Lansing, Ramona

Abstract Introduction: Barrett’s esophagus (BE) confers increased cancer risk. Endoscopic surveillance is recommended to distinguish non-dysplastic BE (NDBE), low grade dysplasia (LGD), high grade dysplasia (HGD), or adenocarcinoma (EAC) for endoscopic intervention. However, this paradigm is limited by sampling error, leading to missed/interval EAC. Herein, we aimed to classify these samples more accurately by performing whole genome methylation sequencing (WGMS) and utilizing multiple genomic features on endoscopic brush samples. Materials and methods: Endoscopic brush samples from patients with NDBE (n=18), HGD (18), EAC (18), and normal controls (18) were collected and underwent WGMS (discovery dataset). Sequence reads were processed through internal pipeline to extract CpG methylation and read level methylation ratio (alpha value). Copy number aberration (CNA) detection was performed using both CNVkit and ichorCNA. Differential methylated regions (DMRs) were identified through comparing EAC/HGD with NDBE and normal samples. Targeted methylation sequencing for selected DMRs and shallow WGMS were performed on EAC (41), HGD (26), LGD (23), NDBE (42), and normal controls (37) as validation. Ratio of the reads with alpha value>0.6 for each DMR was used for further analysis. Detected CNA segments were summarized into chromosome arm loss or gain (aneuploidy score or AS representing the number of chromosome arms gained or lost in a sample) and trimmed median absolute deviation (tMAD). Classification models were developed using the discovery set and the best performing one was applied to the validation dataset. Results: Genome-wide DNA methylation largely segregated different groups of samples as diagnosed; however, some unexpected group crossings were observed. Top 200 DMRs were identified between EAC/HGD and NDBE and normal samples. CNA changes were widespread in EAC, very common in HGD but rare in NDBE (17/18 EACs,14/18 HGDs, 5/18 NDBEs with AS>0). With tMAD > 0.015, 16/18 EACs, 8/18 HGDs but none of 18 NDBEs passed the threshold. EAC or HGD samples without detectable CNAs had very low “tumor” fraction as determined by methylation-based cell type deconvolution or ichorCNA. Similar results were found in the validation dataset. A multi-omics (AS, tMAD, estimated tumor fraction, and DMR methylation) model could predict EAC, HGD, or NDBE with 0.81 accuracy through cross validation (10X) and 0.73 in the independent dataset. This increased to 0.98 and 0.90 in separating high risk EAC/HGD from NDBE, respectively. Conclusion: Multi-omics data obtained from single WGMS allow for a comprehensive analysis of BE samples with varying dysplasia levels. Using classification modeling, these characteristics can be effectively used to accurately identify and distinguish between EAC and HGD from NDBE, suggesting their utility as adjuncts to endoscopic surveillance biopsies. Citation Format: Zhifu Sun, Caroline L. Matchett, Seth W. Slettedahl, William R. Taylor, Panwen Wang, Calise Berger, Caryn E. Anderson, Melissa A. Passe, Ramona Lansing, Collin E. Chalmers, Patrick H. Foote, Jeanette E. Eckel Passow, Doug W. Mahoney, John B. Kisiel, Prasad G. Iyer. Multi-omics cancer risk assessment of Barrett’s esophagus with DNA methylation sequencing from endoscopic brush samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB249.

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.

INTRODUCTION: We assessed if obesity perturbs the esophageal epithelial barrier function independent of promotion of gastroesophageal reflux (GER). METHODS: Thirty-eight participants were divided into 4 groups: Obesity−/GER–, Obesity+/GER−, Obesity−/GER+, and Obesity+/GER+. Esophageal intercellular space and desmosome density (structural integrity) and fluorescein leak (functional integrity) were measured. RESULTS: The Obesity+/GER− group demonstrated increased intercellular space, reduced desmosome density, and increased fluorescein leak compared with control subjects. These changes were similar but not additive to findings seen in Obesity−/GER + and Obesity+/GER+ patients. DISCUSSION: Central obesity impairs structural and functional integrity of the esophageal barrier independent of GER, likely predisposing to esophageal injury.

