Multistage study design from marker discovery to validation. Workflow overview of initial marker discovery using WGMS on DNA extracted from endoscopic brushing samples with cross-reference to an archival RRBS dataset from DNA extracted from independent tissue samples, and independent validation comparing paired endoscopic brushings and sponge-on-string samples. EAC, esophageal adenocarcinoma; EM-seq, enzymatic methyl sequencing; methyl-seq, methyl sequencing; WGBS, whole genome bisulfite sequencing.

Aberrant DNA methylation and copy-number alterations (CNA) drive Barrett's esophagus progression to esophageal adenocarcinoma; however, their combined utility for early detection is unclear. We aimed to identify and validate methylated DNA markers (MDM) and CNAs to distinguish esophageal adenocarcinoma/high-grade dysplasia (HGD) from nondysplastic Barrett's esophagus (NDBE). In this multiphase, multicenter study, we discovered and validated MDMs and quantified CNAs utilizing whole-genome methylation sequencing of esophageal brushings. DNA biomarkers identified from discovery were further validated in independent patients with paired esophageal brushing and swallowed capsule sponge samples. MDMs were filtered against a reduced representation bisulfite sequencing dataset obtained from independent tissue samples to advance only concordant candidates. CNA burden was quantified using ichorCNA-derived aneuploidy scores (AS). Two hundred MDMs discovered in HGD (N = 18) and esophageal adenocarcinoma (N = 18) versus NDBE brushing samples (N = 18) were tested in independent samples (N = 146). A 52-MDM panel achieved a cross-validated AUC of 0.88 [95% confidence interval (CI), 0.82-0.95]; the addition of AS improved discrimination of HGD/esophageal adenocarcinoma from NDBE to 0.91 (95% CI, 0.86-0.97) AUC. At 80% specificity, the combined model detected 93% of esophageal adenocarcinoma and 88% of HGD cases. In paired capsule sponge samples, a 58-MDM panel achieved a cross-validated AUC of 0.77 (95% CI, 0.66-0.88); a combined 58-MDM and AS model achieved AUC 0.80 (95% CI, 0.7-0.9). MDMs and AS discerned HGD/esophageal adenocarcinoma from normal esophagus/NDBE in endoscopic brushing and capsule sponge samples. This approach may improve Barrett's esophagus surveillance.Prevention Relevance: This study demonstrates that combining epigenetic and genomic biomarkers across minimally invasive sampling methods can accurately distinguish HGD/esophageal adenocarcinoma from nondysplastic Barrett's esophagus, offering promising, less invasive strategies to improve BE surveillance and enable endoscopic therapy for esophageal adenocarcinoma prevention and treatment.

ROC curves for MDM performance in paired endoscopic brush and sponge samples. A, AUC analysis demonstrating discrimination of HGD/esophageal adenocarcinoma (EAC) from NDBE using the MDM panel, AS, and the combination in endoscopic brush samples. B, Corresponding AUC analysis showing marker performance in paired available (Table 1) sponge-on-string samples from the same patients.

Methylation and copy-number profiles in discovery and validation case and control sample sets. For methylation, each row is a marker from the filtered set of 52 MDMs and each column a patient sample. For copy number, each row is a chromosomal arm. Methylation marker levels measured in deciles above the 95th percentile value in NDBE are denoted by the increasing intensity of yellow to red color spectrum. Copy-number analysis shows amplifications (red) or deletions (blue) referent to neutral (white) as estimated by the AS method. NDBE referent to NE (gray, neutral by definition). EAC, esophageal adenocarcinoma.

AUC of the 52-MDM model in cytology brush validation data comparing NDBE vs. HGD/EAC, stratified by covariates.

Supplemental Table 1: Study adherence to the STARD (Standards for Reporting Diagnostic Accuracy Studies) 2015 checklist.

BACKGROUND & AIMS:Endoscopic Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) detection is invasive and expensive. Nonendoscopic BE/EAC detection tools are guideline-endorsed alternatives. We previously described a 5-methylated DNA marker (MDM) panel assayed on encapsulated sponge cell collection device (CCD) specimens. We aimed to train a new algorithm using a 3-MDM panel and test its performance in an independent cohort. METHODS:Algorithm training and test samples were from 2 prospective multicenter cohorts. All BE cases had esophageal intestinal metaplasia (with or without dysplasia/EAC); control subjects had no endoscopic evidence of BE. The CCD procedure was followed by endoscopy. From CCD cell lysates, DNA was extracted, bisulfite treated, and MDMs were blindly assayed. The algorithm was set and locked using cross-validated logistic regression (training set) and its performance was assessed in an independent test set. RESULTS:Training (N = 352) and test (N = 125) set clinical characteristics were comparable. The final panel included 3 MDMs (NDRG4, VAV3, ZNF682). Overall sensitivity was 82% (95% CI, 68%-94%) at 90% (79%-98%) specificity and 88% (78%-94%) sensitivity at 84% (70%-93%) specificity in training and test sets, respectively. Sensitivity was 90% and 68% for all long- and short-segment BE, respectively. Sensitivity for BE with high-grade dysplasia and EAC was 100% in training and test sets. Overall sensitivity for nondysplastic BE was 82%. Areas under the receiver operating characteristic curves for BE detection were 0.92 and 0.94 in the training and test sets, respectively. CONCLUSIONS:A locked 3-MDM panel algorithm for BE/EAC detection using a nonendoscopic CCD demonstrated excellent sensitivity for high-risk BE cases in independent validation samples. (Clinical trials.gov: NCT02560623, NCT03060642.).

