Esophageal Adenocarcinoma Research Articles

High proportion of PD-1 and CD39 positive CD8+ tissue resident T lymphocytes correlates with better clinical outcome in resected human oesophageal adenocarcinoma

ObjectiveTo understand the CD8+ tumour infiltrating lymphocyte (TIL) compartment of oesophageal adenocarcinoma (OAC) with regards to markers of lymphocyte exhaustion, tissue residency and to identify possible reasons behind differential responses to therapy.DesignTumour samples from 44 patients undergoing curative resection for OAC were assessed by flow cytometry for presence of antigen-experienced TILs and markers of activation and exhaustion. Populations of PD-1 and CD39 positive OAC TILs were sorted, and bulk RNA sequencing undertaken using a modified SmartSeq2 protocol. Flow cytometric assessment of functionality was completed.ResultsA higher proportion of antigen experienced CD8+ OAC TILs was associated with improved survival following surgery; while, high double positivity (DP) for PD-1 and CD39 among these TILs also correlated significantly with outcome. These DP TILs possess a minority population which is positive for the markers of exhaustion TIM3 and LAG3. Transcriptomic assessment of the PD-1 and CD39 DP TILs demonstrated enrichment for a tissue resident memory T lymphocyte (TRM) phenotype associated with improved survival in other cancers, reinforced by positivity for the canonical TRM marker CD103 by flow cytometry. This population demonstrated maintained functional capacity both in their transcriptomic profile, and on flow cytometric assessment, as well as preserved proliferative capacity.ConclusionResected OAC are variably infiltrated by PD-1 and CD39 DP TILs, an abundance of which among lymphocytes is associated with improved survival. This DP population has an increased, but still modest, frequency of TIM3 and LAG3 positivity compared to DN, and is in keeping with a functionally competent TRM phenotype.

Development of a novel oncolytic adenovirus controlled by CDX2 promoter for esophageal adenocarcinoma therapy.

Prognosis of esophageal adenocarcinoma (EAC) is still poor. Therefore, the development of novel therapeutic modalities is necessary to improve therapeutic outcomes in EAC. Here, we report a novel promoter-controlled oncolytic adenovirus targeting CDX2 (Ad5/3-pCDX2) and its specific anticancer effect for EAC. We used OE19, OE33, HT29, MKN28, RH30, and HEL299 cell lines. To establish CDX2 overexpressing OE19 cells, pCMV-GLI1 plasmid was transfected to OE19 (OE19 + GLI1). The virus replication and cytocidal effect of replication competent Ad5/3-pCDX2 were analyzed in vitro. Antitumor effect of Ad5/3-pCDX2 was assessed in xenograft mouse models by intratumoral injection of the viruses. Finally, efficacy of combination therapy with Ad5/3-pCDX2 and 5FU was evaluated. EAC cells and HT29 showed high mRNA levels of CDX2, but not MKN28, RH30, and HEL299. We confirmed that deoxycholic acid (DCA) exposure enhanced CDX2 expression in EAC cells and OE19 + GLI1 had persistent CDX2 overexpression without DCA. Ad5/3-pCDX2 showed stronger cytocidal effect in OE19 + GLI1 than OE19, whereas Ad5/3-pCDX2 did not kill CDX2-negative cells. Ad5/3-pCDX2 was significantly replicated in EAC cells and the virus replication was higher in OE19 + GLI1 and OE19 with DCA compared to OE19 without DCA exposure. In vivo, Ad5/3-pCDX2 significantly suppressed OE19 tumor growth and the antitumor effect was enhanced in OE19 + GLI1 tumor. In contrast, Ad5/3-pCDX2 did not show significant antitumor effect in MKN28 tumor. Moreover, Ad5/3-pCDX2 significantly increased the efficacy of 5FU in vitro and in vivo. Ad5/3-pCDX2 showed specific anticancer effect for EAC, which was enhanced by bile acid exposure. Ad5/3-pCDX2 has promising potential for EAC therapy in the clinical setting.

Semaphorin 3F (SEMA3F) influences patient survival in esophageal adenocarcinoma

In esophageal adenocarcinoma, the presence of lymph node metastases predicts patients' survival even after curative resection. Currently, there is no highly accurate marker for detecting the presence of lymph node metastasis. The SEMA3F/NRP2 axis was initially characterized in axon guidance and recent evidence has revealed its significant involvement in lymphangiogenesis, angiogenesis, and carcinogenesis. Hence, the objective of this study was to elucidate the roles of SEMA3F and its receptor NRP2 in esophageal adenocarcinoma. We conducted an immunohistochemical evaluation of SEMA3F and NRP2 protein expression in 776 patients with esophageal adenocarcinoma who underwent Ivor-Lewis esophagectomy at the University Hospital of Cologne. Total and positive cancer cell counts were digitally analyzed using QuPath and verified by experienced pathologists to ensure accuracy. Positive expression was determined as a cell percentage exceeding the 50th percentile threshold. In our cohort, patients exhibiting SEMA3F positive expression experience significantly lower pT- and pN-stages. In contrast, positive NRP2 expression is associated with the presence of lymph node metastases. Survival analyses showed that the expression status of NRP2 had no impact on patient survival. However, SEMA3F positivity was associated with a favorable patient survival outcome (median OS: 38.9 vs. 26.5 months). Furthermore, SEMA3F could be confirmed as an independent factor for better patient survival in patients with early tumor stage (pT1N0-3: HR = 0.505, p = 0.014, pT1-4N0: HR = 0.664, p = 0.024, pT1N0: HR = 0.483, p = 0.040). In summary, SEMA3F emerges as an independent predictor for a favorable prognosis in patients with early-stage esophageal adenocarcinoma. Additionally, NRP2 expression is linked to a higher risk of lymph node metastases occurrence. We hypothesize that low SEMA3F expression could identify patients with early-stage tumors who might benefit from more aggressive treatment options or intensified follow-up. Furthermore, SEMA3F and its associated pathways should be explored as potential tumor-suppressing agents.

