Laurence Lovat

Biopsy Site Re-localisation Based on the Computation of Epipolar Lines from Two Previous Endoscopic Images

Tracking biopsy sites in endoscopic images can be useful to provide a visual aid for the guidance of surgical tools, for example when endoscopic guided biopsy is required. A new method for re-localisation of these sites is presented in this paper. It makes use of epipolar geometry properties between three images of the same site observed from different viewpoints with an endoscope. Two epipolar lines are derived from the two first images in the third image where the site needs to be re-localised. Their intersection corresponds to the location of the biopsy site. This method was tested with gastroscopic data from 2 patients with 9 series of three images of the oesophagus. The re-localisation error was estimated at less than 1.5 millimetres by a clinical endoscopist, which is sufficient for most clinical endoscopic applications.

Consensus Statements for Management of Barrett's Dysplasia and Early-Stage Esophageal Adenocarcinoma, Based on a Delphi Process

BACKGROUND & AIMS Esophageal adenocarcinoma (EA) is increasingly common among patients with Barretts esophagus (BE). We aimed to provide consensus recommendations based on the medical literature that clinicians could use to manage patients with BE and low-grade dysplasia, high-grade dysplasia (HGD), or early-stage EA. METHODS We performed an international, multidisciplinary, systematic, evidence-based review of different management strategies for patients with BE and dysplasia or early-stage EA. We used a Delphi process to develop consensus statements. The results of literature searches were screened using a unique, interactive, Web-based data-sifting platform; we used 11,904 papers to inform the choice of statements selected. An a priori threshold of 80% agreement was used to establish consensus for each statement. RESULTS Eighty-one of the 91 statements achieved consensus despite generally low quality of evidence, including 8 clinical statements: (1) specimens from endoscopic resection are better than biopsies for staging lesions, (2) it is important to carefully map the size of the dysplastic areas, (3) patients that receive ablative or surgical therapy require endoscopic follow-up, (4) high-resolution endoscopy is necessary for accurate diagnosis, (5) endoscopic therapy for HGD is preferred to surveillance, (6) endoscopic therapy for HGD is preferred to surgery, (7) the combination of endoscopic resection and radiofrequency ablation is the most effective therapy, and (8) after endoscopic removal of lesions from patients with HGD, all areas of BE should be ablated. CONCLUSIONS We developed a data-sifting platform and used the Delphi process to create evidence-based consensus statements for the management of patients with BE and early-stage EA. This approach identified important clinical features of the diseases and areas for future studies.

Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus

Barretts esophagus is an example of a pre-invasive state, for which current endoscopic surveillance methods to detect dysplasia are time consuming and inadequate. The prognosis of cancer arising in Barretts esophagus is improved by early detection at the stage of mucosal carcinoma or high-grade dysplasia. Molecular imaging methods could revolutionize the detection of dysplasia, provided they permit a wide field of view and highlight abnormalities in real time. We show here that cell-surface glycans are altered in the progression from Barretts esophagus to adenocarcinoma and lead to specific changes in lectin binding patterns. We chose wheat germ agglutinin as a candidate lectin with clinical potential. The binding of wheat germ agglutinin to human tissue was determined to be specific, and we validated this specific binding by successful endoscopic visualization of high-grade dysplastic lesions, which were not detectable by conventional endoscopy, with a high signal-to-background ratio of over 5.

Population-Based Study Reveals New Risk-Stratification Biomarker Panel for Barrett's Esophagus

