Esophageal Samples Research Articles

Single-Cell Sequencing Reveals Heterogeneity and Interactions Between Epithelial Cells and Fibroblasts in Post-ESD Oesophageal Stricture.

Oesophageal stricture, especially circumferential lesions, is a common complication of endoscopic submucosal dissection (ESD). However, the exact mechanisms underlying its development remain unclear. Consequently, understanding tissue microenvironment changes is crucial for identifying therapeutic targets. To address this, single-cell RNA sequencing (scRNA-seq) was performed on oesophageal stricture samples and normal controls. Alterations in cellular composition were observed, particularly in epithelial, endothelial, fibroblast and immune cells. A notable increase was observed in the number of differentiating suprabasal cell_2 (DFSC_2), which displayed pro-keratinizing traits. Detailed investigations revealed augmentation in a subset of these cells, characterised by elevated FTH1 and ECM1 expression, indicating their role in epithelial remodelling. Furthermore, fibroblast heterogeneity was demonstrated, with significant activation of myofibroblasts within stricture tissues. MDK-NCL, CXCL5/6-CXCR2, and TGFA-EGFR ligand-receptor pairs were enhanced in stricture tissues, mediating epithelial-stromal interactions. This study dissected the transcriptional landscape of postoperative oesophageal stricture tissues, providing valuable insights into stricture mechanisms and potential preventive strategies.

Discovery of Methylated DNA Biomarkers for Potential Non-Endoscopic Detection of Barrett's Esophagus and Esophageal Adenocarcinoma.

We sought to develop a minimally-invasive, robust, accessible nonendoscopic strategy to diagnose Barrett's esophagus (BE), esophageal adenocarcinoma (EAC), and its immediate precursor lesion, high-grade dysplasia (HGD) based on methylated DNA biomarkers applied to a retrievable sponge-capsule device in a cohort representative of the BE population (i.e., mostly short-segment, non-dysplastic BE, NDBE). We identified 12 candidate methylation markers to distinguish normal vs. abnormal esophagus. These 12 markers were first assayed in 21-paired matched NDBE-normal esophageal tissues, then assessed in a case-control study of 234 esophageal samples collected using a sponge-capsule device. A classification algorithm was developed using Least Absolute Shrinkage and Selection Operator (LASSO) in a 199-patient training set and tested in an independent 35-patient test set. Twelve markers (A1BG, C9orf50, cg00720137, FLI1, GRAMD1B, HOXB13, IRF4, KCNQ3, NTNG1, SPX, TBC1D30, and USP44) were significantly hypermethylated (i.e., all P<0.05) in BE vs. matched normal esophageal biopsies. A discriminatory three-gene LASSO panel (USP44, TBCD1D30, NELL1), adjusted for age and sex, accurately distinguished HGD or EAC from normal control patients in both training (AUC=0.911, 95%CI=0.863-0.959) and test (AUC=0.969, 95%CI=0.911-1.00) sets. In normal vs. NDBE/LGD/HGD/EAC patients, this algorithm exhibited AUCs of 0.862 (95%CI=0.812-0.912) and 0.864 (95%CI=0.745-0.982) in training and test sets, respectively. In normal vs. NDBE patients, the algorithm yielded AUCs of 0.819 (95%CI=0.748-0.889) and 0.776 (95%CI=0.583-0.968) in training and test sets, respectively. This discriminatory biomarker panel algorithm exemplifies a practical nonendoscopic strategy to diagnose BE, HGD, and EAC using a minimally-invasive sponge-capsule device coupled with DNA methylation markers.

Higher T-bet and IFN-γ expression in advanced chagasic megaesophagus indicates Th1 response in the chronic phase.

