Esophageal Adenocarcinoma Tissues Research Articles

Abstract 4776: NRF2 is overexpressed in esophageal adenocarcinoma and its targeting sensitizes tumor cells to cisplatin through induction of ferroptosis and apoptosis

Abstract Background: Esophageal adenocarcinoma (EAC) is the predominant type of esophageal cancer in the United States, with the 5-year survival rate is below 20%. EAC develops through Barrett’s esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease (GERD), where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator, involving in many cancer hallmarks. Methods and Results: Using western blotting and immunohistochemistry technologies, we detected high NRF2 protein levels in EAC cell lines and primary EAC tissues, as compared with normal esophagus and non-neoplastic Barrett’s esophagus samples. The knockdown of NRF2 using NRF2 specific siRNAs significantly increased oxidative stress in response to cellular stress stimuli by bile salts or cisplatin. This was associated with an increase in DNA damage and inhibition of EAC cell growth. Brusatol, a NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with CDDP induced significant lipid peroxidation and ferroptosis as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. Moreover, the combination of Brusatol and CDDP significantly inhibited EAC tumor xenografts growth in vivo. We confirmed the in vitro data showing ferroptosis as an important mechanism in the xenografted tumors treated with Brusatol or Brusatol and CDDP combination using IHC staining. Conclusion: Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC. Citation Format: Farah Ballout, Heng Lu, Zheng Chen, Tianling Hu, Lei Chen, Wael El-Rifai, Dunfa Peng. NRF2 is overexpressed in esophageal adenocarcinoma and its targeting sensitizes tumor cells to cisplatin through induction of ferroptosis and apoptosis. [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 4776.

Construction of the Interaction Network of Hub Genes in the Progression of Barrett's Esophagus to Esophageal Adenocarcinoma.

Esophageal adenocarcinoma (EAC) is one of the histologic types of esophageal cancer with a poor prognosis. The majority of EAC originate from Barrett's esophagus (BE). There are few studies focusing on the dynamic progression of BE to EAC. R software was used to analyze differentially expressed genes (DEGs) based on RNA-seq data of 94 normal esophageal squamous epithelial (NE) tissues, 113 BE tissues and 147 EAC tissues. The overlapping genes of DEGs between BE and EAC were analyzed by Venn diagram tool. The hub genes were selected by Cytoscape software based on the protein-protein interaction network of the overlapping genes using STRING database. The functional analysis of hub genes was performed by R software and the protein expression was identified by immunohistochemistry. In the present study, we found a large degree of genetic similarity between BE and EAC, and further identified seven hub genes (including COL1A1, TGFBI, MMP1, COL4A1, NID2, MMP12, CXCL1) which were all progressively upregulated in the progression of NE-BE-EAC. We have preliminarily uncovered the probable molecular mechanisms of these hub genes in disease development and constructed the ceRNA regulatory network of hub genes. More importantly, we explored the possibility of hub genes as biomarkers in the disease progression of NE-BE-EAC. For example, TGFBI can be used as biomarkers to predict the prognosis of EAC patients. COL1A1, NID2 and COL4A1 can be used as biomarkers to predict the response to immune checkpoint blockade (ICB) therapy. We also constructed a disease progression risk model for NE-BE-EAC based on CXCL1, MMP1 and TGFBI. Finally, the results of drug sensitivity analysis based on hub genes showed that drugs such as PI3K inhibitor TGX221, bleomycin, PKC inhibitor Midostaurin, Bcr-Abl inhibitor Dasatinib, HSP90 inhibitor 17-AAG, and Docetaxel may be potential candidates to inhibit the progression of BE to EAC. This study is based on a large number of clinical samples with high credibility, which is useful for revealing the probable carcinogenic mechanism of BE to EAC and developing new clinical treatment strategies.

Mir-421 and mir-550a-1 are potential prognostic markers in esophageal adenocarcinoma