BACKGROUND & AIMS The risk of progression of Barretts esophagus (BE) to esophageal adenocarcinoma (EAC) is low and difficult to calculate. Accurate tools to determine risk are needed to optimize surveillance and intervention. We assessed the ability of candidate biomarkers to predict which cases of BE will progress to EAC or high-grade dysplasia and identified those that can be measured in formalin-fixed tissues. METHODS We analyzed data from a nested case-control study performed using the population-based Northern Ireland BE Register (1993-2005). Cases who progressed to EAC (n = 89) or high-grade dysplasia ≥ 6 months after diagnosis with BE were matched to controls (nonprogressors, n = 291), for age, sex, and year of BE diagnosis. Established biomarkers (abnormal DNA content, p53, and cyclin A expression) and new biomarkers (levels of sialyl Lewis(a), Lewis(x), and Aspergillus oryzae lectin [AOL] and binding of wheat germ agglutinin) were assessed in paraffin-embedded tissue samples from patients with a first diagnosis of BE. Conditional logistic regression analysis was applied to assess odds of progression for patients with dysplastic and nondysplastic BE, based on biomarker status. RESULTS Low-grade dysplasia and all biomarkers tested, other than Lewis(x), were associated with risk of EAC or high-grade dysplasia. In backward selection, a panel comprising low-grade dysplasia, abnormal DNA ploidy, and AOL most accurately identified progressors and nonprogressors. The adjusted odds ratio for progression of patients with BE with low-grade dysplasia was 3.74 (95% confidence interval, 2.43-5.79) for each additional biomarker and the risk increased by 2.99 for each additional factor (95% confidence interval, 1.72-5.20) in patients without dysplasia. CONCLUSIONS Low-grade dysplasia, abnormal DNA ploidy, and AOL can be used to identify patients with BE most likely to develop EAC or high-grade dysplasia.

Gastrin-Induced Cyclooxygenase-2 Expression in Barrett's Carcinogenesis

Purpose: Cyclooxygenase (COX)-2 has been causally implicated in carcinogenesis. The evidence for increased COX-2 in the malignant progression of Barrett’s esophagus is contradictory. We hypothesize that COX-2 expression may be causally affected by the gastrin status via the cholecystokinin 2 (CCK2) receptor. Experimental Design: COX-2 and prostaglandin E2 expression were evaluated by Western blotting and enzyme-linked immune assay in samples of squamous esophagus, Barrett’s esophagus with varying degrees of dysplasia to adenocarcinoma, and normal duodenum. Differentiation status was evaluated by histopathology and villin expression. A longitudinal case-control study compared COX-2 in patients who progressed to adenocarcinoma with nonprogressors matched for age and length of follow-up. Messenger RNA levels of gastrin and CCK2 receptor in biopsies and cell lines were evaluated by reverse transcription-PCR, and in vitro gastrin stimulation was conducted with and without inhibitors for CCK2 (YM022) and COX-2 (NS-398). Cell proliferation was evaluated using minichromosome maintenance protein 2 (Mcm2) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Results: COX-2 expression is significantly increased in Barrett’s esophagus before dysplasia development. Expression is highly variable within Barrett’s dysplasia and adenocarcinoma samples independent of differentiation status. In a longitudinal case-control study, the expression levels within patients increased over time, regardless of the degree of malignant progression. Biopsies from nondysplastic Barrett’s esophagus expressed increased gastrin mRNA levels compared with other biopsies. Gastrin significantly induced COX-2, prostaglandin E2, and cell proliferation in biopsies and cell lines. Gastrin-induced proliferation can be inhibited by YM022 and NS-398. Conclusions: COX-2 is up-regulated early in the Barrett’s metaplasia sequence. During carcinogenesis, gastrin is a significant determinant of COX-2 activity levels via the CCK2 receptor.

Evaluation of a Minimally Invasive Cell Sampling Device Coupled with Assessment of Trefoil Factor 3 Expression for Diagnosing Barrett's Esophagus: A Multi-Center Case-Control Study

