Progression To Esophageal Adenocarcinoma Research Articles

Regulation of pro-apoptotic and anti-apoptotic factors in obesity-related esophageal adenocarcinoma

BackgroundObesity is a risk factor for esophageal adenocarcinoma (EAC). It was reported that obesity -associated inflammation correlates with insulin resistance and increased risk of EAC. The objective of the study is to investigate the role of obesity associated inflammatory mediators in the development of EAC.MethodsWe included 23 obese and nonobese patients with EAC or with or without Barrett’s esophagus (BE) after IRB approval. We collected 23 normal, 10 BE, and 19 EAC tissue samples from endoscopy or esophagectomy. The samples were analyzed for the expression levels of pro-apoptotic and anti-apoptotic factors, PKC-δ, cIAP2, FLIP, IGF-1, Akt, NF-kB and Ki67 by immunofluorescence and RT-PCR. We compared the expression levels between normal, BE, and EAC tissue using Students’ t-test between two groups.ResultsOur results showed decreased gene and protein expression of pro-apoptotic factors (bad, bak and bax) and increased expression of anti-apoptotic factors (bcl-2, Bcl-xL) in BE and EAC compared to normal tissues. There was increased gene and protein expression of PKC-δ, cIAP2, FLIP, NF-kB, IGF-1, Akt, and Ki67 in BE and EAC samples compared to normal esophagus. Further, an increased folds changes in mRNA expression of proapoptotic factors, antiapoptotic factors, PKC-δ, IGF-1, Akt, and Ki-67 was associated with obesity.ConclusionPatients with EAC had increased expression of cIAP2 and FLIP, and PKC-δ which is associated with inhibition of apoptosis and possible progression of esophageal adenocarcinoma.

Abstract 6184: A novel approach to Barrett's esophagus risk stratification: Whole-slide image analysis for aneuploidy detection

Abstract Background Barrett’s esophagus (BE) is the only known precursor of esophageal adenocarcinoma (EAC) and there is a need for biomarkers in BE for risk stratification for cancer progression. Aneuploidy has been suggested as a factor in the development, initiation or progression of EAC in patients with BE, and it has been found predictive for EAC progression (Hadjinicolaou, et al. 2020, Sikkema, et al. 2009). However, current assays for detecting aneuploidy require valuable tissue, hence validation studies are limited. We hypothesise that routine histology images can be used to detect ploidy status. We aim to develop a sensitive, accurate, and easy-to-use deep learning tool for this purpose. Methods We used a weakly supervised deep learning-based approach called clustering-constrained-attention multiple-instance learning (CLAM) (Lu et al., 2021) to detect aneuploidy on hematoxylin and eosin-stained whole slide images of endoscopical biopsies from BE, for which the ploidy status had been determined by flow cytometry. To benchmark the model’s performance, we also trained a traditional fully supervised algorithm, ResNet50 (He et al., 2016), with the same dataset. The models were trained on 388 slides (51 aneuploid) from the Seattle Barrett’s Esophagus Annotated Resource (BEAR) and then applied to an independent test cohort of BEAR patients, consisting of 279 slides (36 aneuploid). Results The multi-attention-branch CLAM model achieved AUC of 0.82 and 76.4% balanced accuracy for aneuploidy on the internal test subset (10% of the cohort). On the independent test dataset, the model achieved AUC of 0.85 and 79.3% balanced accuracy, while performance of the fully supervised model was AUC of 0.65 and 33.1 balanced accuracy. In a challenging subset for image-based diagnosis, including 253 samples (29 aneuploid) without dysplasia or atypical mitosis (a major feature of aneuploidy samples) the model achieved a 67.6% accuracy and an AUC of 0.83. Both the flow results (p=2.84e-7) and the model's predictions (p=2.18e-3) revealed a correlation between progression to EAC and ploidy status. Conclusion We developed a deep learning model for predicting aneuploidy in BE biopsies. The weakly-supervised approach can perform much better compared to the traditional fully-supervised model. Aneuploidy is correlated with cancer progression and stands as a promising candidate for the prediction of cancer progression in BE patients. Although atypical mitosis is the primary histological indicator of aneuploidy, our model can still make aneuploid predictions in the absence of atypical mitosis, as it doesn't rely solely on a single parameter. Our model is efficient, capable of processing hundreds of samples resource-effectively, and thus an ideal adjunct to standard histologic evaluation. This classifier may facilitate molecular-based improvements in identifying individuals at high risk of progression. Citation Format: Caner Ercan, Xiaoxi Pan, Thomas G. Paulson, Matthew D. Stachler, Carlo C. Maley, Yinyin Yuan. A novel approach to Barrett's esophagus risk stratification: Whole-slide image analysis for aneuploidy detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6184.