764. PREDICTING AGE-SPECIFIC PREVALENCE OF BARRETT'S OESOPHAGUS IN AUSTRALIAN AND UNITED STATES

Abstract Background Barrett’s esophagus is the precursor to esophageal adenocarcinoma (EAC). EAC detected from endoscopic surveillance programs accounts for <10% of all EAC, suggesting a large number of patients with Barrett’s esophagus are likely unaccounted for. Several studies have estimated the observed prevalence of Barrett’s esophagus to be approximately 1%, but this may be an underestimate. The aim of this study is to use population incidence of EAC and rate of progression from Barrett’s esophagus from large cohort studies to estimate a realistic prevalence of Barrett’s esophagus. Methods A simple tree cohort model was created for progression to EAC from birth to death (100 years) for American and Australian population. Lifetime risk of esophageal cancer and adenocarcinoma were endpoints. This was then used to back-calculate age-specific and overall prevalence of Barrett’s esophagus using an optimisation algorithm. Results Lifetime risk of esophageal cancer and adenocarcinoma in US Whites is 0.56% and 0.36% respectively, while in Australian population is slightly higher at 0.81% and 0.61% (range 0.57% - 0.65%). Estimated overall prevalence of Barrett’s esophagus is ~3% (±0.3%) and ~5.4% (±0.6%) in US White and Australian populations, while in males is 5.3% (±0.6%) and 7.4% (±0.3%), respectively. Female cohorts were seen to have lower chance of developing esophageal cancer, adenocarcinoma, and Barrett’s esophagus. Conclusion Cohort studies of Barrett’s esophagus are likely to underestimate its prevalence of this silent disease. Computational models based on progression from Barrett’s esophagus to EAC accounting for undetected disease reveal higher estimates, which can help in future screening options.

281. PROTEOMIC, TRANSCRIPTOMIC AND GENOMIC ANALYSIS OF ESOPHAGEAL ADENOCARCINOMA UNCOVERS CANDIDATE THERAPEUTIC TARGETS AND CANCER-SELECTIVE POST-TRANSCRIPTIONAL REGULATION

Abstract Background Addressing the poor prognosis associated with esophageal adenocarcinoma (EAC) has been hindered by the absence of biomarkers for early disease detection and therapeutic targets. Despite extensive research on the somatic mutations associated with EAC, the impact of genomic aberrations on protein expression and cancer phenotype remains unclear. Methods We conducted a quantitative proteomic analysis using tumour tissue and patient-matched adjacent normal esophageal and gastric tissues from 23 patients undergoing resection for EAC. We further examined the relationship between transcript and protein levels using patient-matched whole transcriptome RNA sequencing and proteomic data from 7 patients and integrated these findings with data from a cohort of 264 EAC patients with RNA sequencing and 454 patients with whole-genome sequencing, as well as external published datasets. Results We quantified protein expression from 5897 genes in EAC and normal tissues. Several potential biomarkers showing selective expression in EAC, such as the transmembrane protein GPA33, were identified. We validated the EAC-specific expression of GPA33 in an external cohort of 115 patients, suggesting its utility as a diagnostic and therapeutic target. Additionally, integrated analysis of protein and RNA expression in EAC and normal tissues revealed genes with discordant protein and RNA levels, indicating post-transcriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, exhibited rare somatic mutations, suggesting post-transcriptional mechanisms driving this EAC-specific phenotype. AGMAT, as an example, was found to be overexpressed at the protein level in EAC compared to adjacent normal tissues, with a proposed EAC-selective, post-transcriptional mechanism regulating protein abundance. Conclusion Through quantitative proteomic analysis, we identified GPA33 as a candidate EAC biomarker. Integrated analysis of the proteome, transcriptome, and genome in EAC uncovered genes with tumor-selective post-transcriptional regulation of protein expression, offering potential exploitable vulnerabilities for therapeutic intervention.

463. ISOFORM SWITCHING EVENTS IN ESOPHAGEAL ADENOCARCINOMA MAY INFORM NEW THERAPEUTIC TARGETS THROUGH IDENTIFYING MOLECULAR MECHANISMS CONTRIBUTING TO POOR PATIENT OUTCOMES

