Nondysplastic Barrett's Esophagus Research Articles

OGC P04 Spatial sampling of Barrett's oesophagus reveals that tumour heterogeneity is not a useful predictor of progression to cancer

Abstract Background Intra-tumour heterogeneity has been well-described in oesophageal adenocarcinoma and Barrett's oesophagus (BE) segments also consist of multiple clones (groups of cells which share mutations). In BE there is some suggestion that the number of individual genetic clones within the segment and the diversity of the segment can be predictors of progression to cancer. However, this was with pre- whole genome sequencing (WGS) methods. In this study we consider the detailed spatial maps of ten patients with different disease trajectories, using WGS, to determine the heterogeneity and correlate it with their progression. Methods We performed WGS (50x) on 35 high-quality, frozen, endoscopic biopsies taken spatially from ten cases (8 males, 2 females, median age 75 (range 31–83). This included 4 long-term non-dysplastic (ND) patients with long BE segments and 6 patients with dysplasia. We used the copy number and purity information (from Battenberg) and the somatic mutation clustering information (from DPClust) to infer the clonal and subclonal architecture of individual biopsies and looked at the spatial relationship between biopsies within the segment. Results In both ND and dysplastic BE we saw cells with unrelated patterns of mutations, or clones, implying completely separate origins for different parts of the segments. This suggests independent or parallel evolution. There was a higher burden of shared (truncal) mutations in the dysplastic biopsies (p=0.049), indicating a more recent clonal sweep with some clones being outcompeted. Dysplastic samples had more subclonal copy number aberrations and more clonal driver mutations. In addition, most of the driver gene alterations in the dysplastic trees occurred early, whereas CDKN2A, the p16 locus, was the only truncal mutation found on the ND clone trees. We did not find the number of clones or the diversity of the segment to differ between the two groups. Conclusions Each individual patient showed complex, distinct genomic features and evolutionary trajectories. Some cases arose from a single ancestral clone, while others had few shared mutational variants suggesting multiple clones. Importantly, in this sample of cases we did not see any trend that the clonal diversity or number of clones correlated with progression to cancer. This is different to previous studies and highlights the importance of multiple sampling to determine the propensity for cancer progression even if the samples look similar histologically.

Computer-aided characterization of early cancer in Barrett’s esophagus on i-scan magnification imaging: a multicenter international study

We aimed to develop a computer-aided characterization system that could support the diagnosis of dysplasia in Barrett's esophagus (BE) on magnification endoscopy. Videos were collected in high-definition magnification white-light and virtual chromoendoscopy with i-scan (Pentax Hoya, Japan) imaging in patients with dysplastic and nondysplastic BE (NDBE) from 4 centers. We trained a neural network with a Resnet101 architecture to classify frames as dysplastic or nondysplastic. The network was tested on 3 different scenarios: high-quality still images, all available video frames, and a selected sequence within each video. Fifty-seven patients, each with videos of magnification areas of BE (34 dysplasia, 23 NDBE), were included. Performance was evaluated by a leave-1-patient-out cross-validation method. In all, 60,174 (39,347 dysplasia, 20,827 NDBE) magnification video frames were used to train the network. The testing set included 49,726 i-scan-3/optical enhancement magnification frames. On 350 high-quality still images, the network achieved a sensitivity of 94%, specificity of 86%, and area under the receiver operator curve (AUROC) of 96%. On all 49,726 available video frames, the network achieved a sensitivity of 92%, specificity of 82%, and AUROC of 95%. On a selected sequence of frames per case (total of 11,471 frames), we used an exponentially weighted moving average of classifications on consecutive frames to characterize dysplasia. The network achieved a sensitivity of 92%, specificity of 84%, and AUROC of 96%. The mean assessment speed per frame was 0.0135 seconds (SD ± 0.006). Our network can characterize BE dysplasia with high accuracy and speed on high-quality magnification images and sequence of video frames, moving it toward real-time automated diagnosis.

Clinical Relevance of Random Biopsies From the Esophagogastric Junction After Complete Eradication of Barrett’s Esophagus is Low