Abstract 4268: Engineered hydrogel elucidates contributions of matrix mechanics to pathobiology of adenocarcinoma and identify matrix-activated therapeutic targets

Abstract Dysregulation of the extracellular matrix (ECM) properties disturbs tissue homeostasis and contributes to pathological conditions, such as cancer. Recent studies have demonstrated that ECM stiffening is associated with metaplasia-dysplasia-adenocarcinoma sequence in the esophagus. This underscores a compelling need to develop a physiologically-relevant 3D culture model to enable the study of the independent contribution of matrix stiffness to the initiation and progression of esophageal adenocarcinoma (EAC). Such a model will then serve as a platform for pre-clinical drug screening in the context of matrix properties. In this context, we have engineered a hyaluronic acid-based 3D hydrogel platform that supports the growth and differentiation of patient-derived Barrett’s esophagus (BE) organoids, a precursor to EAC, as well as patient-derived EAC organoids. The engineered biomaterial 3D platform allows control over matrix stiffness to better recapitulate the mechanically dynamic esophageal cancer microenvironment and help identify therapeutic targets in EAC.Our data demonstrates that BE and EAC organoid density, growth and proliferation can be controlled by matrix stiffness. RNA sequencing data show that increased matrix stiffness promotes changes in the transcriptional profiles of BE and EAC organoids, revealing enrichment of pathways associated with tumorigenesis and disease progression. Furthermore, we demonstrate through molecular and functional assays that increased matrix stiffness endows stem-like properties to the EAC organoids via Yap-Sox9 mechano-activation. Finally, targeted therapy studies in our in vitro and in vivo engineered environments revealed that Yap inhibition regressed the effects of increased matrix stiffness in EAC organoids. In summary, our data suggest that matrix mechanics have a significant role in activation of EAC-associated signaling pathways in patient-derived BE and EAC organoids. We also demonstrate that the engineered hydrogel serves as a platform to identify potential therapeutic targets to disrupt the contribution of pro-tumorigenic matrix mechanics in EAC. Together, these studies establish an engineered patient-derived organoid culture platform that can be used to elucidate underlying matrix-mediated mechanisms of the metaplasia-dysplasia-adenocarcinoma sequence and inform the development of novel therapeutics that target ECM stiffness in EAC. FUNDING SOURCES: NCI P01CA098101, U54 CA163004 and NIDDK K01 DK133620. Citation Format: Ricardo Cruz-Acuna, Secunda W. Kariuki, Kensuke Sugiura, Spyros Karaiskos, Eleanor M. Plaster, Claudia Loebel, Gizem Efe, Tatiana Karakasheva, Joel T. Gabre, Jianhua Hu, Jason A. Burdick, Anil K. Rustgi. Engineered hydrogel elucidates contributions of matrix mechanics to pathobiology of adenocarcinoma and identify matrix-activated therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4268.

Role of the Microbiome in the Diagnosis and Management of Gastroesophageal Cancers.

Stomach and esophageal cancers are among the highest mortality from cancers worldwide. Microbiota has an interplaying role within the human gastrointestinal (GI) tract. Dysbiosis occurs when a disruption of the balance between the microbiota and the host happens. With this narrative review, we discuss the main alterations in the microbiome of gastroesophageal cancer, revealing its potential role in the pathogenesis, early detection, and treatment. Helicobacter pylori plays a major role the development of a cascade of preneoplastic conditions ranging from atrophic gastritis to metaplasia and dysplasia, ultimately culminating in gastric cancer, while other pathogenic agents are Fusobacterium nucleatum, Bacteroides fragilis, Escherichia coli, and Lactobacillus. Campylobacter species (spp.)'s role in the progression of esophageal adenocarcinoma may parallel that of Helicobacter pylori in the context of gastric cancer, with other esophageal carcinogenic agents being Escherichia coli, Bacteroides fragilis, and Fusobacterium nucleatum. Moreover, gut microbiome could significantly alter the outcomes of chemotherapy and immunotherapy. The gut microbiome can be modulated through interventions such as antibiotics, probiotics, or prebiotics intake. Fecal microbiota transplantation has emerged as a therapeutic strategy as well. Nowadays, it is widely accepted that changes in the normal gut microbiome causing dysbiosis and immune dysregulation play a role gastroesophageal cancer. Different interventions, including probiotics and prebiotics intake are being developed to improve therapeutic outcomes and mitigate toxicities associated with anticancer treatment. Further studies are required in order to introduce the microbiome among the available tools of precision medicine in the field of anticancer treatment.

Prebiotic proanthocyanidins inhibit bile reflux-induced esophageal adenocarcinoma through reshaping the gut microbiome and esophageal metabolome.