ObjectiveTo identify the prognostic indicators of esophageal adenocarcinoma (EAC) for future EAC diagnosis and treatment.MethodsThe EAC dataset from The Cancer Genome Atlas was screened for differentially expressed microRNAs (miRNAs) and mRNAs associated with EAC. Weighted gene coexpression network analysis was performed to cluster miRNAs or mRNA with similar expression patterns to identify the miRNAs or mRNA that are highly associated with EAC. Prognostic miRNAs for overall survival (OS) were identified using Cox proportional-hazards regression analysis and least absolute shrinkage and selection operator based on survival duration and status. Two types of miRNAs were selected to develop a prognostic signature model for EAC using multiple Cox regression analysis. Furthermore, the signature was validated using internal validation sets 1 and 2. The receiver operating characteristic curve and concordance index were used to evaluate the accuracy of the signature and validation sets. The expression of miR-421, miR-550a-3p, and miR-550a-5p was assessed using quantitative polymerase chain reaction (qPCR). The proliferation, invasion, and migration of EAC cells were assessed using CCK8 and transwell assays. The OS of target mRNAs was assessed using Kaplan–Meier analysis. Functional enrichment analysis of the target mRNAs was performed using Metascape.ResultsThe prognostic signature and validation sets comprising mir-421 and mir-550a-1 had favorable predictive power in OS. Compared with the patients with EAC in the high-expression group, those assigned to the low-expression group displayed increased OS according to survival analysis. Differential and qPCR analysis showed that miR-421, miR-550a-3p, and miR-550a-5p were highly expressed in the EAC tissues and cell lines. Moreover, the downregulation of miR-421 and miR-550a-3p with inhibitor markedly suppressed the proliferation, invasion, and migration in OE33 cells compared with the negative control. A total of 20 target mRNAs of three miRNAs were predicted, among which seven target mRNAs—ASAP3, BCL2L2, LMF1, PPM1L, PTPN21, SLC18A2, and NR3C2—had prognostic value; PRKACB, PDCD4, RPS6KA5, and BCL2L2 were enriched in the miRNA cancer pathway.ConclusionPrognostic indicators of EAC may be useful in future EAC diagnosis and treatment.

Comparative transcriptome characterization of esophageal squamous cell carcinoma and adenocarcinoma

BackgroundEsophageal cancers are primarily categorized as esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). While various (epi) genomic alterations associated with tumor development in ESCC and EAC have been documented, a comprehensive comparison of the transcriptomes in these two cancer subtypes remains lacking. MethodsWe collected 551 gene expression profiles from publicly available sources, including normal, ESCC, and EAC tissues or cell lines. Subsequently, we conducted a systematic analysis to compare the transcriptomes of these samples at various levels, including gene expression, promoter activity, alternative splicing (AS), alternative polyadenylation (APA), and gene fusion. ResultsSeven distinct cluster gene expression patterns were identified among the differentially expressed genes in normal, ESCC, and EAC tissues. These patterns were enriched in the PI3K-Akt signaling pathway and the activation of extracellular matrix organization and exhibited repression of epidermal development. Notably, we observed additional genes or unique expression levels enriched in these shared pathways and biological processes related to tumor development and immune activation. In addition to the differentially expressed genes, there was an enrichment of lncRNA co-expression networks and downregulation of promoter activity associated with the repression of epidermal development in both ESCC and EAC. This indicates a common feature between these two cancer subtypes. Furthermore, differential AS and APA patterns in ESCC and EAC appear to partially affect the expression of host genes associated with bacterial or viral infections in these subtypes. No gene fusions were observed between ESCC and EAC, thus highlighting the distinct molecular mechanisms underlying these two cancer subtypes. ConclusionsWe conducted a comprehensive comparison of ESCC and EAC transcriptomes and uncovered shared and distinct transcriptomic signatures at multiple levels. These findings suggest that ESCC and EAC may exhibit common and unique mechanisms involved in tumorigenesis.

OGC O09 The clinical utilisation of tissue imaging Mass Spectrometry as a diagnostic model for Oesophageal Adenocarcinoma

Abstract Background The prevalence of Oesophageal adenocarcinoma (OA) in the United Kingdom has steadily risen since the 1970's. The majority of patients present with advanced disease. Early diagnosis utilising point of care testing may enable an increase in the number of patients managed on a potentially curative pathway. Tissue Imaging Mass Spectrometry by Desorption Electrospray (DESI-MS) is one such tool to differentiate the abundance of lipids in OA. This work presents the novel use of Mass Spectrometry (MS) imaging to identify variations in lipid abundance between OA and normal oesophagus (NO). Methods Tissue was sampled from specimens dissected immediately post-resection from the tumour and at 5-centimetre intervals in the proximal NO. Forty-one unique patient OA and NO tissue samples were analysed in triplicate by DESI-MS tissue imaging to differentiate Glycerophospholipids. A total of 246 DESI-MS images were correlated with paired formalin fixed Haematoxylin and Eosin stained tissue slides, the current gold standard cancer diagnostic test. Principle component analysis utilising a supervised model classified tissue types with a significance of p=0.01. Receiver operator curves (ROC) were used to determine the diagnostic sensitivity and specificity of DESI-MS for the identification of adenocarcinoma. Results Tissue analysis confirmed unsaturated Phosphatidylglycerols and Phosphatidic acids were significantly more abundant in OA compared to NO (p<0.001). Tissue analysis by a Partial Least Square (PLS) supervised linear regression model classified cancer and non-cancer tissue with a confusion matrix classification of 93.3% and 70.8% for OA and NO (Figure 1a-c). A diagnostic sensitivity of 85% and specificity of 88% was determined by ROC curve. The abundance of Phosphatidylglycerol and Phosphatidylserine decreased proportionally in analysed tissue sections at 5cm and 10cm from the primary tumour, as did the degree of lipid unsaturation. Conclusions This work presents a novel tissue imaging MS model to differentiate lipid abundance in OA. An abundance ratio of 1.17:1 (OA:NO) of unsaturated Phosphatidylglycerol was diagnostic of cancer by DESI-MS with a high sensitivity and specificity. This MS tissue analysis model has the potential to enable accurate point of care diagnostics for OA. In addition, we suggest oesophageal cancer induced perturbation of Glycerophospholipid metabolism may be a potential target for future chemotherapeutics.