INTRODUCTION:Endoscopic eradication therapy (EET) is standard of care for T1a esophageal adenocarcinoma (EAC). However, data on outcomes in high-risk T1a EAC are limited. We assessed and compared outcomes after EET of low-risk and high-risk T1a EAC, including intraluminal EAC recurrence, extraesophageal metastases, and overall survival. METHODS:Patients who underwent EET for T1a EAC at 3 referral Barrett's esophagus endotherapy units between 1996 and 2022 were included. Patients with submucosal invasion, positive deep margins, or metastases at initial diagnosis were excluded. High-risk T1a EAC was defined as T1a EAC with poor differentiation and/or lymphovascular invasion, with low-risk disease being defined without these features. All pathology was systematically assessed by expert gastrointestinal pathologists. Baseline and follow-up endoscopy and pathology data were abstracted. Time-to-event analyses were performed to compare outcomes between groups. RESULTS:One hundred eighty-eight patients with T1a EAC were included (high risk, n = 45; low risk, n = 143) with a median age of 70 years, and 84% were men. Groups were comparable for age, sex, Barrett's esophagus length, lesion size, and EET technique. Rates of delayed extraesophageal metastases (11.1% vs 1.4%) were significantly higher in the high-risk group ( P = 0.02). There was no significant difference in the rates of intraluminal EAC recurrence ( P = 0.79) and overall survival ( P = 0.73) between the 2 groups. DISCUSSION:Patients with high-risk T1a EAC undergoing successful EET had a substantially higher rate of extraesophageal metastases compared with those with low-risk T1a EAC on long-term follow-up. These data should be factored into discussions with patients while selecting treatment approaches. Additional prospective data in this area are critical.

Background and Aims: Endoscopic eradication therapy (EET) is guideline endorsed for management of early-stage (T1) esophageal adenocarcinoma (EAC). Patients with baseline high-grade dysplasia (HGD) and EAC are at highest risk of recurrence after successful EET, but limited data exist on long-term (>5 year) recurrence outcomes. Our aim was to assess the incidence and predictors of long-term recurrence in a multicenter cohort of patients with T1 EAC treated with EET. Methods: Patients with T1 EAC achieving successful endoscopic cancer eradication with a minimum of 5 years' clinical follow-up were included. The primary outcome was neoplastic recurrence, defined as dysplasia or EAC, and it was characterized as early (<2 years), intermediate (2-5 years), or late (>5 years). Predictors of recurrence were assessed by time to event analysis. Results: A total of 84 T1 EAC patients (75 T1a, 9 T1b) with a median 9.1 years (range, 5.1-18.3 years) of follow-up were included. The overall incidence of neoplastic recurrence was 2.0 per 100 person-years of follow-up. Seven recurrences (3 dysplasia, 4 EAC) occurred after 5 years of EAC remission. Overall, 88% of recurrences were treated successfully endoscopically. EAC recurrence-related mortality occurred in 3 patients at a median of 5.2 years from EAC remission. Complete eradication of intestinal metaplasia was independently associated with reduced recurrence (hazard ratio, .13). Conclusions: Following successful EET of T1 EAC, neoplastic recurrence occurred after 5 years in 8.3% of cases. Careful long-term surveillance should be continued in this patient population. Complete eradication of intestinal metaplasia should be the therapeutic end point for EET.

SUNY Downstate Health Sciences University, USA; Mayo Clinic in Florida, USA; Trinity Health of New England, USA.