The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammation-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barrett's esophagus, and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague-Dawley rats, with or without reflux induction, received water or C-PAC ad libitum (700 μg/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-κB/TP53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Streptococcus parasanguinis, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory, and immune-implicated proteins and genes, including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1b, Lbp, Lcn2, Myd88, Nfkb1, Tlr2, and Tlr4, aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe, promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation, and cellular damage.

On-chip Raman spectroscopy of live single cells for the staging of oesophageal adenocarcinoma progression.

The absence of early diagnosis contributes to oesophageal cancer being the sixth most common cause of global cancer-associated deaths, with a 5-year survival rate of < 20%. Barrett's oesophagus is the main pre-cancerous condition to adenocarcinoma development, characterised by the morphological transition of oesophageal squamous epithelium to metaplastic columnar epithelium. Early tracking and treatment of oesophageal adenocarcinoma could dramatically improve with diagnosis and monitoring of patients with Barrett's Oesophagus. Current diagnostic methods involve invasive techniques such as endoscopies and, with only a few identified biomarkers of disease progression, the detection of oesophageal adenocarcinoma is costly and challenging. In this work, single-cell Raman spectroscopy was combined with microfluidic techniques to characterise the development of oesophageal adenocarcinoma through the progression of healthy epithelial, Barrett's oesophagus and oesophageal adenocarcinoma cell lines. Principal component analysis and linear discriminant analysis were used to classify the different stages of cancer progression. with the ability to differentiate between healthy and cancerous cells with an accuracy of 97%. Whilst the approach could also separate the dysplastic stages from healthy or cancer with high accuracy-the intra-class separation was approximately 68%. Overall, these results highlight the potential for rapid and reliable diagnostic/prognostic screening of Barrett's Oesophagus patients.

The Expression Patterns and Implications of MALAT1, MANCR, PSMA3-AS1 and miR-101 in Esophageal Adenocarcinoma.

Esophageal adenocarcinoma (EAC) is a malignant tumor with poorly understood molecular mechanisms. This study endeavors to elucidate how the long non-coding RNAs (lncRNAs) MALAT1, MANCR and PSMA3-AS1, as well as the microRNA miR-101, exhibit specific expression patterns in the pathogenesis and prognosis of EAC. A total of 50 EAC tissue samples (tumors and lymph nodes) and a control group comprising 26 healthy individuals were recruited. The samples underwent quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The relative expression levels of MALAT1, MANCR, PSMA3-AS1, and miR-101 were ascertained and correlated with various clinicopathological parameters including TNM staging, tumor characteristics (size and grade of the tumor) lymphatic invasion, disease-free (DFS) and overall survival (OS) of EAC patients. Quantitative analyses revealed that MALAT1 and MANCR were significantly upregulated in EAC tumors and positive lymph nodes when compared to control tissues (p < 0.05). Such dysregulations correlated positively with advanced lymphatic metastases and a higher N stage. DFS in the subgroup of patients with negative lymph nodes was higher in the setting of low-MANCR-expression patients compared to patients with high MANCR expression (p = 0.02). Conversely, miR-101 displayed a significant downregulation in EAC tumors and positive lymph nodes (p < 0.05), and correlated negatively with advanced tumor stage, lymphatic invasion and the grade of the tumor (p = 0.006). Also, patients with low miR-101 expression showed a tendency towards inferior overall survival. PSMA3-AS1 did not demonstrate statistically significant alterations (p > 0.05). This study reveals MALAT1, MANCR, and miR-101 as putative molecular markers for prognostic evaluation in EAC and suggests their involvement in EAC progression.

Cranberry Proanthocyanidins Mitigate Reflux-Induced Transporter Dysregulation in an Esophageal Adenocarcinoma Model.

We recently reported that cranberry proanthocyanidins (C-PACs) inhibit esophageal adenocarcinoma (EAC) by 83% through reversing reflux-induced bacterial, inflammatory and immune-implicated proteins and genes as well as reducing esophageal bile acids, which drive EAC progression. This study investigated whether C-PACs' mitigation of bile reflux-induced transporter dysregulation mechanistically contributes to EAC prevention. RNA was isolated from water-, C-PAC- and reflux-exposed rat esophagi with and without C-PAC treatment. Differential gene expression was determined by means of RNA sequencing and RT-PCR, followed by protein assessments. The literature, coupled with the publicly available Gene Expression Omnibus dataset GSE26886, was used to assess transporter expression levels in normal and EAC patient biopsies for translational relevance. Significant changes in ATP-binding cassette (ABC) transporters implicated in therapeutic resistance in humans (i.e., Abcb1, Abcb4, Abcc1, Abcc3, Abcc4, Abcc6 and Abcc10) and the transport of drugs, xenobiotics, lipids, and bile were altered in the reflux model with C-PACs' mitigating changes. Additionally, C-PACs restored reflux-induced changes in solute carrier (SLC), aquaporin, proton and cation transporters (i.e., Slc2a1, Slc7a11, Slc9a1, Slco2a1 and Atp6v0c). This research supports the suggestion that transporters merit investigation not only for their roles in metabolism and therapeutic resistance, but as targets for cancer prevention and targeting preventive agents in combination with chemotherapeutics.