395. EXPRESSION OF MALAT1, MANCR, PSMA3-AS1 AND MIR-101 IN ESOPHAGEAL ADENOCARCINOMA PATIENTS: PRELIMINARY RESULTS OF A CASE–CONTROL STUDY

Abstract Esophageal adenocarcinoma is a leading cause of cancer-related mortality. Ongoing research aims to elucidate the molecular mechanisms underlying the tumorigenesis and progression of esophageal adenocarcinoma and to identify novel therapeutic targets. The abnormal expression of lncRNAs and miRNAs may play an important role in various biological processes in cancer, including immune response, differentiation, angiogenesis, apoptosis, cell proliferation and autophagy. A total of 50 patients who were operated for esophageal adenocarcinoma in our Department from January 2015 to December 2018, were enrolled in the study. Twenty-six healthy individuals that underwent upper gastrointestinal endoscopy were recruited as controls in the study. Total RNA was extracted from esophageal adenocarcinoma tumors, lymph nodes and healthy esophageal tissues using the reverse transcription and qRT-PCR. The expression of different lncRNAs (MALAT1, MANCR, PSMA3-AS1) and miRNA (miR-101) were determined by the 2-ΔΔct method and presented using Fold-Regulation. MALAT1 expression was upregulated in esophageal adenocarcinoma tumors and positive lymph nodes compared to non-cancerous tissues (1.9 and 2.0 times higher than controls respectively). Higher expression of MANCR was also found in esophageal adenocarcinoma tissues and positive lymph nodes compared to controls (2.6 and 2.1 times respectively). MiR-101 was downregulated in esophageal adenocarcinoma tumors and positive lymph nodes compared to controls (16.0 and 7.8 times lower respectively). No significant difference was observed in PSMA3-AS1 expression between esophageal adenocarcinoma tumors or positive lymph nodes and relevant healthy tissues. MALAT1 and MANCR are upregulated in esophageal adenocarcinoma patients while miR-101 is downregulated. Further research in lncRNAs, miRNAs and their interactions with oncogenic pathways may offer new options for the diagnosis, prognosis and therapy of esophageal adenocarcinoma patients.

IL-33 Participates in the Development of Esophageal Adenocarcinoma.

Background: The progression from chronic gastroesophageal reflux disease (GERD) to Barrett esophagus (BE) and esophageal adenocarcinoma (EAC) is an inflammatory-driven neoplastic change. Interleukin-33 (IL-33) has identified as a crucial factor in several inflammatory disorders and malignancies. Methods: The high-density tissue microarray of the human EAC was analyzed with IL-33 immunohistochemistry staining (IHC). By anastomosing the jejunum with the esophagus, the rat model of EAC with mixed gastroduodenal reflux was established. The expression of IL-33 was determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blot (WB), IHC and enzyme-linked immunosorbent assay (ELISA). Esophageal adenocarcinoma cells (OE19 and OE33) and human esophageal epithelial cells (HEECs) were used. Results: In the cytoplasm of human EAC tissue, IL-33 expression was substantially greater than in adjacent normal tissue. In rat model, the expression of IL-33 in the EAC group was considerably greater than in the control group, and this expression increased with the upgrade of pathological stage. In in vitro experiment, the mRNA and protein levels of IL-33 were considerably greater in OE19 and OE33 than in HEECs. The stimulation of IL-33 enhanced the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of OE19 and OE33, but soluble ST2 (sST2) inhibited these effects. IL-33 stimulated the release of IL-6 by OE19 and OE33 cells. Conclusion: This study demonstrated the overexpression of IL-33 in the transition from GERD to EAC and that IL-33 promoted carcinogenesis in EAC cells through ST2. IL-33 might be a possible preventive target for EAC.