Abstract Background Esophageal adenocarcinoma (EAC) represents a growing health problem characterized by rising incidence and poor prognosis due to late-stage diagnosis coupled with therapeutic resistance. Moreover, EAC is a cancer with a high mutational burden but lacks highly prevalent oncogenic drivers that can be successfully targeted. Herein, we investigated whether newly identified isoform switching events, defined as differential usage of gene transcripts between conditions, may reveal new therapeutic targets and inform the molecular mechanisms contributing to EAC resistance. Methods We conducted RNA-sequencing followed by isoform switching analysis using IsoformSwitchAnalyzeR on Barrett’s low-grade dysplasia (BE.LGD) compared to Barrett’s with high-grade dysplasia (BE.HGD)+EAC tissues alone or in combination with TP53 mutation. Patients were stratified into tertiles based on isoform fraction levels, followed by overall and cancer-free survival analysis using the Cox proportional-hazards model. To assess whether mortality-linked isoforms influence cancer cell growth, viability, migration, and response to chemotherapy (Paclitaxel and Carboplatin). Isoform-specific siRNA knockdown experiments targeting HM13 were performed in OE19 and OE33 EAC cell lines, with STRING protein prediction interaction analysis and western blots conducted to assess pathway and protein alterations. Results Twenty-one isoform switched genes were significantly linked with all-cause mortality in BE.LGD versus BE.HGD+EAC and similarly 20 were significantly associated with cancer-specific patient mortality with 14 shared between the two groups. With inclusion of TP53 mutation status, 13 isoform switched genes were significantly linked with cancer-specific mortality. HM13, DANT2, CFDP1, KRAS, SIMM6 were among the most significantly altered. Knockdown of the pro-cancer isoform of HM13 significantly inhibited EAC cell migration and induced EAC cell death and knockdown of HM13 combined with chemotherapeutic treatment exhibited synergistic effects in EAC cell lines. Protein interaction analysis supported that HM13 is linked to ER-stress associated degradation, cellular response to stress, and ER unfolded protein response which were validated using a panel of relevant proteins (i.e., PERK, IRE-1a, ATF4, BIP, P-JNK). Conclusions Our research identified isoform switching events significantly linked to all-cause mortality as well as cancer specific mortality among EAC patients with and without TP53 mutations. Knockdown of the pro-cancer isoform of HM13 isoform significantly inhibited EAC cell migration and induced EAC cell death with synergistic effects observed with chemotherapeutic agent treatment supporting a role for specific isoform switches in therapeutic sensitization and in this case via the ERAD and ER unfolded protein response pathways. Modulation of specific proteins with established roles in ERAD and ER unfolded responses following isoform specific HM13 knockdown lends further support to an important role for this isoform, especially in TP53 mutant EAC. In conclusion, isoform switches may inform molecular mechanisms contributing to poor patient outcomes and reveal new potential therapeutic targets for EAC.

540. INFILTRATION OF RESECTED OESOPHAGEAL ADENOCARCINOMA WITH ANTIGEN-EXPERIENCED PD-1 AND CD39 POSITIVE CD8+ TISSUE RESIDENT MEMORY CELLS CORRELATE WITH IMPROVED SURVIVAL

Abstract Background The development of novel treatments for oesophageal adenocarcinoma (OAC) represents an area of significant unmet need. Despite the application of immune checkpoint blockade alongside chemotherapy as standard of care for some patients with metastatic OAC, responses to immunotherapies are modest and 5-year survival is 17%. The reasons for this remains unclear and while tumour infiltration with CD8+ T lymphocytes (TILs) at the time of resection is associated with improved outcomes, this heterogeneous population remains incompletely characterised in OAC. Tissue resident memory cells (TRM) are increasingly understood as key mediators of immunotherapy response but their role in OAC is poorly understood. Methods Tumour samples were accessed by punch biopsy from the central portion of cancers resected from 44 patients undergoing curative surgery for OAC. Samples were assessed by multi-parametric flow cytometry for the presence of antigen-experienced TILs and markers of activation and exhaustion. Populations of antigen-experienced PD-1 and CD39 positive CD8+ OAC TILs were sorted, and bulk RNA sequencing (RNAseq) undertaken using a modified SmartSeq2 protocol allowing differential gene expression analysis and gene set enrichment analysis. Flow cytometric assessment of functionality was completed. Results Resected OAC are often highly infiltrated with antigen experienced CD8+ TILs expressing high levels of PD-1 and CD39 and an abundance of this population correlates with improved survival (%PD-1/CD39+ >Median, OS HR 0.12). RNAseq of sorted TILs identified this PD-1+ and CD39+ lymphocyte population as enriched for precursor exhausted-like CD8+ TRM (NES 2.01 p=0.00). This is correlated by their CD103+ and TIM3- predominance on flow cytometry in keeping with TRMsassociated with immunotherapy response in other cancers. This population demonstrated a maintained proliferative potential, an ability to degranulate and produce the key effector molecules IFN-γ, TNF-α and Granzyme B. Conclusions An abundance of a PD-1 and CD39 positive CD8+ TIL population, consistent with a precursor exhausted-like TRM population with maintained proliferative and functional capacity, is observed to correlate with improved outcomes following curative surgery for OAC. This TRM population is consistent with analogous populations observed to be responsible for immunotherapy response in other cancers and their identification may predict benefit from such approaches in OAC and allow better patient selection for treatment with immune checkpoint inhibitors.

507. LONG BARRETT´S ESOPHAGUS IN MONOZYGOTIC TWINS WITH ADENOCARCINOMA DEVELOPMENT

Abstract Background Familial aggregation of Barrett's esophagus (BE) and esophageal adenocarcinoma (EA) suggests a genetic pattern in fewer than 10% of patients with the former diseases. In 1983, BE was described in identical twins, underlining the hereditary influence on the development of mucosal dysplasia in some patients with this epithelial alteration. At present, some studies have attempted to identify chromosomal alterations among patients with familial aggregation of BE or EA, but there is not a clear pattern. Methods Description of the case report of a male patient who underwent esophagectomy due to EA on a long BE, who had a monozygotic twin without gastroesophageal reflux symptoms and no upper GI endoscopies before. Results A 53-year-old man was diagnosed with EA and a long BE (8 cm length) in 2017. He underwent CROSS neoadjuvant treatment and minimally invasive Ivor-Lewis esophagectomy. The pathological report indicated a GI T3N1M0 adenocarcinoma. Unfortunately, the patient developed lung metastases and passed away in 2019. Before that, the patient recalled that he had an identical twin brother with no gastroesophageal reflux symptoms, but we decided to scope him in 2017. The gastroscopy report revealed a 2 cm hiatal hernia with a long BE (C4M6 Prague Classification) but with no dysplasia. Nowadays, the twin patient is under close follow-up with proton pump inhibitors (PPI) and has not developed dysplasia. Conclusion Monozygotic twins provide a perfect model to study the possible influence of genetic predisposition to develop BE and EA. The development of EA in a patient guided us to diagnose and treat a long BE in his asymptomatic identical twin. More studies should be done to find the possible genetic alterations linked to familial aggregation of BE and EA.