An Immune-Related Gene Signature for Predicting Survival and Immunotherapy Efficacy in Esophageal Adenocarcinoma.

BACKGROUND Immune checkpoint inhibitor (ICI) therapy has attracted wide attention in the treatment of malignant tumors. This study was designed to build a prognostic model based on immune-related genes for esophageal adenocarcinoma (EAC). MATERIAL AND METHODS The expression of immune-related differentially-expressed genes (IRDEGs) between EAC and normal samples from The Cancer Genome Atlas database was analyzed. Univariate and multivariate Cox regressions were used to identify the prognostic IRDEGs and construct an immune-related gene signature (IRGS) to predict the overall survival (OS) of EAC patients. Then, the molecular mechanisms and immune characteristics were comprehensively analyzed. RESULTS A total of 111 IRDEGs were obtained from the weighted gene co-expression network analysis. Univariate Cox regression analysis showed that 12 IRDEGs (P<0.05 for all) were linked with OS in the EAC patients. Four genes were used to construct the IRGS based on the multivariate Cox regression analysis. Patients in the high-risk group showed worse OS than those in the low-risk group (P<0.001). A high-risk score was related to DNA replication relevant pathways, an increase in mutation rate, and an increase in activated mast cell infiltration. Patients with high-risk scores had lower tumor immune dysfunction and exclusion scores (P<0.001). CONCLUSIONS IRDEGs may be involved in the progression of EAC. The high-risk group is more suitable for immunotherapy, which may provide a reference value for the treatment of clinical EAC patients. Therefore, it is possible to identify the patients who are better suited for ICI therapy.

Abstract 1232: Gastroesophageal reflux disease promotes E-cadherin cleavage and activates EMT via APE1-redox function in esophageal adenocarcinoma

Abstract The incidence of esophageal adenocarcinoma (EAC) has increased rapidly over the past four decades. Chronic gastroesophageal reflux disease (GERD), where acidic bile salts (ABS) abnormally refluxate into the esophagus, is the leading risk factor for the development of a metaplastic condition known as Barrett’s esophagus (BE) and its progression to EAC. We used 3D organotypic culture, mouse and human tissue samples to assess the role of ABS in Epithelial-to-Mesenchymal Transition (EMT) in EAC. Analysis of public databases revealed significant enrichment of EMT signaling in EAC progression. RNA-seq analysis of EAC cells showed activation of EMT pathway in response to ABS exposure. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of Vimentin, activation of ß-catenin and EMT-transcription factors (EMT-TFs), cell morphological changes, and enhancement of cell migration and invasion capabilities. Mechanistically, we discovered that ABS induced E-cadherin cleavage via MMP14 proteolytic cascade. APE1 silencing, or APE1-redox-specific inhibitor (E3330), abrogated the ABS-induced EMT process and signaling by downregulating MMP14. Furthermore, APE1 and MMP14 co-expression levels were inversely correlated with E-cadherin expression in gastroesophageal junctions of human EAC tissues, and the L2-IL1ß transgenic mouse model of BE/EAC. EAC patients with high APE1 and EMT signatures had worse relapse free survival than those with low signature. In a summary, this study demonstrates the role of ABS in promoting EMT via the redox-sensitive signaling axis of APE1/MMP14/E-cadherin/ß-catenin. Pharmacological inhibition of APE1-redox function could be a potential therapeutic approach to effectively reduce the risk of Barrett’s carcinogenesis. Citation Format: Heng Lu, Longlong Cao, Farah Ballout, Abbes Belkhiri, Dunfa Peng, Lei Chen, Mohammed Soutto, Oliver McDonald, Alexander Zaika, Jianwen Que, Wael El-Rifai. Gastroesophageal reflux disease promotes E-cadherin cleavage and activates EMT via APE1-redox function in esophageal adenocarcinoma [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 1232.