Abstract 2805: Mass spectrometry detects lower frequency of routine markers and consistent overexpression of novel disease drivers in Barrett's-related esophageal cancer

Abstract Introduction: The incidence of esophageal adenocarcinoma (EAC) has increased more than any other cancer in our lifetime. Barrett’s esophagus (BE) is the only known precursor to EAC. At present, HER-2 and anti-PD-L1 therapies are FDA-approved for EAC, and off-label use of EGFR-directed therapy has been reported. Our study aims to use liquid chromatography mass spectrometry (LC/MS) to measure the expression of oncoproteins that are currently approved-drug targets in EAC and explore new promising targets for drug development and diagnostic applications for EAC and BE. Methods: EAC patients who underwent esophagectomy without chemo-radiation were identified. BE patients with subsequent progression to EAC and those without progression for >10 years were identified. Normal esophageal mucosa tissues with no history of progression were analyzed for baseline expression. Tissue sections were microdissected to isolate pure EAC, BE and normal mucosal cells from stroma. These biopsies were solubilized for LC/MS-based quantification of 80+ clinically relevant tumor markers including novel markers which are believed to play a role in the disease progression from BE to EAC. The expression trends of these markers were scrutinized by immunofluorescence (IF) in a subset of specimens/biomarkers to confirm mass spec results. Results: A total of 159 tissues were analyzed via LC-MS. In 55 EAC tissues - HER2, PD-L1 and EGFR were overexpressed in 16.3%, 0%, and 0% of patients, respectively. New markers DAD1 and S100P were noted to have high (7,634.0 and 27,222.6 attomoles/ug) and consistent (98.1% and 94.5%) overexpression in EAC samples. Expression of HER2, S100P and DAD1 were significantly higher in EAC compared to 23 normal esophageal mucosa specimens. There was no statistical difference in the expression of PD-L1 and EGFR between EAC and healthy mucosal samples. 45 BE biopsies progressed to EAC within a mean of 380 days, while the remaining 36 BE specimens did not progress over a 10-year period. 8 novel biomarkers were expressed at a higher level in BE tissue that progressed to cancer when compared to esophageal tissue that remained stable over 10 years. The expression trends observed via LC/MS were confirmed via IF in DAD1, S100P and two other novel markers. Discussion: LC-MS revealed low prevalence of target proteins in currently approved EAC therapies and high levels of newly discovered disease drivers that should be explored as possible therapy targets or diagnostic markers. The differential overexpression of the novel markers in progressive and non-progressive BE tissues, as well as the consistent overexpression of these markers in EAC tumors, demonstrates the potential for early detection of esophageal carcinogenesis and/or reveals potential therapeutic programs for an indication that lacks highly efficacious targeted therapy options. Citation Format: Christopher Hartley, Ajay Bansal, Catherine Hagen, Olivia Driscoll, Seyun Oh, Joe Abdo, Sumeet K. Mittal. Mass spectrometry detects lower frequency of routine markers and consistent overexpression of novel disease drivers in Barrett's-related esophageal cancer [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 2805.

Oestrogen Receptor Isoforms May Represent a Therapeutic Target in Oesophageal Adenocarcinoma.

Simple SummaryOesophageal adenocarcinoma is a lethal malignancy with limited treatment options. Recent studies have identified oestrogen receptors (ERs) in this cancer, which could represent a new target for therapy. In this study, we used laboratory models of oesophageal adenocarcinoma to look for the presence of variant forms of ERs. We also assessed the response to treatment with a drug that acts through these ERs. We found that variant forms of ERs do exist in this malignancy and that some of the variants appear to be important in order for the cells to respond to treatment. This could be due to interactions between different ERs, or between ERs and other molecules that are known to be important in cancer growth. Our findings are encouraging in that drugs that act through ERs might be useful for patients with oesophageal adenocarcinoma in the future.Oesophageal adenocarcinoma is a rapidly increasing problem in which treatment options are limited. Previous studies have shown that oesophageal adenocarcinoma cells and tissues express oestrogen receptors (ERs) and show growth suppression and apoptosis in response to ER modulator agents such as tamoxifen. ERs are known to be expressed in a number of isoforms that act together to regulate cell growth and cell death. In this study, we used western blotting to profile the expression of ERα and ERβ isoforms, and expression of the oncologically related molecules p53, HER2, and EGFR, in a panel of oesophageal adenocarcinoma cell lines. The cytotoxicity of tamoxifen in the cell lines was determined with Annexin V-FITC flow cytometry, and correlations between cytotoxicity and receptor expression were assessed using Spearman’s rank-order correlation. Oesophageal adenocarcinoma cell lines showed varying cytotoxicity in response to tamoxifen. The ER species ERα90, ERα50, and ERα46, as well as p53, were positively associated with a cytotoxic response. Conversely, ERα74, ERα70, and ERβ54 were associated with a lack of cytotoxic response. The ER species detected in oesophageal adenocarcinoma cells may work together to confer sensitivity to ER modulators in this disease, which could open up a new avenue for therapy in selected patients.