387. VALIDATION OF PROMARKERESO, A DIAGNOSTIC BLOOD TEST TO IDENTIFY ESOPHAGEAL ADENOCARCINOMA

Abstract Background Esophageal adenocarcinoma (EAC) has become the predominant type of esophageal cancer in Western countries. Early diagnosis and intervention of EAC significantly reduces the disease mortality. A previous study identified a panel of novel serum glycoprotein biomarkers that were associated with EAC, enabling the development of a prototype diagnostic blood test, PromarkerEso. The current study aimed to validate PromarkerEso in an independent cohort of patients with EAC. Methods Blood samples from an independent cohort of 165 participants (n=99 negative controls, n=66 diagnosed with EAC) were analyzed. Five candidate biomarkers were measured after lectin pulldown using a targeted mass spectrometry assay. Model performance was assessed by the area under the receiver operating characteristics curve (AUC-ROC) with sensitivity and specificity determined. Results After adjusting for clinical variables, diagnostic models showed good discrimination to distinguish participants with EAC from negative control participants. Conclusions This study validates a panel of serum glycoprotein biomarkers for the diagnosis of EAC. These biomarkers have potential utility as a simple blood test, PromarkerEso, that could be used for screening EAC in a large population.

365. METHYLATION BIOMARKERS TO STRATIFY BARRETT’S ESOPHAGUS PATIENTS PROGRESSING AND NON-PROGRESSING TO ESOPHAGEAL ADENOCARCINOMA

Abstract Background Over the last forty years, North America and Europe have witnessed a notable surge in cases of esophageal adenocarcinoma (EAC). This malignancy typically arises from Barrett esophagus (BE), a condition characterized by the replacement of normal squamous epithelium with specialized metaplastic cells. Our study aims to identify specific methylation markers capable of distinguishing between BE patients that will and will not progress to EAC. Methods We conducted differential expression analysis on RNASeq public dataset GSE210647 to distinguish BE progressors from non-progressors. Reads were aligned to hg38 genome using STAR aligner, normalized and analyzed with DESeq2. Significant genes were selected by |log2 fold change| ≥ 0.2 and padj < 0.01. GO, pathway enrichment, and transcription factor (TF) network analyses were performed to uncover biological disparities. Integration with previous methylation data (N.R. Dehkordi et al., 292. Specific methylation markers associated with changes in gene expression distinguish EAC and sub-types of BE patients, ISDE 2023) allowed the identification of overlapping biomarkers, aiding in better discrimination between the two groups. Results Differential expression analysis of GSE210647 identified 2261 significative DEGs between BE progressors and non-progressors. Pathway enrichment analysis in BE progressors revealed significant associations with cellular morphogenesis and reprogramming, alongside the induction of the bile acid pathway, known for its implications in invasiveness and cancer stem cell expansion. TF network analysis identified key regulators such as TBX20, FOXS1, and ZNF454, shedding light on their roles in esophageal carcinogenesis. Among the 505 CpG markers acquired in prior analyses, 127 genomic regions were linked with overexpressed genes either in progressors or in non-progressors BE patients. Leveraging this marker panel, BE samples were stratified into discrete subtypes, aligning either with normal esophageal tissue or EAC, and suggesting a potential stratification by disease progression. Conclusions The integration of expression and methylation analyses allows the identification of key genomic and epigenomic loci able to distinguish BE patients progressing or not-progressing towards EAC. The identified biomarkers are pivotal for personalized risk assessment and for the design of targeted interventions, holding promises for enhanced surveillance protocols, refined early EAC detection approaches, and improved therapeutic strategies aimed at mitigating the risk of EAC development in BE population.

292. CHEMOTHERAPY AND CHEMORADIOTHERAPY FOR ADENOCARCINOMA OF THE OESOPHAGUS AND JUNCTION WITH OLIGOMETASTASES: RESULTS OF THE TNT-OES-1 TRIAL