Background Barretts esophagus (BE) is a commonly undiagnosed condition that predisposes to esophageal adenocarcinoma. Routine endoscopic screening for BE is not recommended because of the burden this would impose on the health care system. The objective of this study was to determine whether a novel approach using a minimally invasive cell sampling device, the Cytosponge, coupled with immunohistochemical staining for the biomarker Trefoil Factor 3 (TFF3), could be used to identify patients who warrant endoscopy to diagnose BE. Methods and Findings A case–control study was performed across 11 UK hospitals between July 2011 and December 2013. In total, 1,110 individuals comprising 463 controls with dyspepsia and reflux symptoms and 647 BE cases swallowed a Cytosponge prior to endoscopy. The primary outcome measures were to evaluate the safety, acceptability, and accuracy of the Cytosponge-TFF3 test compared with endoscopy and biopsy. In all, 1,042 (93.9%) patients successfully swallowed the Cytosponge, and no serious adverse events were attributed to the device. The Cytosponge was rated favorably, using a visual analogue scale, compared with endoscopy (p < 0.001), and patients who were not sedated for endoscopy were more likely to rate the Cytosponge higher than endoscopy (Mann-Whitney test, p < 0.001). The overall sensitivity of the test was 79.9% (95% CI 76.4%–83.0%), increasing to 87.2% (95% CI 83.0%–90.6%) for patients with ≥3 cm of circumferential BE, known to confer a higher cancer risk. The sensitivity increased to 89.7% (95% CI 82.3%–94.8%) in 107 patients who swallowed the device twice during the study course. There was no loss of sensitivity in patients with dysplasia. The specificity for diagnosing BE was 92.4% (95% CI 89.5%–94.7%). The case–control design of the study means that the results are not generalizable to a primary care population. Another limitation is that the acceptability data were limited to a single measure. Conclusions The Cytosponge-TFF3 test is safe and acceptable, and has accuracy comparable to other screening tests. This test may be a simple and inexpensive approach to identify patients with reflux symptoms who warrant endoscopy to diagnose BE.

Cyclin A Immunocytology as a Risk Stratification Tool for Barrett's Esophagus Surveillance

Purpose: Endoscopic surveillance of Barretts esophagus (BE) by histopathologic biopsy assessment is suboptimal. A proliferation marker, minichromosome maintenance protein 2, has potential as a biomarker but lacks specificity. We hypothesized that cyclin A, which detects a proportion of proliferating cells, would be more specific. Because cytologic sampling has clinical advantages, we also evaluated the efficacy of cyclin A in endoscopic brushing samples. Experimental Design: A cross-sectional cyclin A immunostaining study was done in 77 patients attending for BE surveillance and 17 patients undergoing evaluation of esophageal adenocarcinoma. The control tissues were as follows: 30 squamous esophagus, 20 gastric antrum, and 13 duodenum. A nested case-control study was done within the same surveillance cohort (16 progressors compared with 32 matched controls) to determine the relative risk for progression. Immunocytology was done for endoscopic brushings collected prospectively from 75 BE ± dysplasia and 33 esophageal adenocarcinomas. Results: Surface expression of cyclin A in BE samples correlated with the degree of dysplasia (P = 0.016). In the case-control cohort, patients with biopsies expressing cyclin A at the surface were more likely to progress to adenocarcinoma than those who did not (odds ratio, 7.5; 95% confidence interval, 1.8-30.7). The sensitivity and specificity of cyclin A expression in brushings for the detection of high-grade dysplasia and cancer patients were 97.8% and 58.7%, respectively. The associated negative predictive value was 97.4%. Conclusions: Cyclin A immunopositivity correlates with cancer risk. Application of this marker to endoscopic brushings could be used as a first step to identify BE patients with the highest risk of progression.

Image cytometry accurately detects DNA ploidy abnormalities and predicts late relapse to high-grade dysplasia and adenocarcinoma in Barrett's oesophagus following photodynamic therapy

Background and aims:DNA ploidy abnormalities (aneuploidy/tetraploidy) measured by flow cytometry (FC) are strong predictors of future cancer development in untreated Barretts oesophagus, independent of histology grade. Image cytometric DNA analysis (ICDA) is an optical technique allowing visualisation of abnormal nuclei that may be undertaken on archival tissue. Our aim was to determine the accuracy of ICDA vs FC, and evaluate DNA ploidy as a prognostic biomarker after histologically successful treatment with photodynamic therapy (PDT).Methods:Nuclei were extracted from 40 μm sections of paraffin-embedded biopsies and processed for ICDA at UCL and FC at UW using standardised protocols. Subsequently, DNA ploidy was evaluated by ICDA on a cohort of 30 patients clear of dysplasia 1 year after aminolaevulinic acid PDT for high-grade dysplasia (HGD). The results were correlated with long-term outcome.Results:In the comparative study, 93% (41 out of 44) of cases were classified identically. Errors occurred in the near-diploid region by ICDA and the tetraploid region by FC. In the cohort study, there were 13 cases of late relapse (7 cancer, 6 HGD) and 17 patients who remained free of dysplasia after a mean follow-up of 44 months. Aneuploidy post-PDT was highly predictive for recurrent HGD or cancer with a hazard ratio of 8.2 (1.8–37.8) (log-rank P=0.001).Conclusions:ICDA is accurate for the detection of DNA ploidy abnormalities when compared with FC. After histologically successful PDT, patients with residual aneuploidy are significantly more likely to develop HGD or cancer than those who become diploid. DNA ploidy by ICDA is a valuable prognostic biomarker after ablative therapy.