Although random histological sampling from the esophagogastric junction (EGJ) after complete eradication of Barrett's esophagus (BE) is recommended, its clinical relevance is questionable. This study aimed to assess the incidence and long-term outcomes of findings from random EGJ biopsies in a nationwide cohort with long-term follow-up. We included all patients with successful endoscopic eradication therapy (EET), defined as complete endoscopic eradication of all visible BE (CE-BE), for early BE neoplasia from the Dutch registry. Patients were treated and followed-up in 9 expert centers according to a joint protocol. Outcomes included the incidence of intestinal metaplasia (IM) at the EGJ (EGJ-IM) and the association between IM and visible (dysplastic) BE recurrence. A total of 1154 patients were included with a median follow-up of 43 months (interquartile range, 22-69 months). At the time of CE-BE, persisting EGJ-IM was found in 7% of patients (78/1154), which was reproduced during further follow-up in 46% of patients (42/78). No significant association existed between persisting EGJ-IM at CE-BE and recurrent non-dysplastic or dysplastic BE (hazard ratio [HR], 1.15; 95% confidence interval [CI], 0.63-2.13 and HR, 0.73; 95% CI, 0.17-3.06, respectively). Among patients with no EGJ-IM at the time of CE-BE (1043/1154; 90%), EGJ-IM recurred in 7% (72/1043) after a median of 21 months (interquartile range, 15-36 months), and was reproduced during further follow-up in 26% of patients (19/72). No association was found between recurrent EGJ-IM and non-dysplastic or dysplastic recurrence (HR, 1.18; 95% CI, 0.67-2.06 and HR, 0.27; 95% CI, 0.04-1.96, respectively). Because EGJ-IM was not associated with a higher risk for recurrent disease, we recommend to consider abandoning random EGJ sampling after successful EET, under the condition that care is provided in expert centers, and the esophagus, including the EGJ, is carefully inspected (Netherlands Trial Register, NL7309).

S2257 Cirrhosis, Varices, and Barrett’s Esophagus: WATS3D-only Surveillance and Band Ligation in the Management of Nondysplastic Barrett’s Esophagus Progressing to High Grade Dysplasia With Underlying Esophageal Varices

Introduction: Wide-area transepithelial sampling with 3-dimensional computer-assisted analysis (WATS3D) is an emerging technique used to collect esophageal samples via transepithelial brushing. WATS3D can be a safe and useful tool in Barrett’s esophagus (BE) management in patients with underlying small esophageal varices (EV) where traditional 4 quadrant cold forceps biopsies (CFB) would be contraindicated due to the risk of adverse events such as bleeding. We describe the utility of WATS3D-only surveillance combined with band ligation in the management and eradication of BE which progressed to high grade dysplasia (HGD) in a patient with underlying cirrhosis and EV. Case Description/Methods: We present a 66-year-old male with a history of nondysplastic (ND) BE (C0-M1), small Grade 1 EV and cirrhosis who presented for BE surveillance. Due to underlying EV and associated risk of bleeding, CFB was not performed and sampling with WATS3D-only was undertaken. WATS3D was first performed with surveillance esophagogastroduodenoscopy (EGD) in 2015 and demonstrated ND BE. On further EGD surveillance, BE histology remained ND until early 2021 where WATS3D revealed low grade dysplasia (LGD). Repeat EGD with WATS3D brushing was performed 4 months later, which demonstrated progression to HGD. Interval surveillance EGD with WATS3D brushing was, again, performed 4 months later and demonstrated LGD with rare foci of HGD. Of note, in both instances band ligation of the dysplastic segments was done. EGD with WATS3D 3 months later revealed endoscopic clearance of BE post-banding and pathology report revealed, ND, non-goblet cell metaplasia. The final interval surveillance EGD with WATS3D, 3 months later, showed complete eradication of BE with no evidence of intestinal metaplasia. Table 1 provides a thorough chronological description from first EGD to complete resolution of BE, 8 years later. Discussion: WATS3D has been increasingly shown to be an effective BE surveillance and screening tool. However, given the lack of data regarding appropriate surveillance windows for WATS3D-only BE surveillance, we performed sampling at shorter intervals than what would otherwise be recommended, to confirm the lack of dysplasia. This case presents a unique approach to WATS3D-only surveillance combined with band ligation therapy of dysplasia in the management of BE in a cirrhotic patient with underlying EV. Additionally, this case is unique in clearly documenting progression of a short segment of ND BE to HGD with WATS3D sampling alone. Table 1. - Summary of findings and intervention from first EGD to complete resolution of BE (8 year window) Date Variceal Grade Prague Score Number of BE Islands Sampling done by WATS3D Pathology Findings Banding therapy (# of bands) 04/09/14 2 C0-M1 3 No N/A 0 07/29/15 2 C0-M1 Scattered No N/A 0 12/2315 1 C0-M1 Scattered Yes No dysplasia 0 06/15/16 1 C0-M1 Scattered Yes No dysplasia 0 02/08/17 2 C0-M1 Scattered No N/A 4 01/22/18 1 C0-M1 1 No N/A 0 01/30/19 2 C0-M1 2 No N/A 0 01/29/20 1 C0-M1 2 No N/A 0 02/01/21 1 C0-M1 2 Yes Low-grade dysplasia 0 06/01/21 1 C0-M1 2 Yes High-grade dysplasia 3 10/25/21 1 C0-M1 2 Yes Low-grade with rare foci of high-grade dysplasia 2 01/25/22 1 N/A 3 Yes Non-goblet cell metaplasia, no dysplasia 0 04/25/22 1 N/A 0 Yes Squamocolumnar, no dysplasia or metaplasia 0