INTRODUCTION: Nonendoscopic Barrett's esophagus (BE) screening may help improve esophageal adenocarcinoma outcomes. We previously demonstrated promising accuracy of methylated DNA markers (MDMs) for the nonendoscopic diagnosis of BE using samples obtained from a capsule sponge-on-string (SOS) device. We aimed to assess the accuracy of these MDMs in an independent cohort using a commercial grade assay. METHODS: BE cases had ≥ 1 cm of circumferential BE with intestinal metaplasia; controls had no endoscopic evidence of BE. The SOS device was withdrawn 8 minutes after swallowing, followed by endoscopy (the criterion standard). Highest performing MDMs from a previous study were blindly assessed on extracted bisulfite-converted DNA by target enrichment long-probe quantitative amplified signal (TELQAS) assays. Optimal MDM combinations were selected and analyzed using random forest modeling with in silico cross-validation. RESULTS: Of 295 patients consented, 268 (91%) swallowed the SOS device; 112 cases and 89 controls met the pre-established inclusion criteria. The median BE length was 6 cm (interquartile range 4–9), and 50% had no dysplasia. The cross-validated sensitivity and specificity of a 5 MDM random forest model were 92% (95% confidence interval 85%–96%) and 94% (95% confidence interval 87%–98%), respectively. Model performance was not affected by age, gender, or smoking history but was influenced by the BE segment length. SOS administration was well tolerated (median [interquartile range] tolerability 2 [0, 4] on 10 scale grading), and 95% preferred SOS over endoscopy. DISCUSSION: Using a minimally invasive molecular approach, MDMs assayed from SOS samples show promise as a safe and accurate nonendoscopic test for BE prediction.

INTRODUCTION: Endoscopic surveillance guidelines in Barrett’s esophagus (BE) to detect progression to high grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) are based solely on dysplasia grade. Given low rates of progression, identification of predictors (and scores) of progression may help to focus surveillance or intervention in those at higher risk. We aimed to assess predictors of progression and externally validate the Progression in Barrett’s (PIB) score in an independent multicenter prospective BE and EAC cohort. METHODS: The EA and Barrett’s esophagus (EABE) registry is a prospective cohort of patients recruited from three medical centers. Progressors were defined as those with the development of HGD or EAC more than 12 months after initial diagnosis. Demographic and clinical variables were abstracted. The annual incidence rate and predictors of progression were assessed using univariate and multivariable Cox proportional hazards analysis. The ability of the PIB score (incorporating weighted scores for male gender, smoking (ever versus never), BE length and confirmed LGD) to risk stratify the cohort into low (0-10 points), intermediate (11-20 points) and high risk (> 20 points) groups was assessed. RESULTS: 429 patients were included. 84% were men, with a mean (SD) age of 61.5 (9.9) years. 65% were ever smokers and most (76.7%), had NDBE at diagnosis. Mean (SD) maximal BE segment length was 5.9 (3.2) cm. Eighty-two patients (19.1%) progressed to HGD (n = 65) or EAC (n = 17) over a median (IQR) follow up of 7.5 (3.7, 12.5) years. Annual incidence rates of progression to EAC and EAC/HGD were 0.5% and 2.0% respectively. Univariate and multivariable predictors of progression are displayed in Table 1. The PIB score divided patients into three groups (Table 2) with progressively increasing rates of progression. The risk in the high risk group was three fold higher than the low risk group ( P = 0.024). The risk in the intermediate group was twofold higher but not statistically significantly higher ( P = 0.13). The PIB score had a c-statistic score of 0.76 in the original cohort. In this cohort the c-statistic value was lower at 0.64, suggesting moderate performance. CONCLUSION: Male sex and BE length predicted progression in this independent multicenter cohort. The PIB score was also able to risk-stratify patients in this validation cohort with the high risk cohort being 3x more likely to progress compared to the low risk cohort.