Supplementary Figure Legends 1-2 from A Multicenter, Double-Blinded Validation Study of Methylation Biomarkers for Progression Prediction in Barrett's Esophagus

Purpose:When treating esophageal cancer with radiation therapy, it is critical to limit the dose to surrounding structures, such as the lung and/or heart, as much as possible. Proton radiation therapy allows a reduced radiation dose to both the heart and lungs, potentially reducing the risk of cardiopulmonary toxicity. Here, we report disease control, survival, and toxicity outcomes among patients with esophageal cancer treated with proton radiation therapy and concurrent chemotherapy (chemoradiation therapy; CRT) with or without surgery.Materials and Methods:We enrolled 17 patients with thoracic esophageal carcinoma on a prospective registry between 2010 and 2021. Patients received proton therapy to a median dose of 50.4-GyRBE (range, 50.4-64.8) in 1.8-Gy fractions.Acute and late toxicities were graded per the Common Terminology Criteria for Adverse Events, version 4.0 (US National Cancer Institute, Bethesda, Maryland). In addition, disease control, patterns of failure, and survival outcomes were collected.Results:Nine patients received preoperative CRT, and 8 received definitive CRT. Overall, 88% of patients had adenocarcinoma, and 12% had squamous cell carcinoma. With a median follow-up of 2.1 years (range, 0.5-9.4), the 3-year local progression-free, disease-free, and overall survival rates were 85%, 66%, and 55%, respectively. Two patients (1 with adenocarcinoma and 1 with squamous cell carcinoma) recurred at the primary site after refusing surgery after a complete clinical response to CRT. The most common acute nonhematologic and hematologic toxicities, respectively, were grades 1 to 3 esophagitis and grades 1 to 4 leukopenia, both affecting 82% of patients. No acute cardiopulmonary toxicities were observed in the absence of surgical resection. Reagarding surgical complications, 3 postoperative cardiopulmonary complications occurred as follows: 1 grade 1 pleural effusion, 1 grade 3 pleural effusion, and 1 grade 2 anastomotic leak. Two severe late CRT toxicities occurred: 1 grade 5 tracheoesophageal fistula and 1 grade 3 esophageal stenosis requiring a feeding tube.Conclusion:Proton radiation therapy is a safe, effective treatment for esophageal cancer with increasing evidence supporting its role in reducing cardiopulmonary toxicity.

BACKGROUND & AIMS: Tethered capsule endomicroscopy (TCE) involves swallowing a small tethered pill that implements optical coherence tomography (OCT) imaging, procuring high resolution images of the whole esophagus. Here, we demonstrate and evaluate the feasibility and safety of TCE and a portable OCT imaging system in patients with Barrett's esophagus (BE) in a multi-center (5-site) clinical study. METHODS: Untreated patients with BE as per endoscopic biopsy diagnosis were eligible to participate in the study. TCE procedures were performed in unsedated patients by either doctors or nurses. After the capsule was swallowed, the device continuously obtained 10-um-resolution cross-sectional images as it traversed the esophagus. Following imaging, the device was withdrawn through mouth, and disinfected for subsequent reuse. BE lengths were compared to endoscopy findings when available. OCT-TCE images were compared to volumetric laser endomicroscopy (VLE) images from a patient who had undergone VLE on the same day as TCE. RESULTS: 147 patients with BE were enrolled across all sites. 116 swallowed the capsule (79%), 95/114 (83.3%) men and 21/33 (63.6%) women (P = .01). High-quality OCT images were obtained in 104/111 swallowers (93.7%) who completed the procedure. The average imaging duration was 5.55 +/- 1.92 minutes. The mean length of esophagus imaged per patient was 21.69 +/- 5.90 cm. A blinded comparison of maximum extent of BE measured by OCT-TCE and EGD showed a strong correlation (r = 0.77-0.79). OCT-TCE images were of similar quality to those obtained by OCT-VLE. CONCLUSIONS: The capabilities of TCE to be used across multiple sites, be administered to unsedated patients by either physicians or nurses who are not expert in OCT-TCE, and to rapidly and safely evaluate the microscopic structure of the esophagus make it an emerging tool for screening and surveillance of BE patients.