Novel epigenetic network biomarkers for early detection of esophageal cancer

BackgroundEarly detection of esophageal cancer is critical to improve survival. Whilst studies have identified biomarkers, their interpretation and validity is often confounded by cell-type heterogeneity.ResultsHere we applied systems-epigenomic and cell-type deconvolution algorithms to a discovery set encompassing RNA-Seq and DNA methylation data from esophageal adenocarcinoma (EAC) patients and matched normal-adjacent tissue, in order to identify robust biomarkers, free from the confounding effect posed by cell-type heterogeneity. We identify 12 gene-modules that are epigenetically deregulated in EAC, and are able to validate all 12 modules in 4 independent EAC cohorts. We demonstrate that the epigenetic deregulation is present in the epithelial compartment of EAC-tissue. Using single-cell RNA-Seq data we show that one of these modules, a proto-cadherin module centered around CTNND2, is inactivated in Barrett’s Esophagus, a precursor lesion to EAC. By measuring DNA methylation in saliva from EAC cases and controls, we identify a chemokine module centered around CCL20, whose methylation patterns in saliva correlate with EAC status.ConclusionsGiven our observations that a CCL20 chemokine network is overactivated in EAC tissue and saliva from EAC patients, and that in independent studies CCL20 has been found to be overactivated in EAC tissue infected with the bacterium F. nucleatum, a bacterium that normally inhabits the oral cavity, our results highlight the possibility of using DNAm measurements in saliva as a proxy for changes occurring in the esophageal epithelium. Both the CTNND2/CCL20 modules represent novel promising network biomarkers for EAC that merit further investigation.

Telomere shortening accelerates tumor initiation in the L2-IL1B mouse model of Barrett esophagus and emerges as a possible biomarker.

Barrett’s esophagus (BE) is a precursor of the esophageal adenocarcinoma (EAC). BE- development and its progression to cancer is associated with gastroesophageal reflux disease. However, there is currently no molecular risk prediction model that accurately identifies patients at high risk for EAC. Here, we investigated the impact of shortened telomeres in a mouse model for Barrett esophagus (L2-IL1B). The L2-IL1B mouse model is characterized by IL-1β-mediated inflammation, which leads to a Barrett-like metaplasia in the transition zone between the squamous forestomach and glandular cardia/stomach. Telomere shortening was achieved by mTERC knockout. In the second generation (G2) of mTERC knockout L2-IL1B.mTERC−/− G2 mice exhibited telomere dysfunction with significantly shorter telomeres as measured by qFISH compared to L2-IL1B mice, correlating with stronger DNA damage in the form of phosphorylation of H2AX (γH2AX). Macroscopically, tumor area along the squamocolumnar junction (SCJ) was increased in L2-IL1B.mTERC−/− G2 mice, along with increased histopathological dysplasia. In vitro studies indicated increased organoid formation capacity in BE tissue from L2-IL1B.mTERC−/− G2 mice. In addition, pilot studies of human BE-, dysplasia- and EAC tissue samples confirmed that BE epithelial cells with or without dysplasia (LGD) had shorter telomeres compared to gastric cardia tissue. Of note, differentiated goblet cells retained longer telomeres than columnar lined BE epithelium. In conclusion, our studies suggest that shortened telomeres are functionally important for tumor development in a mouse model of BE and are associated with proliferating columnar epithelium in human BE. We propose that shortened telomeres should be evaluated further as a possible biomarker of cancer risk in BE patients.

ADP-ribosylation factor 6 expression increase in oesophageal adenocarcinoma suggests a potential biomarker role for it.

ADP-ribosylation factor 6 small GTPase plays an important role in cell migration, invasion and angiogenesis, which are the hallmarks of cancer. Although alterations in ARF6 expression and activity have been linked to metastatic cancer in one or two tissues, the expression of ARF6 in cancers over a wide range of tissues has not been studied so far. In this report, we analysed the expression of ARF6 mRNA in cancers and corresponding healthy controls from 17 different tissues by real-time qualitative polymerase chain reaction (RT-qPCR). We further evaluated ARF6 protein expression in oesophageal adenocarcinoma (EAC) tissue microarray cores by immunohistochemistry. The ARF6 gene expression levels are highly variable between healthy and cancer tissues. Our findings suggest that the ARF6 gene expression is up-regulated highest in oesophageal cancer. In EAC TMAs, ARF6 protein expression increase correlated with EAC progression. This is the first study to investigate ARF6 gene expression in a wide array of cancer tissues and demonstrate that ARF6 expression, at both mRNA and protein levels, is significantly upregulated in higher grades of EAC, which may be useful in targeting ARF6 for cancer diagnostic and therapeutic purposes.