Abstract 3964: APE1 protects SMAD3 against ROC1 ubiquitin mediated degradation in esophageal adenocarcinoma cells

Abstract Background: The incidence of esophageal adenocarcinoma (EAC) has increased more than six-fold over the past three decades and continues to rise in the Western world. The 5-year survival rate for EAC patients is less than 20% which underscores the need to better understand the underlying mechanisms to identify new therapeutic approaches. This study aimed at investigating the potential role of APE1 in regulating SMAD3 and promoting EAC progression. Methods and Results: Western blot data showed that APE1 and SMAD3 were highly expressed in EAC cell lines. APE1 silencing reduced SMAD3 nuclear expression and downregulated its downstream targets SERPINE1 and c-myc. These results were confirmed by immunofluorescence staining showing loss of nuclear accumulation of SMAD3 after APE1 knockdown. Mechanistically, immunoprecipitation and proximity ligation assays revealed a direct binding between APE1 and SMAD3 in the nucleus. Further investigation showed that APE1 binds to the C-terminal MH2 domain of SMAD3, and this binding protects SMAD3 from ubiquitin mediated proteasomal degradation by blocking its interaction with the RING finger protein, ROC1. Interestingly, APE1-redox-specific inhibition (APX2009) downregulated SMAD3 expression and the APE1 redox-deficient mutant (C65A) disrupted APE1-SMAD3 binding indicating that this regulation depends on APE1 redox activity. Conclusion: Our findings establish a role of APE1 in regulating SMAD3 in EAC. These findings provide a potential therapeutic approach for the treatment of EAC by the pharmacological inhibition of APE1. Citation Format: Farah Ballout, Heng Lu, Dunfa Peng, Wael El-Rifai. APE1 protects SMAD3 against ROC1 ubiquitin mediated degradation in esophageal adenocarcinoma cells. [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 3964.

Antibiotic Exposure is Associated With a Risk of Esophageal Adenocarcinoma

Antibiotic exposure leads to changes in the gut microbiota. Our objective was to evaluate the association between antibiotic exposure and esophageal adenocarcinoma (EAC) risk. We performed a nested case-control study using data from the Veterans Health Administration from 2004 through 2020. The case group consisted of patients who received an incident diagnosis of EAC. For each case, up to 20 matched controls were selected using incidence density sampling. Our primary exposure of interest was any oral or intravenous antibiotic use. Our secondary exposures included cumulative number of days of exposure and classification of antibiotics by various subgroups. Conditional logistic regression was used to estimate the crude and adjusted odds ratios (aORs) for the risk of EAC associated with antibiotic exposure. The case-control analysis included 8226 EAC cases and 140,670 matched controls. Exposure to any antibiotic was associated with an aOR for EAC of 1.74 (95% confidence interval [CI], 1.65-1.83) vs no antibiotic exposure. Compared with no antibiotic exposure, the aOR for EAC was 1.63 (95% CI, 1.52-1.74; P < .001) for cumulative exposure to any antibiotic for 1 to 15 days; 1.77 (95% CI, 1.65-1.89; P < 0 .001) for 16 to 47 days; and 1.87 (95% CI, 1.75-2.01; P < .001) for ≥48 days, respectively (P for trend < .001). Exposure to any antibiotic is associated with an increased risk of EAC, and this risk increases as the cumulative days of exposure increase. This novel finding is hypothesis-generating for potential mechanisms that may play a role in the development or progression of EAC.

2.5D mass spectrometry imaging of N-glycans in esophageal adenocarcinoma and precursor lesions

Glycosylation plays an important role in the progression of esophageal adenocarcinoma (EAC). Being able to image these glycosylation changes directly in endoscopic resection specimens could provide useful insights into the molecular mechanisms of the disease progression and potential markers for EAC staging. For this purpose, both 3D and 2.5D matrix-assisted laser/desorption ionization (MALDI) mass spectrometry imaging (MSI) have been employed in this study to investigate glycosidase-cleaved N-glycans in a total of 24 formalin-fixed paraffin-embedded esophageal local excision specimens spanning all stages of disease progression, namely from non-dysplastic Barrett’s esophagus to metastatic EAC. 3D-MSI was first used to estimate the number of sections needed to sufficiently cover the molecular heterogeneity of each stage of progression. This analysis showed that a total of four sections out of 20 were sufficient. This subset of four sections was measured for all remaining specimens and is called 2.5D-MSI. Subsequent analyses of the 2.5D-MSI datasets revealed significant elevations of five high-mannose N-glycans (Man3, Man4, Man6, Man7, and Man8) in EAC and three complex (Hex6HexNAc5, Hex6HexNAc5NeuAc1, Hex7HexNAc6) N-glycans in metastatic EAC as compared to previous stages of the disease. The augmented levels of these glycans in EAC could be explained by publically available gene expression data of enzymes involved in glycan synthesis and processing. As the role of glycosylation is gaining more interest in MSI and cancer research, our results show the added value of combining localized N-glycan levels, as provided by MSI, with gene expression to gain a deeper understanding of the mechanisms behind N-glycan changes. This gives evidence at multiple levels that specific N-glycosylation plays an important role during progression of dysplasia to EAC and could play a role in patient surveillance.

