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Emerging evidence indicates the potential clinical significance of specific microbial signatures as diagnostic and prognostic biomarkers, in multiple cancers. However, to date, no studies have systematically interrogated circulating metagenome profiling in oesophageal adenocarcinoma (EAC) patients, particularly as novel non-invasive, early detection, surveillance and prognostic classifiers.Metagenome sequencing was performed on 81 serum specimens collected across EAC spectrum, with sequencing reads classified using Bracken and MetaPhlAn3. Followed by the Linear Discriminant Analysis effect size (LEfSe) method to identify microbial profiles between groups. Logistic regression and Kaplan-Meier analyses were used to build classifiers.A significant loss of alpha and beta diversity was identified in serum specimens from EAC patients. We observed a shift in microbial taxa between each group-at the phylum, genus, and species level-with Lactobacillus sakei as the most prominent species in gastroesophageal reflux (GERD) vs other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus (L. Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 (95% CI: 0.78-0.95; P 0.001) and this panel in conjunction with the TNM stage was a robust predictor of overall survival (≥24 months; AUC = 0.84 (95% CI: 0.66-0.92; P = 0.006)).This study firstly describes unique blood-based microbial profiles in patients across EAC carcinogenesis, that are further utilised to establish a novel circulating diagnostic and prognostic metagenomic signature for EAC.Accumulating data indicates the clinical relevance of specific microbial signatures as diagnostic and prognostic biomarkers, in multiple cancers. However, to date, no studies have systematically interrogated circulating metagenome profiling in patients with oesophageal adenocarcinoma (EAC). Herein, we performed metagenome sequencing in serum specimens from EAC patients 81 collected across EAC spectrum and observed a significant loss of alpha and beta diversity, with a shift in microbial taxa between each group-at the phylum, genus, and species level-with Lactobacillus sakei as the most prominent species in gastroesophageal reflux (GERD) vs other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus (L. Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 and this panel, in conjunction with the TNM stage, was a robust predictor of overall survival. This study for the first time describes unique blood-based microbial profiles in patients across EAC carcinogenesis, that are further utilised to establish a novel circulating diagnostic and prognostic metagenomic signature for EAC.

Table S1. 15 Image Analysis Features Utilized by the Risk Classifier Table S2. Performance of Risk Classes Predicted by Test vs. Pathologic Diagnosis in Stratifying BE Patients with Prevalent HGD/EAC from Non-Progressor BE Patients. Figure S1. Representative Whole Slide Scan and Image Analysis Masks.

Table S1. Summary of Progression Endpoints in Incident Progressor Patients. Table S2. Correlation Among Quantitative Image Analysis Features Derived from the Same Candidate Biomarkers. Table S3. Comparison of Predictive Performance of Risk Classes Predicted by Test vs. Clinical Variables in Training Set of BE Patients. Table S4. Comparison of Predictive Performance of Risk Score as a Continuous Variable vs. Clinical Variables in Training Set. Table S5. Comparison of Predictive Performance of Risk Score as a Continuous Variable vs. Clinical Variables in Validation Set. Figure S1. Flowchart of Steps to Train and Validate 3-Tier 15-Feature/Measure Classifier for Risk Prediction in Barrett's Esophagus Biopsies. Figure S2. Multiplexed Biomarker Labeling and Imaging in Incident Progressor BE Cases. Supplementary Methods.

Background: There is a need for improved tools to detect high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus. In previous work, we demonstrated that a 3-tier classifier predicted risk of incident progression in Barrett's esophagus. Our aim was to determine whether this risk classifier could detect a field effect in nondysplastic (ND), indefinite for dysplasia (IND), or low-grade dysplasia (LGD) biopsies from Barrett's esophagus patients with prevalent HGD/EAC.Methods: We performed a multi-institutional case–control study to evaluate a previously developed risk classifier that is based upon quantitative image features derived from 9 biomarkers and morphology, and predicts risk for HGD/EAC in Barrett's esophagus patients. The risk classifier was evaluated in ND, IND, and LGD biopsies from Barrett's esophagus patients diagnosed with HGD/EAC on repeat endoscopy (prevalent cases, n = 30, median time to HGD/EAC diagnosis 140.5 days) and nonprogressors (controls, n = 145, median HGD/EAC-free surveillance time 2,015 days).Results: The risk classifier stratified prevalent cases and non-progressor patients into low-, intermediate-, and high-risk classes [OR, 46.0; 95% confidence interval, 14.86-169 (high-risk vs. low-risk); P < 0.0001]. The classifier also provided independent prognostic information that outperformed the subspecialist and generalist diagnosis.Conclusions: A tissue systems pathology test better predicts prevalent HGD/EAC in Barrett's esophagus patients than pathologic variables. The results indicate that molecular and cellular changes associated with malignant transformation in Barrett's esophagus may be detectable as a field effect using the test.Impact: A tissue systems pathology test may provide an objective method to facilitate earlier identification of Barrett's esophagus patients requiring therapeutic intervention. Cancer Epidemiol Biomarkers Prev; 26(2); 240–8. ©2016 AACR.

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

Purpose: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.Experimental Design: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.Results: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%.Conclusions: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.

Background: Better methods are needed to predict risk of progression for Barrett's esophagus. We aimed to determine whether a tissue systems pathology approach could predict progression in patients with nondysplastic Barrett's esophagus, indefinite for dysplasia, or low-grade dysplasia.Methods: We performed a nested case–control study to develop and validate a test that predicts progression of Barrett's esophagus to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), based upon quantification of epithelial and stromal variables in baseline biopsies. Data were collected from Barrett's esophagus patients at four institutions. Patients who progressed to HGD or EAC in ≥1 year (n = 79) were matched with patients who did not progress (n = 287). Biopsies were assigned randomly to training or validation sets. Immunofluorescence analyses were performed for 14 biomarkers and quantitative biomarker and morphometric features were analyzed. Prognostic features were selected in the training set and combined into classifiers. The top-performing classifier was assessed in the validation set.Results: A 3-tier, 15-feature classifier was selected in the training set and tested in the validation set. The classifier stratified patients into low-, intermediate-, and high-risk classes [HR, 9.42; 95% confidence interval, 4.6–19.24 (high-risk vs. low-risk); P < 0.0001]. It also provided independent prognostic information that outperformed predictions based on pathology analysis, segment length, age, sex, or p53 overexpression.Conclusion: We developed a tissue systems pathology test that better predicts risk of progression in Barrett's esophagus than clinicopathologic variables.Impact: The test has the potential to improve upon histologic analysis as an objective method to risk stratify Barrett's esophagus patients. Cancer Epidemiol Biomarkers Prev; 25(6); 958–68. ©2016 AACR.