Myenteric plexus injury is responsible for the morpho-functional alterations observed in chagasic megaesophagus (CME). The inflammatory response, characterized by elevated synthesis of IFN-γ, TNF-α, and IL-4, contributes to the persistence of parasitism and inflammation. This study assessed the mRNA expression of cytokines, transcription factors, and metalloproteases in subjects with CME. From 2011 to 2017, esophageal samples were collected from 54 subjects with CME (38 advanced and 16 nonadvanced) and eight subjects with idiopathic megaesophagus (IME). The quantitative mRNA expression of TNF-α, IFN-γ, IL-4, IL-10, IL-17, IL-22, IL-23, IL-27, T-bet, ROR-γT, GATA-3, MMP-1, MMP-2, and TIMP-3 genes was analyzed using SYBR Green systems. T-bet expression was significantly higher in the CME group compared to the IME group and the GATA-3 and ROR-γT expression in the CME group, corroborating the higher IFN-γ expression observed in subjects with advanced CME. The increased T-bet and IFN-γ expression in advanced CME reflects the maintenance of a Th1 response in situ and the morpho-functional changes seen in the organ.

Long-term outcome of oesophageal atresia in adolescence (TransEAsome): a national French cohort study protocol

IntroductionThe TransEAsome project, funded by the Agence Nationale de la Recherche, aims to evaluate the long-term outcomes of patients with oesophageal atresia (OA) between 13 and 14 years old and...

Infection survey, molecular, pathogenicity, and morphological characteristics of Sarcocystis species naturally infected water buffaloes (Bubalus bubalis) in Egypt

Abstract Background Sarcocystosis is a parasitic disease found worldwide, resulting from various Sarcocystis species. The current research was carried out in three significant economic areas in Egypt: Greater Cairo, the Nile Delta, and Upper Egypt. It aimed to investigate the occurrence of Sarcocystis spp. in locally bred water buffaloes Bubalus bubalis. Methods To achieve this objective, 317 buffalos were slaughtered in different slaughterhouses in various regions of Egypt. Samples of heart, skeletal muscle, esophagus, and tongue were assessed using macroscopic and microscopic tests. Examination methods included direct optical observation of tissues as well as digestion and examination of the sediment obtained from the tissues. Additionally, ultrastructural features were analyzed using scanning and transmission electron microscopy. Molecular characterization was conducted through PCR, followed by nucleotide sequencing and phylogenetic analysis. Results A total of 317 slaughtered buffaloes were examined for Sarcocystis during the period from September 2021 to October 2023. The prevalence of infection was recorded with 229 out of 317 (72.2%) infected with Sarcocystis spp. The results also showed that the prevalence of Sarcocystis species in females was higher than males. Based on the age of carcasses, adults (&gt; 2 years) had a higher infection rate compared to young ones (&lt; 2 years). Regarding seasonal variation, the highest prevalence of infection was recorded during the summer followed by spring, and then autumn, while winter had the lowest prevalence of infection. Additionally, the skeletal muscle was the most susceptible organ to sarcocystosis (87.3%) followed by the esophageal muscle (8.3%), the tongue (4.4%), and no infection in the heart muscle. The use of scanning and transmission electron microscopy allowed the identification of S. fusiformis and S. cruzi in buffaloes in Egypt. Furthermore, the Sarcocystis 18 S rRNA genes from skeletal tissue samples were cloned and sequenced under accession numbers OQ507387, OQ507388, and OQ507389 for S. fusiforms, and one OQ507391 for S. cruzi. Conclusion The findings revealed a notably high prevalence of Sarcocystis infection (72.2%) in buffaloes from Egypt, with skeletal muscle identified as the organ most susceptible to the parasite. Two Sarcocystis species were detected: S. fusiformis and S. cruzi.

Tissue source may affect the esophageal flora in patients with esophageal squamous cell carcinoma.