Quantitative proteomic profiling of esophageal adenocarcinoma tumors to assess prevalence of approved targets and elucidate novel biomarkers.

343 Background: Esophageal adenocarcinoma (EAC) continues to have extremely poor prognosis despite advances in surgical and oncological regimens. Targeted and immune therapy hold promise to improve outcomes in solid tumors including EAC. At present HER-2 and anti-PD-L1 therapies are approved by the FDA for EAC. Additionally, off-label use of EGFR directed therapy has been reported. The aim of this study is to measure expression of 80+ oncoproteins in EAC using multiplexed mass spectrometry to elucidate expression of currently approved-drug targets and explore promising new targets for drug development. Methods: After Institutional Review Board approval (Mayo Clinic and KUMC), EAC patients whose tumors were resected via esophagectomy before chemotherapy and/or radiation were identified. Normal esophageal mucosa samples with no history of EAC were also retrieved for analysis. Tissue sections were microdissected to exclude stroma from adenocarcinoma cells. Isolated EAC cells were solubilized for mass spectrometry-based quantitation of more than 80 clinically relevant tumor markers. Results: 55 EAC tissues were analyzed. HER2, PD-L1 and EGFR were overexpressed only in 16.3%, 0%, and 0% of patients, respectively. New oncoproteins #2 and #6 were noted to have high (7,634.0 and 27,222.6 attomoles/ug) and consistent (98.1% and 94.5%) overexpression in EAC samples. Expression of HER2, oncoproteins #2 and #6 were significantly higher in EAC compared to normal esophageal mucosa. There was no statistical difference in the expression of PD-L1 and EGFR between EAC and normal samples. Conclusions: Mass spectrometry revealed a very low prevalence of target oncoproteins in EAC for currently used drugs. Our study potentially reveals alternate markers which are near universally present at high levels and should be further explored for targeted treatment.[Table: see text]

Novel evidence of obesity paradox in esophageal adenocarcinoma: perspective on genes that uncouple adiposity from dismal outcomes.

Background: Obesity is a strong risk factor for esophageal adenocarcinoma (EAC). Nevertheless, not all the patients with EAC are obesity, and a substantial proportion of obesity patients don't suffer from poor prognoses. The mechanisms behind the “obesity paradox” that uncouple obesity from dismal outcomes in EAC are unclear. This study aimed to explore the “obesity-guarding” genes (OGG) profiles and their prognostic values in patients with EAC.Methods: Gene expression data and clinical information of patients with EAC were downloaded from The Cancer Genome Atlas (TCGA) database. Enrichment analysis was used to explore the OGG functions and pathways. Cox regression analysis and nomogram model were performed to investigate the OGG prognostic values for overall survival (OS). In addition, relations between OGG and immune cells were assessed by the “CIBERSORT” algorithm and the Tumor IMmune Estimation Resource (TIMER) tool. Finally, the results were experimentally validated in real-world study.Results: A total of 69 OGG were retrieved, and 17 significantly differentially expressed genes (SDEG) were identified between normal and EAC tissues. Enrichment analysis showed the OGG were enriched in the mitochondrion-related and various receptor pathways. Univariate Cox regression results showed that the MCM6, ATXN2 and CSK were significantly associated with OS (P=0.036, 0.039, 0.046, respectively). Multivariate Cox regression analysis showed MCM6 and CSK were independent prognostic genes for OS (P=0.025, 0.041, respectively). Nomogram demonstrated that the OGG had good predictive abilities for the 1-, 2-, and 3-year OS. Immunity analysis demonstrated that OGG were significantly associated with immune cells (P <0.05). In addition, clinical correlation analysis revealed that the OGG had significant relations with clinical parameters (P <0.05). The experiment results confirmed that the SDEG were significantly different between normal and EAC tissues (P <0.05).Conclusions: We identified the OGG expression profiles that may uncouple obesity from poor survival in patients with EAC. They have prognostic values in predicting patients' OS, and may be exploited for prognostic biomarkers.

P-OGC39 The smell of oesophageal adenocarcinoma: opportunities for tests and treatments