488. RADIOFREQUENCY ABLATION OF LOW-GRADE DYSPLASIA IS COST-EFFECTIVE FOR PATIENTS WITH LONG-SEGMENT BARRETT'S OESOPHAGUS

Abstract Barrett’s oesophagus is a precursor to oesophageal adenocarcinoma (OAC), which can be lethal if found at late stages. Stages of Barrett's oesophagus are generally divided into non-dysplasia or dysplasia. There is little doubt that patients with high-grade dysplasia require endoscopic intervention, usually with ablative techniques. Endoscopic treatment of low-grade dysplasia (LGD) has been more controversial, however. Our main aim was to improve cost-effectiveness of LGD ablation using length of Barrett's oesophagus to select high-risk individuals. A cost-utility analysis was undertaken using a Markov cohort model, which simulated progression and regression of non-dysplastic Barrett’s oesophagus (NDBE) to EAC (cycle length of 6 months, time horizon of 35 years). Health states included: NDBE, regression of metaplasia, low-grade dysplasia (LGD), high-grade dysplasia (HGD), EAC, and death. Cost pertaining to investigations and treatments were sourced from local health network. Quality-adjusted life-year (QALY) data were taken from literature values. Initial estimates of transition probabilities between health states were derived from literature and calibrated to lifetime risk of HGD and OAC using standardised methodology. One-way and probabilistic sensitivity analysis were conducted. Several strategies (39 total) were tested in this cost-utility analysis and were broadly divided into selective (length-based) or non-selective LGD ablation. In addition to LGD ablation, surveillance intervals for NDBE were altered to improve cost-effectiveness. In non-selective group of strategies, only 1 was found to be cost-effective, whereas 24 of 28 length-based LGD ablation strategies were found to be cost-effective (at a willingness to pay threshold of AU$50,000/QALY). Probabilistic analysis (1000 simulations) showed that exclusion of individuals with short segment Barrett's oesophagus (SSBE) from surveillance with ablation of LGD in long-segment Barrett's oesophagus (LSBE) was the most cost-effective strategy (AU$33,031/QALY). Patients with long-segment Barrett's oesophagus are at significantly higher risk of progression to adenocarcinoma. Results of this study indicate reducing endoscopic surveillance for SSBE with routine surveillance for LSBE with ablation of LGD when detected is cost-effective in 100% of the simulations. Several studies in recent years have shown similar results. Thus ablation of LGD in Barrett's oesophagus requires serious consideration, especially for high-risk individuals.

335. LONG TERM RISK FOR PROGRESSION TO NEOPLASIA FOR BARRETT’S ESOPHAGUS PATIENTS WITH VALIDATED LOW-GRADE DYSPLASIA OR INDEFINITE FOR DYSPLASIA

Abstract Barrett’s esophagus (BE) with low-grade dysplasia (LGD) confirmed by two pathologists has a high risk of progression to high-grade dysplasia and cancer why ablation or more intense surveillance is recommended. Recent studies suggest that the progression risks for indefinite for dysplasia (IND) and LGD are similar, but the management guidelines are still different. We did a retrospective analysis to calculate the long-term risk for patients with verified LGD and IND. All patients in the Ersta hospital BE-database in Stockholm who at least on one occasion during 1998 to 2012 were diagnosed with dysplasia. The first dysplastic specimen was re-evaluated by two highly experienced GI-pathologist. Clinical data was retrieved from the database and from patient medical records. Incidence rate (IR) was calculated for developing high-grade dysplasia (HGD) or cancer and IR ratio (IRR) was used as relative risk evaluation between the non-dysplastic Barrett’s Esophagus (NDBE) group and the LGD/IND groups and presented with 95% Confidence Intervals (CI). Of 1180 patients with BE, 455 (39%) were diagnosed with dysplasia. 32 were excluded and 3 had HGD. Of the remaining 420 patients, 266 (63%) could be downgraded to NDBE, 82 (20%) were diagnosed as LGD and 71 (17%) as IND. Total follow-up was 4592 person years. The median follow-up time was 12 years. 34 patients developed neoplasia. The yearly IR for HGD or cancer among patients downgraded to NDBE was found to be 0,3% compared to 1,8% for LGD and 1,4% for IND. There was a five-fold increased risk of HGD or cancer among LGD/IND as compared to NDBE. Patients with confirmed IND and LGD were found to have almost similarly elevated long-term risk for progression to neoplasia. Therefore, it seems reasonable that IND patients undergo the same surveillance regimen as non-treated LGD patients. Four patients (one with NDBE, two IND and one LGD) developed cancer more than 20 years after their initial BE diagnosis and therefore, it should be considered to keep BE patients in surveillance programs for a long period of time.