The aim of the present study was to provide a theoretical basis for the selection of standard sampling methods in the study of the esophageal microbiota in patients with esophageal squamous cell carcinoma (ESCC) by comparing the differences in bacterial communities between surgical and endoscopic esophageal mucosal tissues. A total of 72 patients with ESCC who were diagnosed at Taihe Hospital (Shiyan, China) between July 2018 and July 2019 were selected to participate in the present study. The sequence V4 hypervariable region was amplified, and Illumina HiSeq sequencing was performed to analyze the differences between the two groups. The Shannon and Chao1 indices of the postoperative esophageal cancer tissue group samples (Group A) were higher than those of the esophageal mucosa tissue samples (Group B), and the difference was statistically significant (P<0.05). The Simpson index of Group A was higher than that of Group B, but the difference was not significant (P>0.05). The β diversity analysis demonstrated that the overall composition of the flora of the two groups was not significantly different. Linear discriminant analysis effect size analysis showed that the abundance of Megasphaera, Actinobacteria, Enterobacteriaceae and Enterobacteriales in Group A was significantly higher than that in Group B, but the abundance of Mogibacteriaceae in Group B was significantly higher than that in Group A. The top 60 species were selected using the random forest method to establish a model. The error rate of the prediction model constructed using the random forest method was 22.59%. The receiver operating characteristic (ROC) curve analysis confirmed that the present model was reliable and could effectively distinguish between the two groups of samples (area under the curve, 0.86). The source of the sample should be considered in studies investigating the esophageal flora. Considering the increased richness and improved uniformity of postoperative tissue microbiota compared with the mucosal group, it was predicted that postoperative tissue may be more conducive to the study of esophageal cancer microbiota.

Dysbiosis of the Upper Gastrointestinal Tract in Head-and-Neck Cancer Survivors: A Pilot Study Using the Capsule Sponge Device

Background: A non-endoscopic capsule-sponge device allows sampling the entire length of the esophagus. Here, we compared microbiomes of the oral cavity, esophagus, and gastric corpus collected by oral swab, capsule-sponge device, and endoscopic biopsy, respectively, in patients representing three distinct risk profiles for esophageal squamous cell carcinoma (ESCC). Methods: The study enrolled 11 patients with esophageal squamous intraepithelial neoplasia, 21 patients after curative treatment for head and neck squamous cell cancer (HNSCC) (HNSCC survivors), and 40 patients with functional dyspeptic (FD) symptoms. Microbial genomic DNA was analyzed using 16S rRNA gene amplicon sequencing. Results: The Shannon index of the capsule-sponge sample microbiota was significantly higher in FD group than in patients after treatment for HNSCC, and the Chao index of gastric samples differed between HNSCC survivors and FD patients. Analysis of the β-diversity of FD patients, HNSCC, and esophageal squamous intraepithelial neoplasia showed that different genera formed at each location. The abundance of 205, 116, and 9 genera differed between FD patients and HNSCC survivors in the gastric, capsule-sponge, and oral samples, respectively; 33 genera differed between the FD group and patients with esophageal squamous intraepithelial neoplasia in capsule-sponge samples. Conclusions: The bacterial communities of the upper digestive tract were clustered according to the anatomic site. Despite substantial differences in gastric and esophageal microbiota samples between FD patients and HNSCC survivors, the microbial members and diversity showed small differences between FD patients and those with esophageal squamous intraepithelial neoplasia. It remains unclear whether gastric and esophageal dysbiosis is associated with or is a consequence of treatment for HNSCC.

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.

Microbiome of esophageal endoscopic wash samples is associated with resident flora in the esophagus and incidence of cancer