Abstract Background Chemotherapy (FLOT) and chemoradiotherapy (CROSS) are both effective as neoadjuvant regimens for esophageal and junctional cancer. Total Neoadjuvant Therapy (TNT) aims to increase efficacy by combining chemotherapy with chemoradiotherapy. Although TNT is increasingly used in rectal cancer, there are no data on combining e.g. FLOT and CROSS in esophageal cancer. This phase-II study aimed to evaluate the feasibility and safety of the TNT FLOT-CROSS combination in esophageal and junctional adenocarcinoma patients with oligometastatic disease. Methods Patients with oligometastatic (defined as a maximum of four lesions in up to two organs, excluding lymph nodes) esophageal adenocarcinoma were treated with four biweekly cycles of FLOT followed by response evaluation (CT-scan). Patients without disease progression (RECIST v1.1) proceed to CROSS. Afterwards, response was assessed by CT-scan, endoscopy with biopsies and endoscopic ultrasonography with fine-needle aspiration on indication. The multidisciplinary tumor board discussed whether patients could proceed to four additional cycles of FLOT or local therapy of metastases and/or esophagectomy. Primary endpoint was tolerability of TNT FLOT-CROSS, defined as the proportion completing four cycles of FLOT and five cycles of chemoradiotherapy. Secondary endpoints included progression-free survival (PFS), disease control rate (DCR), toxicity and the proportion proceeding to local therapy of metastases and/or esophagectomy. Results Twenty patients (85% males, median age 62.5 years (interquartile range 58.3-66) were included of whom 16 (80%) had a single metastatic lesion. Eighteen patients (90%) successfully completed FLOT and fifteen (75%) completed TNT FLOT-CROSS (Figure 1). Toxicity mainly consisted of grade 1-2 leukopenia/neutropenia during FLOT, leading to treatment delay in twelve patients and dose-limiting of oxaliplatin in one patient. One patient had malaise grade 2. No grade 3-5 toxicities occurred during CROSS. Following TNT FLOT-CROSS, ten patients underwent four additional cycles of FLOT and toxicity resulted in treatment delay and dose reduction in seven patients and hospitalization in two patients. Six patients underwent esophagectomy of whom two underwent local treatment of metastases. The 1- and 2-year PFS was 56% and 48%. Three months after TNT FLOT-CROSS completion, DCR was 73%. Conclusion(s) This study showed that sequencing TNT FLOT-CROSS in patients with oligometastatic esophageal adenocarcinoma is feasible and comes with manageable toxicity and promising efficacy. In follow-up to this study, the TNT-OES-2 study will assess the efficacy and feasibility of TNT FLOT-CROSS and TNT CROSS-FLOT in patients with resectable nodal-positive adenocarcinoma of the esophagus or esophagogastric junction.

PD-1/PD-L1 Inhibitors in Combination With Chemo or as Monotherapy vs. Chemotherapy Alone in Advanced, Unresectable HER2-Negative Gastric, Gastroesophageal Junction, and Esophageal Adenocarcinoma: A Meta-Analysis

AimsAdvanced gastroesophageal cancers are still associated with poor outcomes. We aim to study PD-1/PD-L1 inhibitors in phase III clinical trials that have compared them to chemotherapy in gastric, gastroesophageal junction (GEJ), and esophageal adenocarcinoma. Materials and methodsOn March 28, 2024, we searched: PubMed, Embase, Cochrane Library, Web of Science, Scopus, and ClinicalTrials.gov. We only included randomized clinical trials for PD-1/PD-L1 inhibitors alone or with chemo vs chemotherapy in advanced gastric, GEJ, or esophageal adenocarcinoma. The primary endpoints were overall survival and progression-free survival. A subgroup analysis was conducted for the following variables: treatment line, type of intervention, age group, gender, ECOG Performance Status, combined positive scores (CPS), microsatellite instability (MSI) status, liver metastasis, and primary tumor location. ResultsOnly 10 out of 8,942 articles were included, involving 6,782 patients. PD-1/PD-L1 inhibitors showed a significant improvement in the overall survival compared to chemotherapy alone (hazard ratio (HR): 0.86, 95% CI: 0.80–0.93; p = 0.0002). Combining PD-1/PD-L1 inhibitors with chemotherapy significantly improved overall and progression-free survival compared to monotherapy (combined therapy HR 0.80; p < 0.00001 vs. monotherapy HR 0.98; p = 0.77). CPS ≥1 had an HR of 0.78 (95% CI: 0.73–0.84; p < 0.00001), CPS ≥10 had an HR of 0.67 (95% CI: 0.59–0.76; p < 0.00001), and MSI-high status had an HR of 0.35 (95% CI: 0.24–0.52; p < 0.00001). Esophageal adenocarcinoma, reported in three trials, did not show significant improvement in the overall survival (HR 0.89; 95% CI: 0.69–1.14; p = 0.37). ConclusionPD-1/PD-L1 inhibitors have significantly improved overall survival, and combining them with chemotherapy is more effective than monotherapy. Both CPS ≥10 and MSI-H showed an added benefit to overall survival and should be included in biomarker investigations. Clinical trials are needed for second-line treatments and esophageal adenocarcinoma.

Liquid-liquid phase separation-related signature predicts prognosis and therapeutic response in esophageal adenocarcinoma

BackgroundEsophageal adenocarcinoma is a leading cause of mortality worldwide. New evidence indicates that liquid-liquid phase separation is related to malignancies. The current study aims at exploring the functions of liquid-liquid phase separation within esophageal adenocarcinoma. Patients within the TCGA dataset were classified using liquid–liquid phase separation-related genes. Significantly differentially expressed genes and prognostic factors for overall survival have been screened by Cox regression. Based on the liquid-liquid phase separation score, the construction of a prognostic model and liquid-liquid phase separation signature was constructed. Tumor mutation burden and drug sensitivity were analyzed in two groups: high liquid-liquid phase separation scores, and low liquid-liquid phase separation scores. According to liquid-liquid phase separation, some small-molecule compounds targeting esophageal adenocarcinoma were screened. The results were verified in vitro with an external cohort. Results87 samples are involved, and 61 liquid-liquid phase separation-related genes may influence esophageal adenocarcinoma by changing DNA conformation and metabolism. Meanwhile, based on a high liquid-liquid phase separation score and low score group including 43 patients, it is found that the result significantly lowered the 5-year overall survival to 32.6 %, compared to 64.8 % in the low-score group of 44 patients with p < 0.001. The high score group had an average TIDE score of 0.27 versus 0.14 in the low-score group, with p = 0.003. The median tumor mutation burden was 9.1 mutations/Mb in the high-score group versus 6.4 mutations/Mb in the low-score group, with p = 0.011. The predictive model worked very well, with area under the curve values of 0.82, 0.79, and 0.76 for 1-, 3-, and 5-year survival, respectively. Liquid-liquid phase separation has been validated as an effective prognostic biomarker and drug sensitivity predictor. SignificanceLiquid-liquid phase separation is potentially implicated in esophageal adenocarcinoma and works as a prognostic biomarker assessment of vulnerability to LLPS, which could help develop individualized therapies by showing how one is situated about various medications where responses vary across the body.