Introduction: Sedated endoscopy for Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC) detection is invasive and expensive. Non-endoscopic BE/EAC detection tools have been guideline-endorsed to facilitate higher patient participation at lower cost. We previously described a promising panel of 5 methylated DNA markers (MDMs) assayed on esophageal specimens obtained by a sponge-on-a-string (SOS) cell collection device in phase II studies. We aimed to train an algorithm (establishing marker cut offs, to adjudicate samples as positive/negative) using a final MDM panel followed by testing in an independent sample set. Methods: Algorithm training samples (N=352) were prospectively collected from patients seen at 6 US medical centers. Test samples (N=125) were obtained from an independent, NIH-funded study conducted at 3 US medical centers. Cases had columnar metaplasia with intestinal metaplasia; controls had no endoscopic evidence of BE. Histology was reviewed by expert GI pathologists. The SOS device (25 mm, 10 ppi) was swallowed and withdrawn after 6-8 minutes followed by criterion standard endoscopy within 24 hours. DNA was extracted and bisulfite treated. Five MDMs were blindly assayed using the long probe quantitative amplified signal method. The algorithm was set using cross-validated logistic regression. The locked algorithm was applied to assay results from the test set. Results: Baseline characteristics of patients in training and test sets were comparable (Table). The final panel included 3 MDMs (NDRG4, VAV3, and ZNF682) and a reference marker (B3GALT6). Overall sensitivity for BE/EAC detection in the training set was 81% (95% CI 76-88%) at 90% (85-94%) specificity. Overall BE/EAC sensitivity in the test set was 88% (78-94%) at 84% (70-93%) specificity. Sensitivity for HGD and EAC was 100% in the training and test sets. Sensitivity for short segment NDBE in the test set was 63% (38-84%). Areas under the receiver operating characteristic (AUROC) curve for BE/EAC detection were 0.92 (95% CI 0.89-0.95) and 0.94 (0.90-0.98) in the training and test sets, respectively (Figure). The algorithm was not influenced by age, sex, or smoking history. 97% of participants in the training set and 85% in the test set successfully swallowed the SOS device, which was well tolerated and safe. Conclusion: A 3-MDM panel for BE/EAC detection demonstrated excellent sensitivity for high risk BE cases in multi-center case control training and test sets.Figure 1.: Area under the receiver operating characteristics curves for a 3-marker methylated DNA panel assayed from cytology specimen extracted DNA in training and test sets. Table 1. - Baseline characteristics of patients and performance characteristics of SOS test (overall sensitivity and specificity, and stratified by BE dysplasia grade) in training and test sets Variable Training Set (N=198 controls,154 cases) Test Set(N= 44 controls,81 cases) P value (comparing training and test sets) Control Case Control Case Mean (SD) age 55 (13) 65 (10) 52 (15) 65 (11) 0.312 Male Sex (%) 102 (52) 119 (77) 17 (39) 64 (79) 0.992 Mean (SD) BMI 29 (7) 30 (6) 30 (7) 30 (6) 0.283 Ever Smokers % 77 (39) 87 (56) 18 (41) 47 (58) 0.733 Mean *SD) BE length, cm - 4 (3) - 5 (3) 0.070 Long segment BE, N, (%) - 97 (63) - 56 (69) 0.426 Short segment BE, N (%) - 57 (37) - 25 (31) BE dysplasia grade, N (%) EAC - 12 (8) - 2 (2) HGD - 18 (12) - 11 (14) LGD - 7 (4) - 10 (12) IND - 18 (12) - 14 (17) NDBE (long segment) - 57 (37) - 25 (31) NDBE (short segment) - 42 (27) - 19 (24) Training Set% Positive (95% CI) Test Set% Positive (95% CI) Overall 81% (76-88%) 88 (78-94) Dysplasia grade EAC 100 (100-100) 100 (16-100) HGD 100 (100-100) 100 (72-100) LGD 71 (0-100) 90 (55-100) IND 74 (0-100) 93 (66-100%) NDBE (long segment) 91 (73-100) 96 (80-100%) NDBE (short segment) 61 (25-100) 63 (38-84%) Control (No BE) 10 (2-21) 16 (7-30)

BACKGROUND & AIMS:Recommended surveillance intervals after complete eradication of intestinal metaplasia (CE-IM) after endoscopic eradication therapy (EET) are largely not evidence-based. Using recurrence rates in a multicenter international Barrett's esophagus (BE) CE-IM cohort, we aimed to generate optimal intervals for surveillance.METHODS:Patients with dysplastic BE undergoing EET and achieving CE-IM from prospectively maintained databases at 5 tertiary-care centers in the United States and the United Kingdom were included. The cumulative incidence of recurrence was estimated, accounting for the unknown date of actual recurrence that lies between the dates of current and previous endoscopy. This cumulative incidence of recurrence subsequently was used to estimate the proportion of patients with undetected recurrence for various surveillance intervals over 5 years. Intervals were selected that minimized recurrences remaining undetected for more than 6 months. Actual patterns of post-CE-IM follow-up evaluation are described.RESULTS:A total of 498 patients (with baseline low-grade dysplasia, 115 patients; high-grade dysplasia [HGD], 288 patients; and intramucosal adenocarcinoma [IMCa], 95 patients) were included. Any recurrence occurred in 27.1% and dysplastic recurrence occurred in 8.4% over a median of 2.6 years of follow-up evaluation. For pre-ablation HGD/IMCa, intervals of 6, 12, 18, and 24 months, and then annually, resulted in no patients with dysplastic recurrence undetected for more than 6 months, comparable with current guideline recommendations despite a 33% reduction in the number of surveillance endoscopies. For pre-ablation low-grade dysplasia, intervals of 1, 2, and 4 years balanced endoscopic burden and undetected recurrence risk.CONCLUSIONS:Lengthening post-CE-IM surveillance intervals would reduce the endoscopic burden after CE-IM with comparable rates of recurrent HGD/IMCa. Future guidelines should consider reduced surveillance frequency.