Esophageal adenocarcinoma (EAC) is a leading cause of cancer deaths. Pexidartinib, a multi-gene tyrosine kinase inhibitor, through targeting colony-stimulating factor 1 (CSF-1) receptor (CSF-1R), down modulates macrophage-mediated pro-survival tumor signaling. Previously, CSF-1R inhibitors have successfully shown to enhance antitumor activity of PD-1/PD-L1 inhibitors by suppressing tumor immune evasion, in solid tumors. In this study, we investigated the antitumor activity of pexidartinib alone or in combination with blockade of PD-1 in a de novo EAC rat model. Here, we showed limited toxicity with significant tumor shrinkage in pexidartinib treated animals compared to controls, single agent and in combination with a PD-1 inhibitor, AUNP-12. Suppression of CSF-1/CSF-1R axis resulted in enhanced infiltration of CD3 + CD8 + T cells with reduced M2 macrophage polarization, in the tumor microenvironment (TME). Endpoint tissue gene expression in pexidartinib treated animals demonstrated upregulation of BAX, Cas3, TNFα, IFNγ and IL6 and downregulation of Ki67, IL13, IL10, TGFβ and Arg1 (P

Introduction: There is a critical need to incorporate molecular assessments of minimal residual disease (MRD) during neoadjuvant immunotherapy, in order to identify individuals at high risk for disease recurrence based on analyses of circulating cell-free tumor DNA (ctDNA) landscapes. Here we employed longitudinal liquid biopsies to dynamically assess clinical outcomes with neoadjuvant immuno-chemoradiotherapy in patients with esophageal/gastroesophageal junction (E/GEJ) cancer. Methods: We utilized targeted error-correction sequencing to perform high-depth ctDNA next-generation sequencing for 141 serial plasma and 32 matched white blood cell (WBC) DNA samples from 32 patients with operable stage II/III E/GEJ cancer that received neoadjuvant immune checkpoint blockade (ICB) with chemoradiotherapy prior to surgery (NCT03044613). ctDNA analyses were performed at baseline, post-ICB induction, after completion of chemoradiotherapy (pre-op), and post-operatively (post-op). Using a tumor-agnostic WBC DNA-informed panel NGS approach we determined the cellular origin of plasma variants, filtering out germline and clonal hematopoiesis (CH) variants and evaluated ctDNA clonal dynamics over time. Molecular MRD was evaluated post-ICB, pre-op and post-op and correlated with recurrence-free (RFS) and overall survival (OS). Results: Twenty out of 32 patients had detectable ctDNA at any timepoint. Of the 12 patients with undetectable ctDNA, 9 had only CH- and/or germline-derived variants, while 3 patients had no detectable variants of any origin. ctDNA clearance post-ICB was correlated with tumor regression >80% at the time of resection (Fischer’s exact p=0.04). The subset of patients that did not attain complete pathologic response was heterogeneous with respect to ctDNA dynamics; such that ctDNA clearance pre-op identified patients with longer OS despite residual tumor of >0% at the time of resection (log rank p=0.06). Patients with undetectable ctDNA or ctDNA clearance pre-op had a longer RFS (log rank p=0.007) and OS (log rank p=0.03). Molecular MRD was associated with RFS and OS such that patients with ctDNA clearance post-op had longer RFS (log-rank p=0.007) and OS (log-rank p=0.017). Conclusion: ctDNA clearance post-ICB, pre-op and post-op reflects differential clinical outcomes for patients with E/GEJ cancer receiving neoadjuvant immuno-chemoradiotherapy. Understanding ctDNA dynamics and their relationship with pathological response and long-term outcomes can help identify patients at higher risk for recurrence and open a therapeutic window for future intervention. Citation Format: Blair V. Landon, Ronan J. Kelly, Ali H. Zaidi, Archana Balan, Jenna V. Canzoniero, Gavin Pereira, Zineb Belcaid, Russell K. Hales, K Ranh Voong, Richard J. Battafarano, Blair A. Jobe, Stephen C. Yang, Stephen Broderick, Jinny Ha, Kellie N. Smith, Elizabeth Thompson, Fyza Y. Shaikh, James R. White, Cynthia L. Sears, Eun J. Shin, Ali I. Amjad, Benny Weksler, Josephine L. Feliciano, Chen Hu, Vincent K. Lam, Valsamo Anagnostou. Circulating cell-free tumor DNA dynamics capture minimal residual disease with neoadjuvant immune checkpoint blockade plus chemoradiotherapy for patients with operable esophageal/gastroesophageal junction cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3374.

Introduction: Targeted next-generation sequencing (NGS) of cell-free DNA in plasma, referred to as liquid biopsy, has become a valuable diagnostic tool in clinical oncology. However, detection of variants related to clonal hematopoiesis (CH) is a major confounder that significantly impairs the clinical utility of liquid biopsies. Here we developed a machine-learning model to determine tumor versus CH origin of variants identified in plasma-only NGS. Methods: We assembled a training cohort of 352 variants identified by targeted deep plasma sequencing from 199 patients with stage I-IV breast, colorectal, esophageal, lung, and ovarian cancer, coupled with matched white blood cell (WBC) and tumor tissue NGS to allow determination of the reference origin for each plasma variant. We employed Extreme Gradient Boosting (XGBoost) to integrate fragment, variant, gene, and patient level features to predict tumor versus CH plasma variant origin, evaluating the performance of this approach within the training cohort using 10-fold cross-validation. We applied the fixed model to two independent validation cohorts: a small cell lung cancer (SCLC) cohort comprising of 74 variants from targeted plasma NGS from 26 patients and a multi-cancer cohort of 409 variants detected using the MSK-Impact panel from 74 patients with breast, colorectal, and prostate cancer. Results: Variant allele frequencies (VAF) did not differentiate tumor from CH variants, as the VAFs between tumor (median VAF 0.53%) and CH (median VAF 0.409%) variants in the training cohort were largely overlapping (area under the ROC curve-AUC 0.54, 95% confidence interval-CI 0.48-0.61). Similarly, individual fragmentomic features (mutant fragment length, cut points, and endpoint motifs) had limited ability to distinguish tumor from CH variants (AUC range 0.51-0.76). Using serial plasma samples, we identified stable statistical measures of differences in fragment feature distributions between mutant and wild type fragments; these were subsequently incorporated into an XGBoost machine-learning model along with variant, gene and patient features to predict tumor versus CH variant origin. Our model predicted variant origin with an AUC of 0.95 (95% CI 0.87-1) from 10-fold cross validation in the training cohort. The performance of the model was tested in independent SCLC and multi-cancer validation cohorts; the fixed model predicted plasma variant origin with an AUC of 0.87 (95% CI 0.73-1) and 0.89 (95% CI 0.86-0.92) respectively. Conclusion: We developed a machine-learning model that integrates patient, gene, variant and fragment features to predict tumor versus CH origin of plasma variants across solid tumors and NGS sequencing platforms. The ability to identify bona fide tumor variants in plasma-only sequencing fills a critical need in the clinical implementation of liquid biopsy-guided cancer therapy by reducing misinterpretation due to CH contamination. Citation Format: Jenna V. Canzoniero, Archana Balan, Jillian Phallen, Blair V. Landon, Lavanya Sivapalan, Benjamin Green, Zineb Belcaid, Susan C. Scott, Gavin Pereira, Vincent K. Lam, Ali H. Zaidi, Ronan J. Kelly, Christine L. Hann, Wade T. Iams, Christine M. Lovly, Patrick M. Forde, Gerrit A. Meijer, Geraldine R. Vink, Remond J. Fijneman, The MEDOCC Group, Victor E. Velculescu, Robert B. Scharpf, Valsamo Anagnostou. A machine learning approach to determine the cellular origin of variants in liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3366.