533. OUTCOMES OF RADIOFREQUENCY ABLATION TO TREAT BARRETT’S OESOPHAGUS: AN IRISH MULTICENTRE STUDY

Abstract The standard two-step approach of endoscopic mucosal resection (EMR) followed by radiofrequency ablation (RFA) has shown to be both safe and effective in achieving complete eradication of intestinal metaplasia (CEIM). Despite confidence in the regimen, the long-term sustainability of CEIM has not been well-described, nor has the ideal number of RFA sessions and frequency and length of surveillance. This study used the RIBBON database in Ireland to analyse these factors. Analysis was performed on 398 eligible patients who underwent at least one session of RFA to treat BE from 2009 to 2021 in major centres across Ireland. Patients included had a minimum of 12 months follow-up (FU), with a total 1433.76 patient-years of FU analysed. CEIM was defined as histological and endoscopic absence of BE at post-RFA FU. Patient outcomes were categorised according to recurrence status at FU: sustained CEIM, primary recurrence, treatment failure and unknown outcomes. They were also grouped according to the extent of dysplasia using the Vienna Grading System. Of the 398 patients, 78.14% were male, the mean age at diagnosis was 61.55 years, mean length of Barrett’s segment was 5.36cm, and baseline dysplasia was 19.3% intramucosal carcinoma (IMC), 19% high-grade dysplasia (HGD), 23.3% low-grade dysplasia (LGD) and 37.1% non-dysplastic BE (NBDE). Of these patients, 321 (80.65%) achieved CEIM, at similar proportions. Of those, 67 (20.87%) experienced recurrence over 404.97 patient-years of FU. Further recurrence following subsequent treatment was rare (6 cases). The most common type of recurrence was NDBE (95.64%), and dysplastic recurrence was uncommon (4.36%). The mean time to recurrence was 647.21 days. The results of this study showed that CEIM was achieved by RFA in the majority of cases, with dysplastic recurrence occurring at a very low rate, equating to significantly reduced disease progression. There was a significant difference between the mean number of treatments for those who achieved sustained CEIM (4.15) and those who experienced recurrence (3.29). It is clear that some adjustments to the existing treatment and surveillance regimen could result in improvements in outcome.

An Endoscopic and Histologic Study on Healing of Radiofrequency Ablation Wounds in Patients With Barrett's Esophagus.

Radiofrequency ablation (RFA) of Barrett's esophagus (BE) inflicts a wound spanning 3 epithelial types (stratified squamous, Barrett's metaplasia, gastric epithelium), yet the esophageal injury heals almost completely with squamous epithelium. Knowledge of how this unique wound heals might elucidate mechanisms underlying esophageal metaplasia. We aimed to prospectively and systematically characterize the early endoscopic and histologic features of RFA wound healing. Patients with nondysplastic BE had endoscopy with systematic esophageal photographic mapping, biopsy, and volumetric laser endomicroscopy performed before and at 1, 2, and 4 weeks after RFA. Seven patients (6 men; mean age 56.1 ± 10.9 years) completed this study. Squamous re-epithelialization of RFA wounds did not only progress exclusively through squamous cells extending from the proximal wound edge but also progressed through islands of squamous epithelium sprouting throughout the ablated segment. Volumetric laser endomicroscopy revealed significant post-RFA increases in subepithelial glandular structures associated with the squamous islands. In 2 patients, biopsies of such islands revealed newly forming squamous epithelium contiguous with immature-appearing squamous cells arising from esophageal submucosal gland ducts. Subsquamous intestinal metaplasia (SSIM) was found in biopsies at 2 and/or 4 weeks after RFA in 6 of 7 patients. RFA wounds in BE are re-epithelialized, not just by squamous cells from the proximal wound margin but by scattered squamous islands in which esophageal submucosal gland duct cells seem to redifferentiate into the squamous progenitors that fuel squamous re-epithelialization. SSIM can be found in most patients during the healing process. We speculate that this SSIM might underlie Barrett's recurrences after apparently successful eradication.