Abstract Background Exhaled breath analysis is a promising approach for oesophageal adenocarcinoma (OAC) early detection. The biomarkers of interest are low molecular weight metabolites including volatile aldehydes. In this translational study we investigated whether these metabolites originated from a tumoral source, and how this might impact the diagnosis and treatment of OAC patients. Methods The investigative strategy was directed by an unbiased informatics screen of metabolic reprogramming in OAC, and validated using complimentary gene expression assays (n = 638, including controls). Mass spectrometric methods were used to quantify corresponding metabolites and putative source compounds at a tissue level (n = 158), and also in exhaled breath for correlative purposes. Targeted in vitro experiments were performed to demonstrate the cause and effect of the proposed model of metabolic reprogramming in OAC. Results The unbiased screen and subsequent validation found that reduced aldehyde detoxification is an OAC hallmark. In vitro and in vivo this was associated with endogenous aldehyde accumulation. OAC tissue was generally enriched for volatile aldehydes, including the genotoxins formaldehyde, acetaldehyde, 4-hydroxy-2-nonenal and 2-butenal, and the exhaled biomarker decanal (all P &amp;lt; 0.0001). Decanal concentrations correlated with exhaled concentrations. Considering potential aldehyde sources, the OAC phospholipidome was characterised by desaturated and longer lipid acyls, and these spontaneously generated biomarker aldehyde species at ambient conditions. Enriched genotoxic aldehydes were detectable in base-pairing positions in DNA; this genotoxicity was therapeutically targetable with aldehyde scavengers in vitro. Conclusions These data support a model for enriched exhaled aldehydes based on increased production from an altered lipid phenotype, and reduced detoxification. Some aldehydes are non-reactive and thus support non-invasive detection. Others react with DNA and increase local genotoxicity; this process is druggable. These findings have implications for OAC early diagnosis and chemoprevention.

850 OBJECTIVE IDENTIFICATION OF OESOPHAGEAL ADENOCARCINOMA USING MASS SPECTROMETRY IMAGING

Abstract Outcomes for oesophageal adenocarcinoma continue to be poor, as patients are often diagnosed too late for curative treatment. New tools to assist early and rapid diagnosis are needed. Tissue phospholipids quantified by mass spectrometry imaging (MSI) can objectively diagnose non-oesophageal cancers from control tissue in minutes. However, whether MSI can objectively identify oesophageal adenocarcinoma is unknown and represents a significant challenge, as this microenvironment is complex with phenotypically similar tissue-types. Methods Desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) and bespoke chemometrics were used to assess the phospholipidomes of oesophageal adenocarcinoma and relevant control tissues, in surgical (discovery) and endoscopic (validation) cohorts. Structural annotations verified by tandem MS fragmentation patterns were performed to understand the characteristics of the key differential phospholipids. Bioinformatics, quantitative polymerase chain reaction, and immunohistochemistry were performed to describe the reprogramming of phospholipogenesis in oesophageal adenocarcinoma. Finally, genetic silencing experiments were performed to understand whether this genetic reprogramming impacts the oesophageal adenocarcinoma phospholipid signature. Results Multivariable models based on tissue phospholipids from 117 patients were highly discriminant for oesophageal adenocarcinoma both in discovery (area-under-curve = 0.97) and validation cohorts (AUC = 1). Cancer samples were enriched for polyunsaturated phosphatidylglycerols with longer acyl chains, with stepwise enrichment in pre-malignant tissues. Expression of fatty acid and glycerophospholipid synthesis genes was significantly upregulated, and characteristics of fatty acid acyls matched phospholipid acyls. Mechanistically, reducing ACLY expression in oesophageal adenocarcinoma cells shortened GPL chains, therefore linking de novo lipogenesis to the oesophageal adenocarcinoma phospholipidome. Conclusion DESI-MSI (i) objectively identified invasive oesophageal adenocarcinoma from a number of pre-malignant tissues (ii) described the characteristics of cancer phospholipids, which have unknown function (iii) unveiled a new mechanism of phospholipidomic reprogramming. These results call for accelerated diagnosis studies using DESI-MSI in the upper gastrointestinal endoscopy suite, as well as functional studies to determine how polyunsaturated phosphatidylglycerols are generated, and how they contribute to oesophageal carcinogenesis.

C-Myb facilitates immune escape of esophageal adenocarcinoma cells through the miR-145-5p/SPOP/PD-L1 axis.

Esophageal adenocarcinoma (EAC), a subtype of esophageal carcinoma, is a severe health problem associated with high death rate and poor prognosis. Immunotherapy has proven to be effective in many solid tumors, including EAC, but immune escape blocks its effectiveness. Thus, we explored the mechanisms and functional role of c‐Myb in immune escape of EAC cells. Clinical EAC tissues were collected for determining the expression of c‐Myb, speckled POZ protein (SPOP), and miR‐145‐5p. Functional assays were then performed to detect the interactions between c‐Myb and SPOP as well as between SPOP and miR‐145‐5p. EAC cell invasion and migration were assessed. Next, T cells were sorted and cocultured with EAC cells with different treatments followed by detection of T‐cell viability. In addition, a mouse model of EAC was constructed for relevant in vivo assays. c‐Myb and miR‐145‐5p were highly expressed and SPOP had low expressions in EAC. c‐Myb activated the transcription of miR‐145‐5p, which in turn targeted SPOP. Further, SPOP accelerated the ubiquitination of PD‐L1 to enhance its expression. Overexpression of PD‐L1 suppressed T‐cell functions and promoted proliferative and migrative abilities of EAC cells to induce immune escape. The above findings were also confirmed in the ECA mouse model in vivo. Our findings uncovered that c‐Myb can promote the immune escape of EAC cells by favoring the transcription of miR‐145‐5p and inhibiting SPOP‐dependent ubiquitination and degradation of PD‐L1, thus, presenting new target for EAC adjunct therapy.