Background: Esophageal adenocarcinoma (EAC) is a deadly disease with limited treatment options. STING is a transmembrane protein that activates transcription of interferon genes, resulting in stimulation of APCs and enhanced CD8+ T-cell infiltration. The present study evaluates STING agonists, alone and in combination with radiation to determine durable anticancer activity in solid tumors. Materials and Methods: Esophagojejunostomy was performed on rats to induce reflux leading to the development of EAC. At 32 weeks post operatively, rats received intratumorally either 50 μg STING (ADU-S100) or placebo (PBS), +/– 16Gy radiation. Drug activity was evaluated by pre- and post- treatment MRI, histology, immunofluorescence and RT-PCR. Results: Mean MRI tumor volume decreased by 30.1% and 50.8% in ADU-S100 and ADU-S100 + radiation animals and increased by 76.7% and 152.4% in placebo and placebo + radiation animals, respectively (P < 0.0001). Downstream gene expression, pre- to on- and post- treatment, demonstrated significant upregulation of IFNβ, TNFα, IL-6, and CCL-2 in the treatment groups vs. placebo. On- or post- treatment, radiation alone, ADU-S100 alone, and ADU-S100 + radiation groups demonstrated enhanced PD-LI expression, induced by upregulation of CD8+ T-cells (p < 0.01). Conclusions: ADU-S100 +/– radiation exhibits potent antitumor activity and a promising immunomodulatory profile in a de novo EAC.

Introduction No manometric criteria have been defined to select patients for magnetic sphincter augmentation (MSA). The first step to establish such criteria is to measure the outflow resistance at esophagogastric junction (EGJ) imposed by MSA. This resistance needs to be overcome by the esophageal contraction in order for the esophagus to empty and to avoid postoperative dysphagia. This study was designed to measure the outflow resistance caused by MSA in patients free of postoperative dysphagia. Methods Records of the patients who underwent MSA in our institution were reviewed. A group of MSA patients with excellent functional outcome, who were free of clinically significant postoperative dysphagia, were selected. These patients then underwent high-resolution impedance manometry (HRIM) at a target date of 1 year after surgery. The outflow resistance was measured by the esophageal intrabolus pressure (iBP) recorded 2 cm proximal to the lower esophageal sphincter (LES). Results The study population consisted of 43 patients. HRIM was performed at mean of 20.4 (10.4) months after surgery. The mean (SD) amplitude of the iBP was 13.5 (4.3) before surgery and increased to 19.1 (5.6) after MSA (p 15 beads) [19.7 (4.5) vs. 18.4 (5.9), p = 0.35]. There was a significant correlation between the iBP and % incomplete bolus clearance [Spearman R: 0.44 (95% CI 0.15–0.66), p = 0.0032]. The 95th percentile value for iBP after MSA was 30.4 mmHg. Conclusion The EGJ outflow resistance measured by iBP is increased after MSA. The upper limit of normal for iBP is 30 mmHg in this cohort of patients who were free of dysphagia after MSA. This degree of resistance needs to be overcome by distal esophageal contraction and will likely be requisite to prevent persistent postoperative dysphagia.

Radiofrequency ablation is a common treatment for atrial fibrillation, and esophageal complications are exceedingly rare. This report describes the case of a patient with no other known cancer risk factors who had esophageal squamous cell carcinoma that developed at the site of esophageal thermal injury, which occurred during a radiofrequency catheter ablation procedure.

Purpose: 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. Experimental Design: 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. Results: 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%. Conclusions: 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.

Introduction Magnetic sphincter augmentation (MSA) is a promising antireflux surgical treatment. The cost associated with the device may be perceived as a drawback by payers, which may limit the adoption of this technique. There are limited data regarding the cost of MSA in the management of reflux disease. The aims of the study were to report the clinical outcome and quality of life measures in patients after MSA and to compare the pharmaceutical and procedure payer costs and the disease-related and overall expense of MSA compared to laparoscopic Nissen fundoplication (LNF) from a payer perspective. Methods and procedures This prospective observational study was performed in conjunction with the region’s largest health insurance company. Data were collected on patients who underwent MSA over a 2-year period beginning in September 2015 at the study network hospitals. The LNF comparison group was procured from members’ claims data of the payer. Inclusion was predicated by patients having continuous coverage during study period. The total procedural reimbursement and the disease-related and overall medical claims submitted up to 12 months prior to surgery and up to 12 months following surgery were obtained. The payer reimbursement data are presented as allowed cost per member per month (PMPM). These values were then compared between groups. Results There were 195 patients who underwent MSA and 1131 that had LNF. MSA results in comparable symptom control, PPI elimination rate, and quality of life measures compared to values reported for LNF in the literature. The median (IQR) reimbursement of surgery was $13,522 (13,195–14,439) for those who underwent MSA and $13,388 (9951–16,261) for patients with LNF, p = 0.02. In patients who underwent MSA, the median reimbursement related to the upper gastrointestinal disease was $ 305 PMPM, at 12 months prior to surgery and $ 104 at 12 months after surgery, representing 66% decrease in cost. These values were $ 233 PMPM and $126 PMPM for patients who underwent LNF, representing a 46% decrease (p = 0.0001). At 12 months following surgery, the reimbursement for overall medical expenses had decreased by 10.7% in the MSA group and 1.4% in the LNF group when compared to the preoperative baseline reimbursement. The reimbursement for PPI use after surgery showed a 95% decrease in the MSA group and 90% among LNF group when compared to the preoperative baseline (p = 0.10). Conclusion When compared with LNF, MSA results in a reduction of disease-related expenses for the payer in the year following surgery. While MSA is associated with a higher procedural payer cost compared to LNF, payer costs may offset due to reduction in the expenses after surgery.

Introduction Magnetic sphincter augmentation (MSA) is an effective treatment for patients with gastroesophageal reflux disease. In early studies, patients with a hiatal hernia (HH) ≥ 3 cm were excluded from consideration for implantation and initially the FDA considered its use as “precautionary” in this context. This early approach has led to an attitude of hesitance among some surgeons to offer this therapy to patients with HH. This study was designed to evaluate the impact of HH status on the outcome of MSA and to report the rate of HH recurrence after MSA. Methods and procedures This is a retrospective review of prospectively collected data of patients who underwent MSA between June 2013 and August 2017. Baseline clinical and objective data were collected. Patients were divided into four groups based on HH status: no HH, small HH (

Treatment options and risk stratification for esophageal adenocarcinomas (EAC) currently rely on pathological criteria such as tumor staging. However, with advancement in immune modulated treatments, there is a need for accurate predictive biomarkers that will help identify high-risk patients and provide novel therapeutic targets. Hence, we analyzed as prognostic classifiers a host of histopathological parameters in conjunction with novel immune biomarkers. Specifically, gene expression levels for CXCL9, IDO1, LAG3, and TIM3 were established in treatment naive samples. Additionally, PD-L1 and CD8 positivity was determined by immunohistochemical staining. Based on our finding, a Cox model consisting of pathological complete response (CR), LAG3, and CXCL9 provided improved predictability for disease-free survival (DFS) compared to CR alone, and it demonstrated statistical significance for predictability of recurrence (p=0.0001). Likewise, for overall survival (OS), a Cox model constituted of TIM3, CR, and IDO1 performed better than CR alone, and it demonstrated statistical significance for predictability of survival (p = 0.0004). TIM3 was identified as the best predictor for OS (HR=4.43, p=0.0023). In conclusion, given the paucity of treatment options for EAC, evaluation of these biomarkers early in the disease course will lead to better risk stratification of patients and much needed alternatives for improved therapy.