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.

Abstract 4008: Transcriptomic signatures in esophageal adenocarcinoma define distinct subtypes with therapeutic relevance

Abstract Background &amp; Aims: Esophageal cancer is the fifth most common solid cancer globally, and esophageal adenocarcinoma (EAC) is the predominant histological subtype in the western world. Patients often present at an advanced stage and overall-5-year survival rates are less than 15%. In many patients, the response to neoadjuvant therapy is encouraging at first, but most will develop metastatic disease several years later. Recently, we observed plasticity along the epithelial-to-mesenchymal (EMT) axis in EAC tumor cells upon therapeutic pressure. The last decade has seen the discovery of static molecular subtypes in cancers and a poor-prognosis mesenchymal subtype has been reported in multiple gastrointestinal cancers, but not in EAC. Here, we set out to identify transcriptomic subtypes and the frequency of their occurrence during EAC progression, and exposure to therapy in a clinical setting. Methods: A cohort comprising of 174 esophageal cancer cases (n=100 pre-treatment biopsies, n=46 neoadjuvantly-treated resection specimens, and n=28 metastatic biopsies) with 12 esophageal cancer cell lines (8 primary and 4 ATCC cell lines) was RNA-sequenced. To circumvent non-tumor signals confounding the analysis, we leveraged non-negative matrix factorization (NMF) as a virtual microdissection tool. Patients were subtyped by consensus clustering the top 50 exemplar genes per tumor-cell intrinsic signature. Next, the subtypes were validated in two independent cohorts and clinical correlates were analyzed. In addition, we perturbed subtype-specific regulators to functionally assess the existence of the subtypes. Results: Here, we identified 7 biological signatures involved in primary and metastatic EAC. Two of these were unambiguously tumor-intrinsic signals. All seven signatures associated with tumor cellularity scores estimated by an experienced pathologist confirming the reliability of NMF as a virtual microdissection tool. Upon clustering two EAC subtypes emerged: Intestinal-like (IL) and Mesenchymal-like (ML). ML-subtyped patients were observed with increased frequency after neoadjuvant treatment, and following metastatic dissemination. This association was validated in an independent cohort. Lastly, we identified HMGA2 as a key transcription factor for the ML-subtype. Genetic perturbation of HMGA2 induced an IL-associated phenotype. Conclusion: Our study demonstrate for the first time from clinical transcriptomics data that EAC cells can exist in distinct cell states. In addition, the abundance of either cell states, that constitute to tumor subtype, alter when exposed to chemoradiation or following metastatic spread. These subtypes offer a valuable method to design more informed personalized treatment approaches for EAC patients; for existing and future subtype-directed treatments. Citation Format: Mark P.G. Dings, Amber P. Zalm, Marjolein F. Lansbergen, César Oyarce, Sybren L. Meijer, Cynthia Waasdorp, Jan Paul Medema, Hanneke van Laarhoven, Maarten F. Bijlsma. Transcriptomic signatures in esophageal adenocarcinoma define distinct subtypes with therapeutic relevance [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 4008.

Abstract 3838: Engineered hydrogel elucidates contributions of matrix mechanics to esophageal adenocarcinoma and identify matrix-activated therapeutic targets

Abstract INTRODUCTION: Changes in the tumor microenvironment arbitrated by a stiffened ECM are associated with tumor aggression and enable increased propensity towards metastasis. For instance, in vitro (2D) studies have implicated ECM properties in EAC progression. However, these studies are limited by the lack of 3D intercellular interactions, underscoring the need for physiologically relevant 3D culture models, such as patient-derived organoids (PDOs), that better recapitulate human cancer and its microenvironment to elucidate underlying mechanisms. Engineered hydrogels are an evolving and important component of 3D organoid culture systems, especially to introduce tunable physicochemical matrix signals that have been investigated in tumor progression and metastasis. Furthermore, PDOs have become an attractive pre-clinical in vitro model to study cancer biology and evaluate response to therapeutics. METHODS: We have engineered a visible light-mediated hydrogel platform that supports the development of patient derived Barrett's esophagus (BE) organoids, a precursor to esophageal adenocarcinoma (EAC), as well as EAC organoids. This synthetic biomaterial platform allows control over hydrogel stiffness to better recapitulate the mechanically dynamic esophageal cancer microenvironment, and may help identify therapeutic targets in EAC organoids. RESULTS: Our preliminary data have demonstrated that BE and EAC organoid density, size and proliferation can be controlled by synthetic ECM biomechanical properties. Furthermore, our data show that increased matrix stiffness promotes changes in the transcriptional profiles of EAC organoids, as observed via Principal Component Analysis, and gene set enrichment analysis of upregulated genes reveals enrichment of anti-apoptotic pathways. This suggests that the synthetic ECM facilitates activation of mechanotransduction pathways in EAC organoids and that matrix mechanics have a significant role in activation of canonical anti-apoptotic signaling pathways. Ongoing studies involve identifying matrix stiffness-activated therapeutic targets via small molecule inhibition of upregulated genes that are considered prospective biomarkers in GI cancer. SUMMARY: Our work is significant because it establishes a biomaterial platform that overcomes the limitations of current 3D organoid culture methods to elucidate the role of the tumor microenvironment in EAC tumorigenesis and to identify disease-relevant therapeutic targets. This work will also provide an opportunity to further establish the engineered biomaterial as a platform to potentially elucidate the mechanisms of, and therapy targets for, other human adenocarcinomas in the context of changes in matrix biomechanics.FUNDING: NCI P01-CA098101, U54 CA-163004 and Charles H. Revson Senior Fellowships in Biomedical Science. Citation Format: Ricardo Cruz-Acuña, Secunda W. Kariuki, Claudia Loebel, Tatiana Karakasheva, Joel T. Gabre, Jason A. Burdick, Anil K. Rustgi. Engineered hydrogel elucidates contributions of matrix mechanics to esophageal adenocarcinoma and identify matrix-activated therapeutic targets [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 3838.