536. TUMOR-INFORMED “LIQUID BIOPSY” FOR ESOPHAGEAL ADENOCARCINOMA FROM MATCHED CANCER ORGANOID CULTURE

Abstract Background The absence of recurrent mutations in esophageal adenocarcinoma (EAC) poses a challenge in detecting circulating tumor DNA (ctDNA) in plasma and may hinder the advancement of liquid biopsy methods. To address this, we cultured patient-derived EAC organoids (PDOs), speculating that they could serve as a guide for identifying ctDNA in the patient's blood samples. This approach aims to leverage organoids as a potential tool to overcome the complexity of identifying ctDNA in EAC, offering a promising avenue for refining liquid biopsy strategies in clinical practice. Methods PDOs were generated from EAC tumor tissue in Matrigel domes and expanded in suspension culture. To isolate mononucleosomes (147 bp), chromatin from PDOs was extracted and digested with micrococcal nuclease (MNase). Fragments larger than 147 bp were removed through size selection. MNase-sequencing was performed to generate a mutation map with preferential coverage of nucleosome-protected DNA for each sample. Matched whole genome sequencing of the tumor for each respective PDO sample was used as a control. Primers were designed for the identified mutations in nucleosome-protected DNA and used to amplify patient cfDNA for sequencing. Results DNA from five different PDOs were collected and MNase digested. MNase concentration and digestion time were optimized for each sample. MNase digestion produced mononucleosomes of approximately 147 bp for all samples. MNase-sequencing identified 24 mutations in peaks (mononucleosomes) in 24 genes, including known oncogenes. Among these were 16 missense, 2 frameshift, and 1 nonsense mutations, and 5 mutations in splice regions. To date, amplicons of expected size were detected by PCR for six genes using either total PDO DNA or normal cell-free DNA, confirming the detectability of these genes. PCR amplification using patient ctDNA and next-generation sequencing is ongoing. Conclusion These findings show that we are able to isolate and detect somatic mutations in nucleosomes from different PDOs, allowing us to generate a nucleosome SNV map for each sample. Preliminary data indicate these regions can be PCR amplified from normal cfDNA. Amplification and sequence verification of mutated regions from corresponding patient blood ctDNA is ongoing. The mapping of patient-specific variants will enable the development of targeted personalized PCR panels, aiding in recurrence prediction and enhancing drug screening accuracy. This advancement holds promise for early cancer detection and improving prognoses for individuals with EAC by addressing gaps in recurrence prediction.

202. MULTI-ARRAY MIRNA APPROACH AND ASSOCIATED PROTEIN EXPRESSION PROFILING FOR THE DEVELOPMENT OF PREDICTIVE AND PROGNOSTIC BIOMARKERS IN ESOPHAGEAL ADENOCARCINOMA

Abstract Background Esophageal adenocarcinoma (EA) may develop metastases even in early stage (pT1) disease, but molecular bi-omarkers are lacking to guide therapeutic decisions regarding the type of treatment. Endoscopic submucosal dis-section (ESD) is an effective treatment of early esophageal adenocarcinomas. However, ESD only an option for patients without lymph node metastases. The aim of this study is to detect possible biomarkers with which one could estimate lymphatic metastasis. Methods A multi-array miRNA study with consecutive in silico analysis of dependent genes and in situ analysis of selected proteins was chosen to detect possible biomarkers. A retrospective analysis including twenty cases of EA of the distal esophagus/esophagogastric junction after ESD without or after esophagectomy with lymph node metastasis (each n=10) was performed. The DNA of the Formalin-Fixed Paraffin-Embedded (FFPE) material of these patients was analysed using an advanced miRNA Human Card (spotted with 754 unique microRNAs) with consecutive in silico gene regulation and immunohistochemical analysis of the associated proteins. Result The multi-array miRNA of the tumour sample with EA showed an upregulation of 25 and a downregulation of 20 microRNAs, with the highest differences for the miRNAs miR-100-5p, miR-501-3p and miR-576-5p (upregulation) and miR-150-3p, miR-190a-5p and miR-545-3p (downregulation). The in silico analysis of these mirRNAs revealed the downstream paired partners RDX-Rock2, DICER1-AGO2, YAF2-EZF6 and TET2-5HMC, which were significantly differentially expressed by in-situ immunohistochemical staining. Biologically relevant downstream gene and pro-tein targets, which were also significantly differentially expressed in-situ in relation to the tumour stage of EA could be found. Conclusion This molecular study provides evidence for a specific miRNA marker profile when comparing cases with localized and advanced EA. These preclinical experimental biomarker analyses may help to guide therapeutic decisions in cases with EA, although validation in a larger cohort of EA is necessary to draw firm conclusions.

