGD/EC patients and had a sensitivity and specificity for any grade of dysplasia of 93 and 83%, respectively. The Seattle protocol had similar sensitivity for HGD/IMC and any grade of dysplasia (83 and 89%, respectively). For the per-location analysis, a total of 155 endoscopic areas were analysed with pCLE and molecular biomarkers. pCLE had a sensitivity and a specificity for HGD/IMC and any grade of dysplasia of 100/64% and 78/75%, respectively. Overall, 40% of pCLE irregular sequences corresponded to non-dysplastic areas (false positive). We found a statistically significant enrichment (p Conclusion AFI-targeted pCLE has a high diagnostic accuracy for dysplasia in BO. Tissue biomarkers are a useful adjunct to characterise the field of molecular abnormality associated with optical dysplasia. These results suggest that the presence of pCLE irregularity, even in the absence of histological dysplasia, relates to molecular changes and may warrant close follow up. Disclosure of Interest None Declared.