Abstract 5741: Characterizing isoform switching events in esophageal adenocarcinoma

Abstract Esophageal adenocarcinoma (EAC), the major subtype of esophageal cancer in the US, is characterized by increasing incidence rates and high mortality due to ineffective screening, late-stage diagnosis, and poor treatment efficacy. The only known precursor to EAC is Barrett’s Esophagus (BE), but the precise mechanisms and molecular events leading to disease progression are still being unraveled. In recent years, analysis of TCGA data revealed that isoform switching events with predicted functional consequences are common in many cancers, but the characterization of switching events in esophageal adenocarcinoma (EAC) is lacking. Next-generation sequencing was utilized to detect levels of RNA transcripts and specific isoforms in 66 treatment naïve esophageal tissues from 41 patients. Tissues ranged from premalignant Barrett’s esophagus (BE), BE with low or high-grade dysplasia (LGD or HGD) to EAC. Samples were stratified by histopathology, p53 mutation status, and significant isoform switching events with predicted functional consequences, followed by enrichment analysis. Barrett’s with high-grade dysplasia increased risk for EAC progression. Comparing BE with LGD and BE with HGD, isoform switching events were identified in 75 genes including KRAS, APC, RNF-128 and WRAP53. Stratification based on p53 status increased the number of significant isoform switches to 135 suggesting switching events impact cellular functions based on tissue histopathology, as well as p53 status. Analysis of isoforms agnostic to mutant p53, exclusive to mutant p53, and shared between the two groups revealed unique signatures including demethylation, lipid and retinoic acid metabolism, and glucuronidation, respectively. About 25% of isoform switching events were identified without significant alteration of gene-level expression. Importantly, two p53-interacting isoforms, RNF128 and WRAP53, were significantly linked to patient survival. Analysis of global isoform switching consequences and alternative splicing events also revealed significant changes in the fraction of coding transcripts, complete open-reading-frames, signal peptides, alternative 5’ donor sites, and alternative transcription start sites. Thus, analysis of isoform switching events may provide new insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC treatment or prevention. Citation Format: Yun Zhang, Katherine M. Weh, Connor L. Howard, Jean-Jack Riethoven, Jennifer L. Clarke, Kiran H. Lagisetty, Jules Lin, Rishindra M. Reddy, Andrew C. Chang, David G. Beer, Laura A. Kresty. Characterizing isoform switching events in esophageal adenocarcinoma [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 5741.

Deciphering the Immune Complexity in Esophageal Adenocarcinoma and Pre-Cancerous Lesions With Sequential Multiplex Immunohistochemistry and Sparse Subspace Clustering Approach.