Extracellular matrix (ECsM) bioscaffolds have been successfully used to treat five esophageal adenocarcinoma (EAC) patients following resection of neoplastic mucosal tissue. The present study evaluated the in vitro effect of ECM harvested from nonmalignant, decellularized tissue on EAC cell phenotype to understand the molecular mechanisms underlying the clinical findings. Nonmalignant (Het-1A), metaplastic (CP-A), and neoplastic (SK-GT-4, OE33) esophageal epithelial cells were exposed to ECM degradation products (250 μg/mL) prepared from heterologous urinary bladder tissue or homologous esophageal mucosa tissue, and evaluated for cell morphology, cell function, and EAC signaling pathways. Both the ECM sources downregulated neoplastic cell phenotype, but had distinctive tissue-specific effects. Urinary bladder ECM decreased OE33 and SK-GT-4 metabolism and increased CP-A apoptosis. Esophageal ECM decreased SK-GT-4, CP-A, and Het-1A proliferation; robustly downregulated PI3K-Akt-mTOR, cell cycle/DNA replication signaling, and upregulated autophagy signaling in OE33 cells; and increased cell cycle/DNA replication signaling in Het-1A cells. Both ECM sources decreased OE33 proliferation and phosphorylated AKT in OE33 cells, and in contrast, increased phosphorylated AKT in Het-1A cells. The results support the concept that the biochemical signals in nonmalignant ECM can downregulate neoplastic cell phenotype with minimal, and sometimes opposite, effects on normal cells. PI3K-Akt signaling has been implicated in EAC progression and these ECM-mediated effects may be favorable for an esophageal therapy following cancer resection. IMPACT STATEMENT: Extracellular matrix (ECM) biomaterials were used to treat esophageal cancer patients after cancer resection and promoted regrowth of normal mucosa without recurrence of cancer. The present study investigates the mechanisms by which these materials were successful to prevent the cancerous phenotype. ECM downregulated neoplastic esophageal cell function (proliferation, metabolism), but normal esophageal epithelial cells were unaffected in vitro, and suggests a molecular basis (downregulation of PI3K-Akt, cell cycle) for the promising clinical results. The therapeutic effect appeared to be enhanced using homologous esophageal ECM. This study suggests that ECM can be further investigated to treat cancer patients after resection or in combination with targeted therapy.

Background: Preclinical and clinical data indicate that neoadjuvant chemoradiotherapy (CRT) may prime an anti-tumor immunological response in esophageal cancer driven by intratumoral CD8+ T cells and PD-L1 expression. LAG-3 is also highly expressed in esophagogastric cancers. The microbiome, a novel and potentially modifiable, biomarker of IO response, has not yet been examined in the neoadjuvant setting in esophageal cancer and is the goal of our study. Methods: Fecal samples were collected from patients with stage II/III esophageal or gastroesophageal junction carcinoma eligible for curative resection treated with the standard of care regimen of carboplatin paclitaxel (50mg/m2), radiation 50.4 Gy in 28 fractions and an Ivor-Lewis esophagectomy 6-10 weeks after last CRT and immunotherapy (IO) dose. Patients on arm A (n=11) received 2 cycles of induction with nivolumab plus 3 additional cycles on week 1, 3 and 5 of CRT. Patients on arm B (n=8) received nivolumab plus relatlimab on the same schedule (Clinical trial: NCT03044613). We examined longitudinal fecal samples from n=19 patients across both arms (n=90 samples) using 16S rRNA amplicon sequencing. Patients were classified based on pathological response: complete response (CR) and grades 1, 2, and 3 (G1, G2, G3) with increasing residual tumor visible in the resected specimen. Sequencing data was trimmed and filtered for contaminants, followed by high-resolution taxonomic assignment and normalization of reads across all samples. Analysis was performed using multiple metrics for alpha diversity and beta-diversity, with principal coordinates analysis/PERMANOVA, and pathway analysis using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Results: Patients with improved response in the neoadjuvant setting (CR/G1 vs G2/G3) grouped in distinct clusters using Bray-Curtis (p < 0.001). Patients with CR had higher alpha diversity, using both measures of richness and evenness, compared to patients with a G3 responses (p < 0.03). Specifically, family Bacteroidaceae and genus Bacteroides were enriched in patients with CR vs G3 (p < 0.02). At the species level, B. finegoldii, B. ovatus, and B. uniformis were enriched in patients with CR vs G3 (p < 0.02). In contrast, genus Klebsiella and Clostridium termitidis were enriched in patients with a poor response, G3 (p Conclusions: Patients with operable esophageal cancer and improved responses to combined CRT and IO had distinct microbiome profiles enriched in multiple Bacteroides species. Further analyses and validation efforts are underway to confirm metabolomic pathways. Citation Format: Fyza Y. Shaikh, James R. White, Ronan J. Kelly, Ali H. Zaidi, Jenna V. Canzoniero, Josephine L. Feliciano, Russell K. Hales, K Ranh Voong, Richard J. Battafarano, Blair A. Jobe, Stephen C. Yang, Stephen Broderick, Jinny Ha, Kellie N. Smith, Elizabeth Thompson, Eun J. Shin, Ali I. Amjad, Patrizia Guerrieri, Benny Weksler, Chen Hu, Valsamo Anagnostou, Vincent K. Lam, Cynthia L. Sears. Patients with operable esophageal cancer and improved responses to combined chemoradiotherapy and immunotherapy display distinct microbiome profiles enriched in multiple Bacteroides species [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1973.

The aim of this study was to assess the impact of chemoradiation on the immune microenvironment to influence and optimally design future neoadjuvant clinical trials.Programmed death (PD)-1 inhibitors in metastatic gastroesophageal cancer have demonstrated response rates of approximately 25% in programmed death ligand-1 (PD-L1+) tumors. Unfortunately, the majority of patients do not respond. Therefore, a rationale strategy of combining immunotherapeutic agents with chemoradiation in earlier stage esophageal cancer may prevent metastatic disease in patients.To determine the effects of chemoradiation on resected esophageal adenocarcinomas, we examined the immune microenvironment pre- and post-chemoradiation using immunohistochemistry, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and functional analysis of tumor-infiltrating lymphocytes. Additionally, to assess the duration and dependency of radiation-induced PD-L1 upregulation, a surgical rat reflux model of esophageal adenocarcinoma is used. First, tumor-bearing animals were dosed with single-fraction 13Gy or 16Gy radiation to determine safety, dose correlation, and PD-L1 upregulation using qRT-PCR post-radiation. Next, longitudinal PD-L1 expression levels within individual animals were determined using serial endoscopic biopsies at baseline, 1, 5, and 9 weeks post 16Gy radiation.The majority of cancers displayed enhanced interferon γ and activated CD8+ T lymphocytes at the tumor stroma interface. These tumors also demonstrated enhanced upregulation of PD-L1 and multiple other immune checkpoints including TIM3, GITR, IDO1, LAG3, OX40, and KIR. The animal model results indicated PD-L1 upregulation is dose-dependent and transiently elevated post radiation exposure.Collectively, these findings provide insights into the evolving immune landscape after chemoradiation and have significant implications for neoadjuvant trial designs that will combine radiotherapy with immune checkpoint inhibitors.