Barrett Esophagus: Quality of life and factors associated with illness perception

BackgroundHealth‐related Quality of life (HRQoL) in patients with Barrett's esophagus (BE), a premalignant condition, may be influenced by gastroesophageal reflux disease (GERD) symptoms and the risk of developing esophageal adenocarcinoma.MethodsWe aim to investigate HRQoL in non‐dysplastic Barrett Esophagus (NDBE) patients, identify factors associated with a negative illness perception of the diagnosis BE and compare outcomes between patients treated in a specialized BE center with non‐expert centers. In this multi‐center cross‐sectional study, HRQoL of NDBE patients were assessed using the Short Form 36, Hospital Anxiety and Depression Scale, Cancer worry Scale, and Reflux Disease Questionnaire. A multivariable, linear regression analysis was conducted to assess factors associated with illness perception (Illness perception scale) of the BE diagnosis. Outcome parameters of patients from expert centers were compared to non‐expert centers.ResultsA total of 859 NDBE patients (mean age 63.6% and 74.5% male), of which 640 from BE expert centers were included. BE patients scored similar or higher means (i.e. better) on generic HRQoL in comparison with a Dutch norm population. The multivariable regression model showed that cancer worry, GERD symptoms, signs of anxiety and depression, and female gender were associated with a negative illness perception of BE. GERD symptoms were reported in the minority (22.4%) of BE patients. Levels of anxiety symptoms were comparable to a Dutch norm population (mean 3.7 vs. 3.9 p 0.183) and lower for depression symptoms (mean 6.8 vs. 7.6 p < 0.001). Overall, there were no differences found on outcomes between expert centers and non‐expert centers.ConclusionNDBE patients scored similar or better on generic HRQoL, anxiety and depression than an age and gender matched norm population. The presence of cancer worry, gastrointestinal symptoms, anxiety and depression, and female gender are factors associated with a negative illness perception of the diagnosis BE.

Development of a deep learning model for the histologic diagnosis of dysplasia in Barrett’s esophagus

The risk of progression in Barrett's esophagus (BE) increases with development of dysplasia. There is a critical need to improve the diagnosis of BE dysplasia, given substantial interobserver disagreement among expert pathologists and overdiagnosis of dysplasia by community pathologists. We developed a deep learning model to predict dysplasia grade on whole-slide imaging. We digitized nondysplastic BE (NDBE), low-grade dysplasia (LGD), and high-grade dysplasia (HGD) histology slides. Two expert pathologists confirmed all histology and digitally annotated areas of dysplasia. Training, validation, and test sets were created (by a random 70/20/10 split). We used an ensemble approach combining a "you only look once" model to identify regions of interest and histology class (NDBE, LGD, or HGD) followed by a ResNet101 model pretrained on ImageNet applied to the regions of interest. Diagnostic performance was determined for the whole slide. We included slides from 542 patients (164 NDBE, 226 LGD, and 152 HGD) yielding 8596 bounding boxes in the training set, 1946 bounding boxes in the validation set, and 840 boxes in the test set. When the ensemble model was used, sensitivity and specificity for LGD was 81.3% and 100%, respectively, and >90% for NDBE and HGD. The overall positive predictive value and sensitivity metric (calculated as F1 score) was .91 for NDBE, .90 for LGD, and 1.0 for HGD. We successfully trained and validated a deep learning model to accurately identify dysplasia on whole-slide images. This model can potentially help improve the histologic diagnosis of BE dysplasia and the appropriate application of endoscopic therapy.

Novel DNA Methylation Biomarker Panel for Detection of Esophageal Adenocarcinoma and High-Grade Dysplasia.

Current endoscopy-based screening and surveillance programs have not been proven effective at decreasing esophageal adenocarcinoma (EAC) mortality, creating an unmet need for effective molecular tests for early detection of this highly lethal cancer. We conducted a genome-wide methylation screen to identify novel methylation markers that distinguish EAC and high-grade dysplasia (HGD) from normal squamous epithelium (SQ) or nondysplastic Barrett's esophagus (NDBE). DNA methylation profiling of samples from SQ, NDBE, HGD, and EAC was performed using HM450 methylation arrays (Illumina) and reduced-representation bisulfate sequencing. Ultrasensitive methylation-specific droplet digital PCR and next-generation sequencing (NGS)-based bisulfite-sequencing assays were developed to detect the methylation level of candidate CpGs in independent esophageal biopsy and endoscopic brushing samples. Five candidate methylation markers were significantly hypermethylated in HGD/EAC samples compared with SQ or NDBE (P < 0.01) in both esophageal biopsy and endoscopic brushing samples. In an independent set of brushing samples used to construct biomarker panels, a four-marker panel (model 1) demonstrated sensitivity of 85.0% and 90.8% for HGD and EACs respectively, with 84.2% and 97.9% specificity for NDBE and SQ respectively. In a validation set of brushing samples, the panel achieved sensitivity of 80% and 82.5% for HGD and EAC respectively, at specificity of 67.6% and 96.3% for NDBE and SQ samples. A novel DNA methylation marker panel differentiates HGD/EAC from SQ/NDBE. DNA-methylation-based molecular assays hold promise for the detection of HGD/EAC using esophageal brushing samples.