Esophageal adenocarcinoma (EAC) develops from a chronic inflammatory environment across four stages: intestinal metaplasia, known as Barrett’s esophagus, low- and high-grade dysplasia, and adenocarcinoma. Although the genomic characteristics of this progression have been well defined via large-scale DNA sequencing, the dynamics of various immune cell subsets and their spatial interactions in their tumor microenvironment remain unclear. Here, we applied a sequential multiplex immunohistochemistry (mIHC) platform with computational image analysis pipelines that allow for the detection of 10 biomarkers in one formalin-fixed paraffin-embedded (FFPE) tissue section. Using this platform and quantitative image analytics, we studied changes in the immune landscape during disease progression based on 40 normal and diseased areas from endoscopic mucosal resection specimens of chemotherapy treatment- naïve patients, including normal esophagus, metaplasia, low- and high-grade dysplasia, and adenocarcinoma. The results revealed a steady increase of FOXP3+ T regulatory cells and a CD163+ myelomonocytic cell subset. In parallel to the manual gating strategy applied for cell phenotyping, we also adopted a sparse subspace clustering (SSC) algorithm allowing the automated cell phenotyping of mIHC-based single-cell data. The algorithm successfully identified comparable cell types, along with significantly enriched FOXP3 T regulatory cells and CD163+ myelomonocytic cells as found in manual gating. In addition, SCC identified a new CSF1R+CD1C+ myeloid lineage, which not only was previously unknown in this disease but also increases with advancing disease stages. This study revealed immune dynamics in EAC progression and highlighted the potential application of a new multiplex imaging platform, combined with computational image analysis on routine clinical FFPE sections, to investigate complex immune populations in tumor ecosystems.

Barrett's Metaplasia Progression towards Esophageal Adenocarcinoma: An Attempt to Select a Panel of Molecular Sensors and to Reflect Clinical Alterations by Experimental Models.

The molecular processes that predispose the development of Barrett’s esophagus (BE) towards esophageal adenocarcinoma (EAC) induced by gastrointestinal reflux disease (GERD) are still under investigation. In this study, based on a scientific literature screening and an analysis of clinical datasets, we selected a panel of 20 genes covering BE- and EAC-specific molecular markers (FZD5, IFNGR1, IL1A, IL1B, IL1R1, IL1RN, KRT4, KRT8, KRT15, KRT18, NFKBIL1, PTGS1, PTGS2, SOCS3, SOX4, SOX9, SOX15, TIMP1, TMEM2, TNFRSF10B). Furthermore, we aimed to reflect these alterations within an experimental and translational in vitro model of BE to EAC progression. We performed a comparison between expression profiles in GSE clinical databases with an in vitro model of GERD involving a BE cell line (BAR-T) and EAC cell lines (OE33 and OE19). Molecular responses of cells treated with acidified bile mixture (BM) at concentration of 100 and 250 μM for 30 min per day were evaluated. We also determined a basal mRNA expression within untreated, wild type cell lines on subsequent stages of BE and EAC development. We observed that an appropriately optimized in vitro model based on the combination of BAR-T, OE33 and OE19 cell lines reflects in 65% and more the clinical molecular alterations observed during BE and EAC development. We also confirmed previous observations that exposure to BM (GERD in vitro) activated carcinogenesis in non-dysplastic cells, inducing molecular alternations in the advanced stages of BE. We conclude that it is possible to induce, to a high extent, the molecular profile observed clinically within appropriately and carefully optimized experimental models, triggering EAC development. This experimental scheme and molecular marker panel might be implemented in further research, e.g., aiming to develop and evaluate novel compounds and prodrugs targeting GERD as well as BE and EAC prevention and treatment.

Simvastatin Inhibits Histologic Changes Associated with Gastroduodenal Reflux in a Murine Model.

Observational studies demonstrate a protective effect of statins on the development and progression of esophageal adenocarcinoma. The role of statins in the prevention of reflux-induced esophageal changes remains unknown. Using a mixed gastroduodenal reflux mouse model, we hypothesized that oral administration of simvastatin would attenuate reflux-induced mucosal changes of the distal esophagus. Human Barrett's (CPB) and esophageal adenocarcinoma (FLO1 and OE19) cells were treated with simvastatin. Cell proliferation and apoptosis were evaluated using the MTS proliferation and annexin V apoptosis assays, respectively. A reflux mouse model was generated by performing a side-to-side anastomosis between the gastroesophageal junction and first portion of the duodenum (duodeno-gastroesophageal anastomosis, DGEA). DGEA mice were fed a standard or simvastatin-containing diet following surgery. Mice were euthanized 6weeks post-operatively. Simvastatin significantly decreased proliferation and increased apoptosis in all cell lines. Compared to control animals, mice undergoing DGEA who were fed a standard diet demonstrated a fourfold increase in mucosal thickness and significant increase in proliferating cells (p < 0.0001). DGEA mice fed a simvastatin-containing diet had an attenuated response to reflux, with a significant reduction in mucosal hyperplasia and proliferation (p < 0.0001). DGEA mice fed a simvastatin-containing diet demonstrated significant upregulation of procaspase-3 (p = 0.009) and cleaved caspase-3 (p = 0.034) in the distal esophagus. We demonstrate for the first time a reduction in reflux-induced histologic changes of the distal esophagus following oral administration of simvastatin in vivo. These findings identify simvastatin as a potential preventative agent to inhibit the development and progression of reflux-induced esophageal injury.