Improvement over time in outcomes for patients undergoing endoscopic therapy for Barrett's oesophagus-related neoplasia: 6-year experience from the first 500 patients treated in the UK patient registry

Background Barretts oesophagus (BE) is a pre-malignant condition leading to oesophageal adenocarcinoma (OAC). Treatment of neoplasia at an early stage is desirable. Combined endoscopic mucosal resection (EMR) followed by radiofrequency ablation (RFA) is an alternative to surgery for patients with BE-related neoplasia. Methods We examined prospective data from the UK registry of patients undergoing RFA/EMR for BE-related neoplasia from 2008 to 2013. Before RFA, visible lesions were removed by EMR. Thereafter, patients had RFA 3-monthly until all BE was ablated or cancer developed (endpoints). End of treatment biopsies were recommended at around 12 months from first RFA treatment or when endpoints were reached. Outcomes for clearance of dysplasia (CR-D) and BE (CR-IM) at end of treatment were assessed over two time periods (2008–2010 and 2011–2013). Durability of successful treatment and progression to OAC were also evaluated. Results 508 patients have completed treatment. CR-D and CR-IM improved significantly between the former and later time periods, from 77% and 56% to 92% and 83%, respectively (p<0.0001). EMR for visible lesions prior to RFA increased from 48% to 60% (p=0.013). Rescue EMR after RFA decreased from 13% to 2% (p<0.0001). Progression to OAC at 12 months is not significantly different (3.6% vs 2.1%, p=0.51). Conclusions Clinical outcomes for BE neoplasia have improved significantly over the past 6 years with improved lesion recognition and aggressive resection of visible lesions before RFA. Despite advances in technique, the rate of cancer progression remains 2–4% at 1 year in these high-risk patients. Trial registration number ISRCTN93069556.

Mass Spectrometric Analysis of Exhaled Breath for the Identification of Volatile Organic Compound Biomarkers in Esophageal and Gastric Adenocarcinoma

Objective: The present study assessed whether exhaled breath analysis using Selected Ion Flow Tube Mass Spectrometry could distinguish esophageal and gastric adenocarcinoma from noncancer controls. Background: The majority of patients with upper gastrointestinal cancer present with advanced disease, resulting in poor long-term survival rates. Novel methods are needed to diagnose potentially curable upper gastrointestinal malignancies. Methods: A Profile-3 Selected Ion Flow Tube Mass Spectrometry instrument was used for analysis of volatile organic compounds (VOCs) within exhaled breath samples. All study participants had undergone upper gastrointestinal endoscopy on the day of breath sampling. Receiver operating characteristic analysis and a diagnostic risk prediction model were used to assess the discriminatory accuracy of the identified VOCs. Results: Exhaled breath samples were analyzed from 81 patients with esophageal (N = 48) or gastric adenocarcinoma (N = 33) and 129 controls including Barretts metaplasia (N = 16), benign upper gastrointestinal diseases (N = 62), or a normal upper gastrointestinal tract (N = 51). Twelve VOCs—pentanoic acid, hexanoic acid, phenol, methyl phenol, ethyl phenol, butanal, pentanal, hexanal, heptanal, octanal, nonanal, and decanal—were present at significantly higher concentrations (P < 0.05) in the cancer groups than in the noncancer controls. The area under the ROC curve using these significant VOCs to discriminate esophageal and gastric adenocarcinoma from those with normal upper gastrointestinal tracts was 0.97 and 0.98, respectively. The area under the ROC curve for the model and validation subsets of the diagnostic prediction model was 0.92 ± 0.01 and 0.87 ± 0.03, respectively. Conclusions: Distinct exhaled breath VOC profiles can distinguish patients with esophageal and gastric adenocarcinoma from noncancer controls.