Oestrogen receptors: A potential therapeutic target in oesophageal adenocarcinoma?

Oesophageal cancer is the seventh most common cancer in the world and adenocarcinoma is the dominant subtype in Western industrialised nations. The global 5-year relative survival rate for oesophageal adenocarcinoma is 12%. Chemotherapy is a standard treatment offered to patients with both resectable and unresectable disease. However, there are only a few established chemotherapeutic drug options and progress in this area is limited. Recent efforts have focused on targeted molecular therapies. Epidemiological evidence points towards hormonal influences on disease development, particularly sex hormones. Several research studies have demonstrated oestrogen receptor (ER) expression in oesophageal adenocarcinoma tissue, making them a possible option for targeting with ER modulating agents. ERs are also present in laboratory models of the disease and experiments in ER-positive cell lines suggest that ER modulator therapy may be effective. A deeper understanding of the roles of ERα and ERβ in this disease would be valuable for future translation into clinical practice. In this review, we discuss the association between oestrogens and the development of oesophageal adenocarcinoma and the potential to modulate ER signalling networks for therapeutic benefit.

DNA methylome and transcriptome analysis established a model of four differentially methylated positions (DMPs) as a diagnostic marker in esophageal adenocarcinoma early detection.

BackgroundEsophageal carcinogenesis involves in alterations of DNA methylation and gene transcription. This study profiled genomic DNA methylome vs. gene expression using transcriptome data on esophageal adenocarcinoma (EAC) tissues from the online databases in order to identify methylation biomarkers in EAC early diagnosis.Materials and MethodsThe DNA methylome and transcriptome data were downloaded from the UCSC Xena, Gene Expression Omnibus (GEO), and The Cancer Genome Atlas (TCGA) databases and then bioinformatically analyzed for the differentially methylated positions (DMPs) vs. gene expression between EAC and normal tissues. The highly methylated DMPs vs. reduced gene expression in EAC were selected and then stratified with those of the corresponding normal blood samples and other common human cancers to construct an EAC-specific diagnostic model. The usefulness of this model was further verified in other three GEO datasets of EAC tissues.ResultA total of 841 DMPs were associated with expression of 320 genes, some of which were aberrantly methylated in EAC tissues. Further analysis showed that four (cg07589773, cg10474350, cg13011388 and cg15208375 mapped to gene IKZF1, HOXA7, EFS and TSHZ3, respectively) of these 841 DMPs could form and establish a diagnostic model after stratified them with the corresponding normal blood samples and other common human cancers. The data were further validated in other three GEO datasets on EAC tissues in early EAC diagnosis.ConclusionThis study revealed a diagnostic model of four genes methylation to diagnose EAC early. Further study will confirm the usefulness of this model in a prospective EAC cases.

A five-gene signature for predicting overall survival of esophagus adenocarcinoma.

Esophageal adenocarcinoma (EAC) is common and aggressive with increasing trend of incidence. Urgent need for an effective signature to assess EAC prognosis and facilitate tailored treatment is required.Differentially expressed mRNAs (DEMs) were identified by analyzing EAC tissues and adjacent normal samples from The Cancer Genome Atlas (TCGA). Then univariate regression analyses were performed to confirm prognostic DEMs. We used least absolute shrinkage and selection operator (LASSO) to build a prognostic mRNA signature whose performance was assessed by Kaplan–Meier curve, receiver operating characteristic (ROC). GSE72874 were used as an external test set. The performances of the signature were also validated in internal TCGA and external test sets. Gene set enrichment analysis (GSEA) and tumor immunity analysis were performed to decipher the biological mechanisms of the signature.A 5-mRNA signature consisted of SLC26A9, SINHCAF, MICB, KRT19, and MT1X was developed to predict prognosis of EAC. The 5-mRNA signature was promising as a biomarker for predicting 3-year survival rate of EAC in the internal test set, the entire TCGA set, and the external test set with areas under the curve (AUC) = 0.849, 0.924, and 0.747, respectively. Patients were divided into low- and high-risk groups based on risk scores of the signature. The high-risk group was mainly associated with cancer-related pathways and low levels of B cell infiltration.The 5-mRNA prognostic signature we identified can reliably predict prognosis and facilitate individualized treatment decisions for EAC patients.