Targeted genetic and epigenetic profiling of esophageal adenocarcinomas and non-dysplastic Barrett’s esophagus

BackgroundDespite the efforts to describe the molecular landscape of esophageal adenocarcinoma (EAC) and its precursor lesion Barrett’s esophagus (BE), discrepant findings are reported. Here, we investigated the prevalence of selected genetic (TP53 mutations and microsatellite instability (MSI) status) and epigenetic (DNA promoter hypermethylation of APC, CDKN2A, MGMT, TIMP3 and MLH1) modifications in a series of 19 non-dysplastic BE and 145 EAC samples. Additional biopsies from adjacent normal tissue were also evaluated. State-of-the-art methodologies and well-defined scoring criteria were applied in all molecular analyses.ResultsOverall, we confirmed frequent TP53 mutations among EAC (28%) in contrast to BE, which harbored no mutations. We demonstrated that MSI and MLH1 promoter hypermethylation are rare events, both in EAC and in BE. Our findings further support that APC, CDKN2A, MGMT and TIMP3 promoter hypermethylation is frequently seen in both lesions (21–89%), as well as in a subset of adjacent normal samples (up to 12%).ConclusionsOur study further enlightens the molecular background of BE and EAC. To the best of our knowledge, this is one of the largest studies addressing a targeted analysis of genetic and epigenetic modifications simultaneously across a combined series of non-dysplastic BE and EAC samples.

Multicenter Randomized Controlled Trial of Surveillance Versus Endoscopic Therapy for Barrett’s Esophagus With Low-grade Dysplasia: The SURVENT Trial: Study Rationale, Methodology, Innovation, and Implications

Multicenter Randomized Controlled Trial of Surveillance Versus Endoscopic Therapy for Barrett’s Esophagus With Low-grade Dysplasia: The SURVENT Trial: Study Rationale, Methodology, Innovation, and Implications

The utility of P53 immunohistochemistry in the diagnosis of Barrett's oesophagus with indefinite for dysplasia.

AimsBarrett's oesophagus with indefinite for dysplasia (BE‐IND) is a subjective diagnosis with a low interobserver agreement (IOA) among pathologists and uncertain clinical implications. This study aimed to assess the utility of p53 immunohistochemistry (p53‐IHC) in assessing BE‐IND specimens.Methods and resultsArchive endoscopic biopsies with a BE‐IND diagnosis from two academic centres were analysed. First, haematoxylin and eosin‐stained slides (H&E) were reviewed by four expert gastrointestinal (GI) pathologists allocated into two groups (A and B). After a washout period of at least 8 weeks, H&E slides were reassessed side‐to‐side with p53‐IHC available. We compared the rate of changed diagnosis and the IOA for all BE grades before and after p53‐IHC. We included 216 BE‐IND specimens from 185 patients, 44.0 and 32.9% of which were confirmed after H&E slide revision by groups A and B, respectively. More than half the cases were reclassified to a non‐dysplastic BE (NDBE), while 5.6% of cases in group A and 7.4% in group B were reclassified to definite dysplasia. The IOA for NDBE, BE‐IND, low‐grade dysplasia (LGD) and high‐grade dysplasia (HGD)/intramucosal cancer (IMC) was 0.31, 0.21, −0.03 and −0.02, respectively. Use of p53‐IHC led to a >40% reduction in BE‐IND diagnoses (P < 0.001) and increased IOA for all BE grades [κ = 0.46 (NDBE), 0.26 (BE‐IND), 0.49 (LGD), 0.35 (HGD/IMC)]. An aberrant p53‐IHC pattern significantly increased the likelihood of reclassifying BE‐IND to definite dysplasia (odds ratio = 44.3, 95% confidence interval = 18.8–113.0).ConclusionP53‐IHC reduces the rate of BE‐IND diagnoses and improves the IOA among pathologists when reporting BE with equivocal epithelial changes.

Validation of Novel Molecular Imaging Targets Identified by Functional Genomic mRNA Profiling to Detect Dysplasia in Barrett's Esophagus.