Pseudoachalasia is mimicking clinical and physiologic manifestations of idiopathic achalasia but results from alternative etiologies that infiltrate or obstruct the esophagogastric junction (EGJ). Anti-reflux surgery is one of the potential etiologies of pseudoachalasia. The majority of cases with persistent dysphagia after a tightly constructed Nissen fundoplication results from EGJ outlet obstruction (EGJOO) and in rare cases progresses to pseudoachalasia. In these extreme cases, endoscopic dilation is not a sufficient treatment and take down of fundoplication would be necessary. In this case report, we present a patient with long-standing GERD symptoms that underwent magnetic sphincter augmentation (MSA) with complete resolution of his reflux symptoms. He did not have dysphagia prior to surgery and his preoperative manometry showed normal peristaltic progression of esophageal contractions. He developed pseudoachalasia 14 months after surgery. Repeated endoscopic dilation in this case resulted in resolution of dysphagia and complete restoration of peristaltic contractions.

Improved methods of diagnosis of laryngopharyngeal reflux (LPR) would enable surgeons to better identify patients who may benefit from antireflux surgery (ARS). The objective of the present study was to assess if hypopharyngeal Pepsin and Sep70 expression combined with hypopharyngeal multichannel intraluminal impedance (HMII) has the potential to increase diagnostic sensitivity of LPR. This study was performed on patients who underwent unsedated transnasal endoscopy with hypopharyngeal biopsy and 24-h HMII to determine abnormal proximal exposure (APE) and DeMeester score (DMS) from 2013 to 2016. Pepsin and Sep70 protein expression was assessed by Western blots of biopsy specimens. The outcomes of ARS were assessed using reflux symptom index (RSI). HMII APE classification, Sep 70, and Pepsin protein levels were compared in normative and symptomatic LPR patients and further analyzed alongside quality of life changes following ARS. Of 30 subjects enrolled, 23 were excluded for abnormal HMII results or endoscopic evidence of esophagitis. Seven subjects and 105 patients were included in the normative and symptomatic groups, respectively. Compared to the normative group, only Pepsin expression was significantly higher in the symptomatic group [APE+/LPR+ (p = 0.000), APE+/LPR− (p = 0.001), and APE− (p = 0.047)]. Further, the ratio of Sep70/Pepsin was significantly lower in the symptomatic group [APE+/LPR+ (p = 0.008), APE+/LPR− (p = 0.000), and APE− (p = 0.050)], and a cutoff ratio for a diagnosis of LPR was established as

PURPOSE/OBJECTIVE(S) Esophageal adenocarcinoma is the sixth leading cause of cancer-related deaths in the United States. For locally advanced patients, neoadjuvant chemoradiotherapy followed by surgery is the standard of care. Risk stratification relies heavily on clinicopathologic features, particularly pathologic response, which is inadequate. This highlights the need for the development of biomarkers that can help improve outcomes. Herein, we have developed 2 distinct biomarker signatures to either predict response to neoadjuvant therapy or stratify patients into risk categories for survival. MATERIALS/METHODS We assembled a cohort of 34 patients with locally advanced esophageal adenocarcinoma, with the majority (21) receiving concurrent chemoradiotherapy with carboplatin/paclitaxel. A capture based targeted sequencing was performed in paired biopsy specimens obtained at baseline, 3- and 6-weeks post-treatment. Differentially mutated gene analysis between responders and non-responders of treatment was performed to determine predictors of response. A univariate Cox proportional hazard regression was used to examine associations between gene mutation status and overall survival. A risk score was then calculated for each patient using a linear combination of univariate coefficient and mutation status of gene. RESULTS 15 patients (71%) receiving the CROSS regimen were classified as responders. Among responders to chemotherapy, the most frequently mutated genes were MKI67, SYNE1, PCLO, MSH3, RECQL4, NOTCH2, ILR7, CIITA, LRRK2 and EML4. Tumor mutation count was significantly reduced for these genes in post-treatment samples (P = 5.89E-03). We have identified a 3 gene signature based on mutations in EPHA5, BCL6, and ERBB2 that robustly predicts response to carboplatin/paclitaxel based neoadjuvant therapy. For this model, sensitivity is 84.6% and specificity is 100%. In the overall cohort, a 9 gene signature using APC, MAP3K6, ETS1, CSF3R, PDGFRB, GATA2, ARID1A, and FGF6, was also created that significantly stratifies patients into risk categories, prognosticating for improved relapse-free (P = 0.005) and overall survival (P = 3.325E-06). Sensitivity for this model is 73.33% and specificity is 94.74%. CONCLUSION We have identified a 3-gene signature (EPHA5, BCL6 and ERBB2) that is predictive of response to neoadjuvant therapy and a separate 9 gene classifier which prognosticates for survival outcomes. These provide significant potential for personalized management of locally advanced esophageal cancer.

Introduction Macrophages are capable of extreme plasticity and their activation state has been strongly associated with solid tumor growth progression and regression. Although the macrophage response to extracellular matrix (ECM) isolated from normal tissue is reasonably well understood, there is a relative dearth of information regarding their response to ECM isolated from chronically inflamed tissues, pre-neoplastic tissues, and neoplastic tissues. Esophageal adenocarcinoma (EAC) is a type of neoplasia driven by chronic inflammation in the distal esophagus, and the length of the esophagus provides the opportunity to investigate macrophage behavior in the presence of ECM isolated from a range of disease states within the same organ. Methods. Normal, metaplastic, and neoplastic ECM hydrogels were prepared from decellularized EAC tissue. The hydrogels were evaluated for their nanofibrous structure (SEM), biochemical profile (targeted and global proteomics), and direct effect upon macrophage (THP-1 cell) activation state (qPCR, ELISA, immunolabeling) and indirect effect upon epithelial cell (Het-1A) migration (Boyden chamber). Results. Nanofibrous ECM hydrogels from the three tissue types could be formed, and normal and neoplastic ECM showed distinctive protein profiles by targeted and global mass spectroscopy. ECM proteins functionally related to cancer and tumorigenesis were identified in the neoplastic esophageal ECM including collagen alpha-1(VIII) chain (COL8A1), lumican, and elastin. Metaplastic and neoplastic esophageal ECM induce distinctive effects upon THP-1 macrophage signaling compared to normal esophageal ECM. These effects include activation of pro-inflammatory IFNγ and TNFα gene expression and anti-inflammatory IL1RN gene expression. Most notably, neoplastic ECM robustly increased macrophage TNFα protein expression. The secretome of macrophages pre-treated with metaplastic and neoplastic ECM increases the migration of normal esophageal epithelial cells, similar behavior to that shown by tumor cells. Metaplastic ECM shows similar but less pronounced effects than neoplastic ECM suggesting the abnormal signals also exist within the pre-cancerous state. Conclusion. A progressively diseased ECM, as exists within the esophagus exposed to chronic gastric reflux, can provide insights into novel biomarkers of early disease and identify potential therapeutic targets.