450. TISSUE RESIDENT MEMORY CELLS: CORRELATING THE PHENOTYPIC AND FUNCTIONAL CAPABILITIES OF A POTENTIAL TUMOUR REACTIVE CELL POPULATION IN OESOPHAGEAL ADENOCARCINOMA

Abstract Background Oesophageal adenocarcinoma (OAC) is a disease with a high mortality and morbidity rate. Immunotherapy has recently demonstrated progress in the treatment of OAC but its impact on long term survival is currently uncertain. Tissue resident memory (TRM) T cells are a subset of lymphocytes within the tumour/tissue infiltrating lymphocyte (TIL) milieu that has attracted considerable interest within cancer immunology. Although TRM cells have been associated with better outcomes in multiple cancer types there is a lack of knowledge of their role in OAC. This study aims to critically correlate the phenotype of TRM with their functional capacity in this disease. Methods 33 patients undergoing surgical resection for OAC were recruited and consented for collection of fresh tumour (T), adjacent normal tissue (AN), and peripheral blood. Peripheral blood mononuclear cell (PBMC) from blood and infiltrating lymphocytes from T and AN were isolated using standard density gradient and enzymatic digestion protocols. Multiparametric flow cytometry was performed on 12 patients using matched PBMC, T and AN infiltrating lymphocytes for phenotypic analysis and 21 patients for functional analysis following cell stimulation assay. BD FACSymphony was used for data acquisition and data analysed on FlowJo version 10.10. Statistical analysis was carried out using GraphPad Prism. Results TRM (CD103+CD69+) dominated the CD8+ T cell population of T and AN TIL. Expression of checkpoint proteins PD1+ and CD39+, a marker of tumour reactivity, was identified on CD8+ TRM and CD8+ non TRM in TIL. The differentiation status of TRM within memory lineages demonstrated that TRM express a higher proportion of effector memory cells (CCR7-CD45RA) compared to those in blood. TRM cells also showed lower expression of the transcription factors EOMES, TBET and TCF compared to CD8+ cells within PBMC. Moreover, tumour CD8+ TRM populations had reduced ability to produce IL-2 following stimulation compared to adjacent normal TRM populations. Conclusion TRM make up a large proportion of CD8+ T cell within the tumour microenvironment (TME) of OAC. TRM generation is associated with the downregulation of T-Box transcription factors EOMES and TBET and expression of an effector memory phenotype with reduction of the stem-like transcription factor, TCF. High levels of CD39+ indicate a strong level of activation but the increased PD1 expression and reduced capability to produce IL2 indicate functional impairment suppressed within the TME. An opportunity may exist to reverse this with novel immunotherapeutic agents.

547. DISEASE SPECIFIC MORTALITY OF EARLY-STAGE ESOPHAGEAL NEOPLASIA IN BARRETT’S ESOPHAGUS TREATED WITH ENDOSCOPIC THERAPY

Abstract Background High-grade dysplasia (HGD) and intramucosal (T1a) esophageal adenocarcinoma (EAC) are indication for endoscopic eradication therapy (EET) with endoscopic resection (ER) and/or radiofrequency ablation (RFA). The goal of the endoscopic therapy is to prevent progression to advanced EAC and cure the underlying Barrett’s esophagus (BE) when present. Previous studies have showed high rate of success of EET in short term, however little is known about long term results of EET. We aimed to evaluate long-term outcome of EET with particular reference to disease specific mortality and progression to major surgery. Methods We conducted a single center retrospective study including patients who underwent EET for HGD and T1a EAC January 2005-December 2022. We excluded patients with gastric disease and evidence of T1b EAC at baseline ER. Primary outcomes assessed disease specific mortality (DSM) of esophageal cancer in this longitudinal cohort, which is defined as death within 12 months of progression to advanced cancer. Secondary outcomes evaluate endoscopic recurrence, progression to esophagectomy, post-procedure complications and 5-year overall survival. Results 299 patients were included in this study. 252 were treated with a combination of ER and RFA, whereas 47 only received primary RFA. Median number of EET sessions in both patient groups is 4(p=0.48). Patients with HGD were more likely to be detected on surveillance compared to those with T1a EAC (p=0.004). We demonstrate a DSM rate of 2.7%(n=8), with a 5-year overall survival of 74.6%(n=223). 5.7%(n=17) of patients sustained severe post-procedure complications (Clavien-Dindo&amp;gt;3). Endoscopic recurrence occurred in 18.7%(n=56) and 6.7%(n=20) developed disease progression that required surgical intervention. Baseline T1a was associated with higher rates of esophagectomy compared to HGD (p=0.0135). Conclusion EET is a safe and effective treatment for patients with early-stage esophageal neoplasia. In comparison to esophago-gastric surgery, EET offers excellent results with a significantly lower morbidity and mortality risk. However, it is resource intensive as patients often require multiple treatments with long-term, routine surveillance. Patients also require strict compliance to maximize therapeutic outcomes and prevent recurrence or progression. Further studies are required to improve risk-stratification in this cohort and determine the optimal surveillance strategy post EET.

567. EPIDEMIOLOGY OF BARRETT'S ESOPHAGUS IN ICELAND; INCIDENCE AND PROGRESSION TO NEOPLASM FROM A LARGE POPULATION-BASED STUDY