Simple SummaryBarrett’s esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE), including low-grade dysplasia (LGD) and high-grade dysplasia (HGD), shows a higher progression risk to EAC compared to non-dysplastic BE (NDBE). If LGD or HGD is detected, more intensive endoscopic surveillance or endoscopic treatment is recommended. This results in a significantly improved prognosis compared to EACs treated by surgery and/or chemoradiotherapy. However, the miss rates for detecting DBE by endoscopy remain high. Fluorescence molecular endoscopy (FME) can fill this gap by targeting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. We identified SPARC, SULF1, PKCι, and DDR1 as promising imaging targets for FME to differentiate DBE from NDBE tissue. We are also the first to develop near-infrared fluorescent tracers, SULF1-800CW and SPARC-800CW, for the endoscopic imaging of DBE tissue. Barrett’s esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE) has a higher progression risk to EAC compared to non-dysplastic BE (NDBE). However, the miss rates for the endoscopic detection of DBE remain high. Fluorescence molecular endoscopy (FME) can detect DBE and mucosal EAC by highlighting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. Publicly available RNA expression profiles of EAC and NDBE were corrected by functional genomic mRNA (FGmRNA) profiling. Following a class comparison between FGmRNA profiles of EAC and NDBE, predicted, significantly upregulated genes in EAC were prioritized by a literature search. Protein expression of prioritized genes was validated by immunohistochemistry (IHC) on DBE and NDBE tissues. Near-infrared fluorescent tracers targeting the proteins were developed and evaluated ex vivo on fresh human specimens. In total, 1976 overexpressed genes were identified in EAC (n = 64) compared to NDBE (n = 66) at RNA level. Prioritization and IHC validation revealed SPARC, SULF1, PKCι, and DDR1 (all p < 0.0001) as the most attractive imaging protein targets for DBE detection. Newly developed tracers SULF1-800CW and SPARC-800CW both showed higher fluorescence intensity in DBE tissue compared to paired non-dysplastic tissue. This study identified SPARC, SULF1, PKCι, and DDR1 as promising targets for FME to differentiate DBE from NDBE tissue, for which SULF1-800CW and SPARC-800CW were successfully ex vivo evaluated. Clinical studies should further validate these findings.

A new artificial intelligence system successfully detectsand localises early neoplasia in Barrett's esophagus by using convolutional neural networks.

Background and aimsSeattle protocol biopsies for Barrett's Esophagus (BE) surveillance are labour intensive with low compliance. Dysplasia detection rates vary, leading to missed lesions. This can potentially be offset with computer aided detection. We have developed convolutional neural networks (CNNs) to identify areas of dysplasia and where to target biopsy.Methods119 Videos were collected in high‐definition white light and optical chromoendoscopy with i‐scan (Pentax Hoya, Japan) imaging in patients with dysplastic and non‐dysplastic BE (NDBE). We trained an indirectly supervised CNN to classify images as dysplastic/non‐dysplastic using whole video annotations to minimise selection bias and maximise accuracy. The CNN was trained using 148,936 video frames (31 dysplastic patients, 31 NDBE, two normal esophagus), validated on 25,161 images from 11 patient videos and tested on 264 iscan‐1 images from 28 dysplastic and 16 NDBE patients which included expert delineations. To localise targeted biopsies/delineations, a second directly supervised CNN was generated based on expert delineations of 94 dysplastic images from 30 patients. This was tested on 86 i‐scan one images from 28 dysplastic patients.FindingsThe indirectly supervised CNN achieved a per image sensitivity in the test set of 91%, specificity 79%, area under receiver operator curve of 93% to detect dysplasia. Per‐lesion sensitivity was 100%. Mean assessment speed was 48 frames per second (fps). 97% of targeted biopsy predictions matched expert and histological assessment at 56 fps. The artificial intelligence system performed better than six endoscopists.InterpretationOur CNNs classify and localise dysplastic Barrett's Esophagus potentially supporting endoscopists during surveillance.

Impact of expert center endoscopic assessment of confirmed low grade dysplasia in Barrett's esophagus diagnosed in community hospitals.

Background The optimal management for patients with low grade dysplasia (LGD) in Barrett’s esophagus (BE) is unclear. According to the Dutch national guideline, all patients with LGD with histological confirmation of the diagnosis by an expert pathologist (i. e. “confirmed LGD”), are referred for a dedicated re-staging endoscopy at an expert center. We aimed to assess the diagnostic value of re-staging endoscopy by an expert endoscopist for patients with confirmed LGD. Methods This retrospective cohort study included all patients with flat BE diagnosed in a community hospital who had confirmed LGD and were referred to one of the nine Barrett Expert Centers (BECs) in the Netherlands. The primary outcome was the proportion of patients with prevalent high grade dysplasia (HGD) or cancer during re-staging in a BEC. Results Of the 248 patients with confirmed LGD, re-staging in the BEC revealed HGD or cancer in 23 % (57/248). In 79 % (45/57), HGD or cancer in a newly detected visible lesion was diagnosed. Of the remaining patients, re-staging in the BEC showed a second diagnosis of confirmed LGD in 68 % (168/248), while the remaining 9 % (23/248) had nondysplastic BE. Conclusion One quarter of patients with apparent flat BE with confirmed LGD diagnosed in a community hospital had prevalent HGD or cancer after re-staging at an expert center. This endorses the advice to refer patients with confirmed LGD, including in the absence of visible lesions, to an expert center for re-staging endoscopy.