We examined the utility of serial liquid biopsies to monitor clonal dynamics and predict pathologic response in patients with esophageal/gastroesophageal junction (E/GEJ) cancer undergoing treatment with neoadjuvant immunotherapy and concurrent chemoradiation (CA209-906; NCT03044613). Methods: Using Targeted Error Correction sequencing (TEC-Seq), we performed high-depth next generation sequencing on 79 serial plasma samples from 16 patients with operable stage II/III E/GEJ cancer undergoing treatment with neoadjuvant nivolumab, followed by nivolumab plus chemo-radiation and surgery as part of the CA209-906 trial. Liquid biopsies were evaluated pre-treatment, after each of two cycles of neoadjuvant nivolumab, and after concurrent nivolumab and chemoradiation immediately prior to surgery, for an average of 4 time points per patient. Deep sequencing of matched leukocyte DNA and whole exome sequencing (WES) of pre-treatment tumors was performed. The origin of plasma variants was determined by comparison to tumor WES (tumor-derived) and leukocyte TEC-Seq (clonal hematopoiesis (CH)-derived or germline) and their clonal dynamics over time were evaluated. Resected tumors were reviewed by central pathology and percent residual tumor was estimated. Patients were followed for an average of 2 years post-surgery. Results: Eight of 16 patients had detectable circulating tumor-derived DNA (ctDNA) at any time point. Additionally, 13 CH-derived mutations were detected in plasma of 8 patients. The number of CH-derived mutations was correlated with increasing patient age. Identification and removal of CH-derived mutations via comparison to matched leukocyte sequencing allowed for accurate assessment of kinetics of bona fide tumor-derived mutations in plasma. Tumor mutation burden as determined by tumor WES was not associated with pathological response (mean baseline mutations per exome 90 vs 81 for patients with residual disease >20% and 20% (50% vs 23% with or without detectable ctDNA respectively). ctDNA clearance, defined as detectable ctDNA at one or more earlier time points that subsequently becomes undetectable before surgery, occurred in 5 patients and was associated with improved pathologic response (80% of patients with ctDNA clearance had residual tumor Conclusion: Comprehensive analyses of ctDNA from E/GEJ patients undergoing neoadjuvant immunotherapy with concurrent chemoradiation revealed ctDNA dynamics that were associated with pathologic response and disease recurrence. These approaches may be used to identify patients at high risk for disease progression. Citation Format: Jenna VanLiere Canzoniero, Vincent Lam, Zineb Belcaid, Mara Lanis, Lamia Rhymee, Blair Landon, Archana Balan, Kristen Marrone, Patrick Forde, Benjamin Levy, David Ettinger, Heather Schneider, Hanika Rodavia, Richard Battafarano, Stephen Yang, Stephen Broderick, Jinny Ha, Blair Jobe, Ali Zaidi, Elizabeth Thompson, Julie Brahmer, Victor Velculescu, Ronan Kelly, Josephine Feliciano, Valsamo Anagnostou. Liquid biopsy approaches for determining pathologic response to neoadjuvant immunotherapy in esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 538.

Esophageal adenocarcinoma (EAC) is a deadly disease with limited therapeutic options. In the present study, we determined the preclinical efficacy of CDK4/6 inhibitor abemaciclib for treatment of EAC. In vitro, apoptosis, proliferation, and pathway regulation were evaluated in OE19, OE33, and FLO1 EAC cell lines. In vivo, esophagojejunostomy was performed on rats to induce EAC. At 36 weeks post-surgery, MRI and endoscopic biopsy established baseline tumor volume and molecular correlates, respectively. Next, the study animals were randomized to 26mg/kg intraperitoneal abemaciclib treatment or vehicle control for 28 days. Pre and post treatment MRIs, histopathology, and qRT-PCR were utilized to determine response. Our results demonstrated treatment with abemaciclib lead to increased apoptosis, and decreased proliferation in OE19 (p=0.185), OE33 (p=0.048), and FLO1 (p=0.043) with anticipated downstream molecular inhibition. In vivo, 78.9% of treatment animals demonstrated >20% tumor volume decrease (placebo 0%). Mean tumor volume changed in the treatment arm by -65.5% (placebo +133.5%) (p

Aim To efficiently replicate the biology and pathogenesis of human esophageal adenocarcinoma (EAC) using the modified Levrat model of end-to-side esophagojejunostomy. Methods End-to-side esophagojejunostomy was performed on rats to induce gastroduodenoesophageal reflux to develop EAC. Animals were randomly selected and serially euthanized at 10 (n = 6), 17 (n = 8), 24 (n = 9), 31 (n = 6), 38 (n = 6), and 40 (n = 6) wk postoperatively. The esophagi were harvested for downstream histopathology and gene expression. Histological evaluation was completed to determine respective rates of carcinogenic development. Quantitative reverse transcription-polymerase chain reaction was performed to determine gene expression levels of MUC2, CK19, and CK20, and results were compared to determine significant differences throughout disease progression stages. Results The overall study mortality was 15%. Causes of mortality included anastomotic leak, gastrointestinal hemorrhage, stomach ulcer perforation, respiratory infection secondary to aspiration, and obstruction due to tumor or late anastomotic stricture. 10 wk following surgery, 100% of animals presented with esophagitis. Barrett's esophagus (BE) was first observed at 10 wk, and was present in 100% of animals by 17 wk. Dysplasia was confirmed in 87.5% of animals at 17 wk, and increased to 100% by 31 wk. EAC was first observed in 44.4% of animals at 24 wk and increased to 100% by 40 wk. In addition, two animals at 38-40 wk post-surgery had confirmed macro-metastases in the lung/liver and small intestine, respectively. MUC2 gene expression was progressively down-regulated from BE to dysplasia to EAC. Both CK19 and CK20 gene expression significantly increased in a stepwise manner from esophagitis to EAC. Conclusion Esophagojejunostomy was successfully replicated in rats with low mortality and a high tumor burden, which may facilitate broader adoption to study EAC development, progression, and therapeutics.

The purpose of the current study is to determine the efficacy of a PI3K/mTOR dual inhibitor, LY3023414, on established EAC in an in vivo model.Esophageal adenocarcinoma (EAC) is a highly lethal cancer with limited treatment options. The PI3K/mTOR pathway is upregulated in EAC and may be a target for novel therapies.Esophagojejunostomy was performed on Sprague-Dawley rats to induce carcinogenesis, and LY3023414 was cyclically administered intraperitoneally between 32 and 40 weeks postsurgery to treatment animals. Magnetic resonance imaging (MRI) and histology were used to determine clinical response. Immunohistochemistry, immunofluorescence, and Western blot were used to validate apoptosis by cleaved caspase-3, proliferation by Ki67, and pathway inhibition, respectively.Mean MRI tumor volume increased by 109.2% in controls (n = 32) and decreased by 56.8% in treatment animals (n=17) (P0.01). Treatment with LY3023414 demonstrated tumor volume increase in 0% (control = 46.4%) (P0.01), decrease in 58.8% (control = 7.1%) (P0.01), and stable volume in 41.2% (control = 46.4%) (P = 0.77). EAC prevalence in controls increased by 25%; whereas, prevalence in treatment animals decreased by 29.4% (P0.01). Approximately, 75% of treatment animals presenting with residual masses on MRI had a histological response50%. Increased apoptosis by cleaved caspase-3 (P = 0.03) and decreased proliferation by Ki67 (P0.01) were demonstrated in the treatment arm, when compared with the control arm. On Western blot analysis of pathway checkpoints, p-mTOR (p=0.03) and PI3K-α (P = 0.04) were downregulated in treatment responsive residual tumors, when compared with controls.LY3023414 demonstrates efficacy against EAC in a preclinical model, establishing the rationale for clinical testing.