Abstract Background Barrett’s esophagus (BE) is the known precursor to esophageal adenocarcinoma (EAC). Previous studies have demonstrated a wide variety of progression rate whereas no nationwide data regarding the progression has been reported. The aims of the study were to evaluate the prevalence of BE and neoplasms in BE, and progression to those in a large population based and nationwide. Methods This was a retrospective study and patients from 7 institutions who were identified through a database of centralized pathology laboratory in an expert center. Demographics and relevant clinicopathological features were obtained from medical records of patients with a pathologically confirmed BE by presence of intestinal metaplasia between 1990 and 2022. Results A total of 811 patients were identified with BE, 551 men (67.9%), male to female ratio 2.1:1, and with a median age at diagnosis of 63 years (IQR 53-73). At the time of BE diagnosis, the prevalence of neoplasm was 13.9%, (56 LGD, 14 HGD, and 43 EAC). Out of 56 LGD, 16.1% progressed to HGD (n=6) or EAC (n=3). Among 698 BE patients without neoplasm at the time of BE diagnosis, the cumulative incidence of neoplasm was 6.9% (29 LGD, 7 HGD, and 12 EAC) with median observation-period of 7 years (IQR: 4-9). In total, 161 cases of neoplasm (65.8% in long-segment, 34.2% in short-segment BE) were diagnosed in this BE cohort; 67.2% at time of BE diagnosis; and 32.8% without dysplasia at diagnosis progressed to neoplasm during follow-up. Conclusion In this large population-based study of patients with BE, around 14% were diagnosed as harboring neoplasm concomitantly with BE, which accounted for approximately 67% of all neoplasms. During the median follow-up of 7 years, about 7% of BE showed dysplastic or malignant progression.

605. MITOCHONDRIAL METABOLISM IN UPPER GI ADENOCARCINOMA DETERMINES PATIENT SURVIVAL

Abstract Background Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for meeting the vast majority of cellular energy demands. Ever since the description of aerobic glycolysis by Warburg in 1927, it has been recognised that cancers have dysfunctional metabolism. Clinically, this Warburg effect is the basis of FDG-PET imaging, increased FDG uptake represents increased tumour glycolysis and translates to worse upper GI adenocarcinoma outcomes. The cause of OXPHOS disruption in upper GI adenocarcinoma is poorly understood, its impact on potential therapeutic strategies is yet to be explored. This is particularly pertinent in upper GI adenocarcinomas as despite recent improvements, 5-year survival remains alarmingly low. Method Oesophageal and gastric adenocarcinoma samples collected in the UK from 175 patients (89 oesophageal adenocarcinoma, 86 gastric adenocarcinoma, 131 male, 44 female, median age=65, IQR=12) who underwent radical resection with curative intent between 1995 and 2009. Quantitative immunofluorescence were used to determine expressions of OXPHOS Complex I and IV at a single cell level. Patients were divided into high vs. low expressors of Complex I and IV using K-means clustering to allow for subsequent subgroup analysis. Result Survival analysis demonstrated significantly worse outcomes for low expressors as compared to high expressors of Complex I at 5 years post-op (5-year survival probability low expressors=0.440, 95%CI=0.358-0.539, high expressors=0.629, 95%CI=0.515-0.769, P-value=0.048). Similarly comparison of Complex IV expression also demonstrates disadvantageous survival for Complex IV low expressors (5-year survival probability low expressors=0.432, 95%CI=0.346-0.538, high expressors=0.603, 95%CI=0.499-0.729, P-value=0.042). The low expressors of Complex I and IV encompassed significantly more Stage III disease as per AJCC 7th edition. The low vs. high expression Complex I and IV groups are similarly matched for baseline patient characteristics. Conclusion These results demonstrate reduced capacity to carry out OXPHOS in oesophageal and gastric adenocarcinoma is characteristic of more advanced cancer and results in worse patient outcomes. Further work is needed to determine if OXPHOS status impacts on neo-adjuvant therapy response and how to exploit OXPHOS deficiency therapeutically in oesophageal and gastric adenocarcinomas. The limitations of this preliminary work include small sample size and tissue derived from historic patients over a long period of time, during which treatment and outcomes for upper GI cancer have significantly changed.

763. TEXTBOOK ONCOLOGIC OUTCOME FOLLOWING TRIMODALITY THERAPY FOR ESOPHAGEAL ADENOCARCINOMA

Abstract Background Although the concept of textbook oncologic outcome (TOO) as an emerging quality benchmark has been validated in the general surgical oncology literature, data regarding the frequency of achieving TOO and its impact on subsequent survival outcomes in patients with esophageal adenocarcinoma (EAC) who undergo trimodality therapy remain undefined. Methods Patients with adenocarcinoma of the esophagus/gastroesophageal junction (GEJ, Siewert I/II) who underwent neoadjuvant chemoradiation and esophagectomy at a single institution (2000-2022) were retrospectively identified. Patients undergoing salvage resection were excluded. TOO was defined according to a published consensus definition: length of stay (LOS) ≤14 days, R0, ≥20 resected lymph nodes, no anastomotic leak (any grade), no Clavien-Dindo (CD) grade ≥3 complications, no hospital or intensive care unit (ICU) readmission, and no reoperation. Multivariable logistic regression was used to identify clinicopathologic features associated with TOO. Multivariable Cox regression and landmark analyses were used to quantify the association between TOO and OS. Results Of 1138 patients, TOO was observed in 38.2% (435): 90.9% (1034) R0, 60.9% (693) ≥20 lymph nodes, 89.9% (1023) no anastomotic leak, and 98.4% (1120) no in-hospital mortality. Features independently associated with reduced odds of TOO included: cervical anastomosis (OR 0.50, P=0.001), preoperative delay ≥90 days after neoadjuvant therapy (OR 0.62, P=0.004), induction chemotherapy (OR 0.74, P=0.039), coronary artery disease (OR 0.67, P=0.043). Among patients who underwent esophagectomy &amp;lt;90 days after neoadjuvant therapy and survived ≥30 days, TOO was independently associated with improved OS (HR 0.80, P=0.018). TOO remained associated with improved OS using 90-day (P=0.014) and 120-day (P=0.037) landmarks. Conclusions Although TOO is difficult to achieve following trimodality therapy for EAC, it is strongly associated with improved long-term overall survival. As such, further efforts are warranted to explore the utility of TOO as a quality benchmark in EAC patients and to validate its impact across varying practice settings.