Agrin Loss in Barrett's Esophagus-Related Neoplasia and Its Utility as a Diagnostic and Predictive Biomarker.

There is an unmet need for identifying novel biomarkers in Barrett's esophagus that could stratify patients with regards to neoplastic progression. We investigate the expression patterns of extracellular matrix (ECM) molecules in Barrett's esophagus and Barrett's esophagus-related neoplasia, and assess their value as biomarkers for the diagnosis of Barrett's esophagus-related neoplasia and to predict neoplastic progression. Gene-expression analyses of ECM matrisome gene sets were performed using publicly available data on human Barrett's esophagus, Barrett's esophagus-related dysplasia, esophageal adenocarcinoma (ADCA) and normal esophagus. Immunohistochemical expression of basement membrane (BM) marker agrin (AGRN) and p53 was analyzed in biopsies of Barrett's esophagus-related neoplasia from 321 patients in three independent cohorts. Differential gene-expression analysis revealed significant enrichment of ECM matrisome gene sets in dysplastic Barrett's esophagus and ADCA compared with controls. Loss of BM AGRN expression was observed in both Barrett's esophagus-related dysplasia and ADCA. The mean AGRN loss in Barrett's esophagus glands was significantly higher in Barrett's esophagus-related dysplasia and ADCA compared with non-dysplastic Barrett's esophagus (NDBE; P < 0.001; specificity = 82.2% and sensitivity = 96.4%). Loss of AGRN was significantly higher in NDBE samples from progressors compared with non-progressors (P < 0.001) and identified patients who progressed to advanced neoplasia with a specificity of 80.2% and sensitivity of 54.8%. Moreover, the combination of AGRN loss and abnormal p53 staining identified progression to Barrett's esophagus-related advanced neoplasia with a specificity and sensitivity of 86.5% and 58.7%. We highlight ECM changes during Barrett's esophagus progression to neoplasia. BM AGRN loss is a novel diagnostic biomarker that can identify patients with NDBE at increased risk of developing advanced neoplasia.

C5b-9 Membrane Attack Complex Formation andExtracellular Vesicle Shedding in Barrett's Esophagus and Esophageal Adenocarcinoma.

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett’s Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

Prediction of Progression in Barrett’s Esophagus Using a Tissue Systems Pathology Test: A Pooled Analysis of International Multicenter Studies

Prediction of progression risk in Barrett's esophagus (BE) may enable personalized management. We aimed to assess the adjunct value of a tissue systems pathology test (TissueCypher) performed on paraffin-embedded biopsy tissue, when added to expert pathology review in predicting incident progression, pooling individual patient-level data from multiple international studies METHODS: Demographics, clinical features, the TissueCypher risk class/score, and progression status were analyzed. Conditional logistical regression analysis was used to develop multivariable models predicting incident progression with and without the TissueCypher risk class (low, intermediate, high). Concordance (c-) statistics were calculated and compared with likelihood ratio tests to assess predictive ability of models. A risk prediction calculator integrating clinical variables and TissueCypher risk class was also developed. Data from 552 patients with baseline no (n= 472), indefinite (n= 32), or low-grade dysplasia (n= 48) (comprising 152 incident progressors and 400 non-progressors) were analyzed. A high-risk test class independently predicted increased risk of progression to high-grade dysplasia/adenocarcinoma (odds ratio, 6.0; 95% confidence interval, 2.9-12.0), along with expert confirmed low-grade dysplasia (odds ratio, 2.9; 95% confidence interval, 1.2-7.2). Model prediction of progression with the TissueCypher risk class incorporated was significantly superior than without, in the whole cohort (c-statistic 0.75 vs 0.68; P < .0001) and the nondysplastic BE subset (c-statistic 0.72 vs 0.63; P < .0001). Sensitivity and specificity of the high risk TissueCypher class were 38% and 94%, respectively. An objective tissue systems pathology test high-risk class is a strong independent predictor of incident progression in patients with BE, substantially improving progression risk prediction over clinical variables alone. Although test specificity was high, sensitivity was modest.