Background: There is a need for improved tools to detect high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus. In previous work, we demonstrated that a 3-tier classifier predicted risk of incident progression in Barrett's esophagus. Our aim was to determine whether this risk classifier could detect a field effect in nondysplastic (ND), indefinite for dysplasia (IND), or low-grade dysplasia (LGD) biopsies from Barrett's esophagus patients with prevalent HGD/EAC. Methods: We performed a multi-institutional case–control study to evaluate a previously developed risk classifier that is based upon quantitative image features derived from 9 biomarkers and morphology, and predicts risk for HGD/EAC in Barrett's esophagus patients. The risk classifier was evaluated in ND, IND, and LGD biopsies from Barrett's esophagus patients diagnosed with HGD/EAC on repeat endoscopy (prevalent cases, n = 30, median time to HGD/EAC diagnosis 140.5 days) and nonprogressors (controls, n = 145, median HGD/EAC-free surveillance time 2,015 days). Results: The risk classifier stratified prevalent cases and non-progressor patients into low-, intermediate-, and high-risk classes [OR, 46.0; 95% confidence interval, 14.86-169 (high-risk vs. low-risk); P < 0.0001]. The classifier also provided independent prognostic information that outperformed the subspecialist and generalist diagnosis. Conclusions: A tissue systems pathology test better predicts prevalent HGD/EAC in Barrett's esophagus patients than pathologic variables. The results indicate that molecular and cellular changes associated with malignant transformation in Barrett's esophagus may be detectable as a field effect using the test. Impact: A tissue systems pathology test may provide an objective method to facilitate earlier identification of Barrett's esophagus patients requiring therapeutic intervention. Cancer Epidemiol Biomarkers Prev; 26(2); 240–8. ©2016 AACR.

Magnetic sphincter augmentation (MSA) is a promising surgical treatment for patients with GERD. The aim of this study was to evaluate the outcomes of MSA in a large cohort of patients with GERD and to determine the factors predicting a favorable outcome.This was a retrospective review of prospectively collected data of 553 patients who underwent MSA at our institution in a 5-year period. Preoperative clinical, endoscopic, manometric, and pH data were used in a univariate analysis. This was followed by a regression multivariable analysis to determine the factors predicting a favorable outcome. Favorable outcome was defined as freedom from proton pump inhibitors and ≥50% improvement in Gastroesophageal Reflux Disease-Health-Related Quality of Life (GERD-HRQL) total score.At a mean (SD) follow-up of 10.3 (10.6) months after MSA, 92.7% of the patients were free of proton pump inhibitor use and 84% reported at least 50% improvement in their GERD-HRQL total score. The GERD-HRQL total score was improved from a mean (SD) baseline value of 33.8 (18.7) to 7.2 (9.0) (p0.001) and 76.1% of the patients had normalization of their esophageal acid exposure. Independent predictors of a favorable outcome after MSA included age younger than 45 years (odds ratio [OR] 4.2; 95% CI, 1.1 to 15.2; p = 0.0305), male sex (OR 2.5; 95% CI, 1.1 to 5.7; p = 0.0301), GERD-HRQL total score15 (OR 7.5; 95% CI, 3.3 to 16.8; p0.0001), and abnormal DeMeester score (OR, 2.6; 95% CI, 1.1 to 5.7; p = 0.0225).In this largest single-institution series, we demonstrate that MSA implantation is associated with very good clinical and objective outcomes. Age younger than 45 years, male sex, GERD-HRQL total score15, and abnormal DeMeester score are the 4 preoperative factors predicting a favorable outcome and can be used in patient counseling and MSA use.

Introduction Magnetic sphincter augmentation (MSA) results in less severe side effects compared with Nissen fundoplication, but dysphagia remains the most common side effect reported by patients after MSA. This study aimed to characterize and review the management of postoperative dysphagia and identify the preoperative factors that predict persistent dysphagia after MSA. Material and Methods This is a retrospective review of prospectively collected data of patients who underwent MSA between 2013 and 2018. Preoperative objective evaluation included upper endoscopy, esophagram, high-resolution impedance manometry (HRIM), and esophageal pH testing. Postoperative persistent dysphagia was defined as a postoperative score of > 3 for the dysphagia-specific item within the GERD-HRQL at a minimum of 3 months following MSA. A timeline of dysphagia and dilation rates was constructed and correlated with the evolution of our patient management practices and modifications in surgical technique. Results A total of 380 patients underwent MSA, at a mean (SD) follow up of 11.5 (8.7) months, 59 (15.5%) patients were experiencing persistent dysphagia. Thirty-one percent of patients required at least one dilation for dysphagia or chest pain and the overall response rate to this procedure was 67%, 7 (1.8%) patients required device removal specifically for dysphagia. Independent predictors of persistent dysphagia based on logistic regression model included (1) absence of a large hernia (OR 2.86 (95% CI 1.08–7.57, p = 0.035)); (2) the presence of preoperative dysphagia (OR 2.19 (95% CI 1.05–4.58, p = 0.037)); and (3) having less than 80% peristaltic contractions on HRIM (OR 2.50 (95% CI 1.09–5.73, p = 0.031)). Graded cutoffs of distal contractile integral (DCI), mean wave amplitude, DeMeester score, sex, and body mass index were evaluated within the model and did not predict postoperative dysphagia. Frequent eating after surgery, avoidance of early dilation, and increase in the size of the LINX device selected decreased the need for dilation. Conclusion In a large cohort of patients who underwent MSA, we report 15.5% rate of persistent postoperative dysphagia. The overall response rate to dilation therapy is 67%, and the efficacy of dilation with each subsequent procedure reduces. Patients with normal hiatal anatomy, significant preoperative dysphagia, and less than 80% peristaltic contractions of the smooth muscle portion of the esophagus should be counseled that they have an increased risk for persistent postoperative dysphagia.

Esophageal adenocarcinoma (EAC) is a lethal cancer requiring improved screening strategies and treatment options due to poor detection methods, aggressive progression, and therapeutic resistance. Emerging circulating tumor DNA (ctDNA) technologies may offer a unique non-invasive strategy to better characterize the highly heterogeneous cancer and more clearly establish the genetic modulations leading to disease progression. The presented review describes the potential advantages of ctDNA methodologies as compared to current clinical strategies to improve clinical detection, enhance disease surveillance, evaluate prognosis, and personalize treatment. Specifically, we describe the ctDNA-targetable genetic markers of prognostic significance to stratify patients into risk of progression from benign to malignant disease and potentially offer cost-effective screening of established cancer. We also describe the application of ctDNA to more effectively characterize the heterogeneity and particular mutagenic resistance mechanisms in real-time to improve prognosis and therapeutic monitoring strategies. Lastly, we discuss the inconsistent clinical responses to currently approved therapies for EAC and the role of ctDNA to explore the dynamic regulation of novel targeted and immunotherapies to personalize therapy and improve patient outcomes. Although there are clear limitations of ctDNA technologies for immediate clinical deployment, this review presents the prospective role of such applications to potentially overcome many of the notable hurdles to treating EAC patients. A deeper understanding of complex EAC tumor biology may result in the progress toward improved clinical outcomes.

4042 Background: Improving immunotherapy efficacy remains an unmet need in esophagogastric cancer and a deeper understanding of tumor and immune system dynamics during therapy may tailor immuno-oncology approaches. Methods: We performed whole exome sequencing (WES) and bulk RNA sequencing (RNAseq) of 70 serial tumor samples from 23 patients with stage II/III esophageal/gastroesophageal junction (E/GEJ) cancer treated on a phase 1B clinical trial with neoadjuvant nivolumab with or without relatlimab (anti-LAG-3) and chemoradiation followed by surgery (NCT03044613; CA209-906). Pathologic response was measured by tumor regression at the time of resection. Median follow up was 23 months post-surgery. Serial tumor samples were collected prior to therapy, after 2 cycles of induction immune checkpoint blockade (ICB), and at the time of resection. Twenty-two baseline tumor/normal DNA pairs were analyzed by WES and 48 serial tumor samples were analyzed by RNAseq. WES data was analyzed to identify somatic mutations, generate tumor mutation burden (TMB) estimates and assess the fraction of expressed mutations in conjunction with RNAseq data. Immune cell subset composition was determined by RNAseq data deconvolution by CIBERSORT and gene set enrichment analyses were performed utilizing GSEA. B-cell density was inferred by immunoglobulin rearrangements detected by RNAseq. Results: Gene set enrichment expression analyses revealed an upregulation of effector pro-inflammatory cytokines after induction ICB. Interferon-gamma, interferon-alpha and TNF-alpha related genes were significantly upregulated after induction ICB compared to baseline (p < 0.0001). In contrast, significant downregulation of E2F targets (p = 0.002), G2M checkpoint genes (p = 0.005) and DNA damage repair genes (p = 0.004) was observed post ICB; enrichment analyses were independent of response to therapy and treatment arm. While TMB was not predictive of pathologic response (p = 0.22), patients with tumors harboring a higher number of expressed mutations were more likely to achieve a pathologic complete response (pCR; p = 0.026). RNAseq deconvolution analyses revealed a higher B-cell density post ICB induction in tumors with pCR (p = 0.018). Furthermore, an increased baseline content of intra-tumoral activated M1 macrophages differentiated tumors from patients achieving a pCR (p = 0.0034), which was further exemplified post induction ICB. Conclusions: Neoadjuvant immunotherapy induces an inflammatory immune response in the tumor microenvironment that is linked with tumor elimination and pathologic response. Our findings highlight the importance of nuanced multi-omics analyses to understand the wiring of response to immunotherapy and guide therapy for E/GEJ cancer.

Esophageal adenocarcinoma (EAC) is an aggressive cancer necessitating the development of improved risk stratification tools for personalized care. Previously, microRNAs have been shown to correlate with the progression and prognosis of various cancer types; however, the value in EAC remains largely unexplored. We performed global microRNA profiling on 32 formalin-fixed, paraffin-embedded EAC specimens to identify microRNAs associated with progression. Literature search and pathway analysis further refined output to five significantly deregulated candidate biomarkers. Four of the five microRNAs (miR-652-5p, miR-7-2-3p, miR-3925-3p, and miR-219-3p) were validated by qRT-PCR. Survival outcomes were evaluated in testing set of 26 stage II/III EAC patients to determine the prognostic relevance of the selected microRNAs. In the testing set, miR-652-5p and miR-7-2-3p expressions were significantly associated with progression-free survival (p-value = .00771 and p-value = .00293). The highest area under receiver operating characteristic (ROC) curve was 0.8212 for the combination of miR-652-5p and miR-7-2-3p. Collectively, our findings demonstrated that the miR-652-5p/miR-7-2-3p signature may serve as a promising prognostic marker in patients with locally advanced EAC.

4571 Background: While neoadjuvant chemoradiotherapy (CRT) has emerged as an important treatment modality in patients with locally advanced esophageal adenocarcinoma (EAC), ~60%-70% of patients do not respond to such treatments; but are exposed to their toxicity nonetheless. This highlights the clinical need for the development of biomarkers that can robustly predict response to CRT and spare others from the toxicity and expense associated with these treatments. Herein, we systematically and comprehensively identified a biomarker signature that predicts response to neoadjuvant therapy in EAC patients. Methods: A cohort of 31 EAC patients treated with chemotherapy or chemoradiotherapy was assembled, with a majority of patients receiving carboplatin, paclitaxel and concurrent radiotherapy. Specifically, we performed a capture based targeted sequencing in paired biopsy specimens obtained at baseline and 3-6 weeks post-treatment. In addition, we also analyzed the predictive potential of a panel of immune-related genes (TIM3, LAG3, IDO1 and CXCL9) in these matched pre- and post-treatment tissues. Results: In our cohort, based upon pathologic response to neoadjuvant CRT, 8 EAC patients were categorized as non-responders, while 23 were deemed as responders to CRT. Among responders, the most frequently mutated genes were MKI67, SYNE1, PCLO, RECQL4, MSH3, NOTCH2, ILR7, CIITA, LRRK2 and EML4, and the overall tumor mutation burden (TMB) was significantly reduced for these genes in post-treatment samples ( P=5.73E-03). Similarly, in non-responders NLRP1, MAP3K1, ASMTL, and ALK harbored frequent mutations, and the TMB was significantly reduced for these genes in post-treatment samples ( P=5.57E-03). We compared responders and non-responders from the pre-treatment samples and identified differentially mutated genes including EPHA5, ZNF217, RELN, PALB2 and MYO18A. Similarly, responders had all four immune-related genes significantly up-regulated in post-treatment samples than pre-treatment samples. We constructed a risk-stratification model that comprised of mutational score from 5 differentially mutated genes, together with 4 immune-related genes, which achieved an AUC of 0.96 in predicting response to CRT in EAC patients ( P=1.03E-04). Conclusions: Using a systematic biomarker discovery approach, we have developed a novel biomarker signature that robustly predicts response to CRT in EAC patients and has a significant potential for personalized management of locally advanced EAC patients.

445 Background: While neoadjuvant chemoradiotherapy (CRT) has emerged as an important treatment modality in patients with locally advanced esophageal adenocarcinoma (EAC), ~60%-70% of patients do not respond to such treatments; but are exposed to their toxicity nonetheless. This highlights the clinical need for the development of biomarkers that can robustly predict response to CRT and spare others from the toxicity and expense associated with these treatments. Herein, we systematically identified a biomarker signature that predicts response to CRT in EAC patients. Methods: Using a clinical-trial driven cohort of 25 EAC patients treated with 5- fluorouracil plus carboplatin and concurrent radiation therapy, we performed whole-exome sequencing (WES) in paired biopsy specimens obtained at baseline and 3-6 weeks post-treatment. In addition, we also analyzed the predictive potential of a panel of immune-related genes (TIM3, LAG3, IDO1 and CXCL9) in these matched tissues. Results: In our cohort, based upon RECIST criteria, 14 EAC patients were categorized as non-responders, while 11 were deemed as responders to CRT. Among responders, the most frequently mutated genes were NOTCH1, NOTCH2, NOTCH3, and MLL2; and the overall tumor mutation burden (TMB) was significantly reduced for these genes in post-treatment specimens (P