Change in mucosal microbiome is associated with various types of cancer in digestive tract. We hypothesized that microbial communities in the esophageal endoscopic wash fluids reflects resident flora in esophageal mucosa that is associated with esophageal carcinoma (EC) risk and/or directly correlates microbiome derived from EC tumor tissue. Studying microbial communities in esophageal endoscopic wash samples would be therefore useful to predict the incidence or risk of EC. We examined microbial communities of the endoscopic wash samples from 45 primary EC and 20 respective non-EC controls using 16S rRNA V3-V4 amplicon sequencing. The result was also compared with microbial communities in matched endoscopic biopsies from EC and non-cancerous esophageal mucosa. Compared with non-EC controls, 6 discriminative bacterial genera were detected in EC patients. Among them, relative abundance ratio of Prevotella and Shuttlewarthia, as well as decrease of genus Prevotella presented good prognostic performance to discriminate EC from controls (area under curve, 0.86, 0.82, respectively). Multivariate analysis showed occurrence of EC was an independent factor associated with decrease of this bacteria. Abundance of genus Prevotella in the esophageal endoscopic wash samples was significantly correlated with the abundance of this bacteria in the matched endoscopic biopsies from non-cancerous esophageal mucosa but not in the EC tissues. Our findings suggest that microbiome composition in the esophageal endoscopic wash samples reflects resident flora in the esophagus and significantly correlates with the incidence of EC.

Analytical Validation of a DNA Methylation Biomarker Test for the Diagnosis of Barrett's Esophagus and Esophageal Adenocarcinoma from Samples Collected Using EsoCheck®, a Non-Endoscopic Esophageal Cell Collection Device.

Barrett's esophagus (BE) is a known precursor to esophageal adenocarcinoma (EAC). Guidelines recommend BE screening in populations with multiple risk factors, for which non-endoscopic esophageal cell collection with biomarker testing is considered as an acceptable alternative to esophagogastroduodenoscopy (EGD). The aim of this study was to evaluate analytical performance characteristics of EsoGuard® (EG), a DNA methylation biomarker assay, as a laboratory-developed test (LDT) in esophageal samples collected with the swallowable EsoCheck® (EC) device. EG is a next-generation sequencing (NGS) assay that evaluates methylated vimentin (VIM) and cyclin A1 (CCNA1), clinically validated biomarkers for the detection of BE and EAC. The studies were conducted according to standards of College of American Pathology (CAP), Clinical Laboratory Improvement Amendments (CLIA), and New York (NY) state requirements for the analytical validation of molecular assays. Comparison to Sanger sequencing showed that EG was 100% accurate at all 31 CpG sites evaluated by the assay. The analytical sensitivity, specificity, and accuracy of the assay were 89%, 100%, and 96%, respectively. Intra- and inter-assay precision was 100%. The limit of detection (LOD) was 1 in 400 methylated cells, and the reference range was 84%. In summary, EsoGuard demonstrates high analytical accuracy, repeatability, and reproducibility in samples collected using the EsoCheck device.

Anti-tumour activity of Panobinostat in oesophageal adenocarcinoma and squamous cell carcinoma cell lines

BackgroundOesophageal cancer remains a challenging disease with high mortality rates and few therapeutic options. In view of these difficulties, epigenetic drugs have emerged as potential alternatives for patient care. The goal of this study was to evaluate the effect and biological consequences of Panobinostat treatment, an HDAC (histone deacetylase) inhibitor already approved for treatment of patients with multiple myeloma, in oesophageal cell lines of normal and malignant origin, with the latter being representative of the two main histological subtypes: adenocarcinoma and squamous cell carcinoma.ResultsPanobinostat treatment inhibited growth and hindered proliferation, colony formation and invasion of oesophageal cancer cells. Considering HDAC tissue expression, HDAC1 was significantly upregulated in normal oesophageal epithelium in comparison with tumour tissue, whereas HDAC3 was overexpressed in oesophageal cancer compared to non-malignant mucosa. No differences between normal and tumour tissue were observed for HDAC2 and HDAC8 expression.ConclusionsPanobinostat exposure effectively impaired malignant features of oesophageal cancer cells. Because HDAC3 was shown to be overexpressed in oesophageal tumour samples, this epigenetic drug may represent an alternative therapeutic option for oesophageal cancer patients.

Molecular Analysis of Persistent and Recurrent Barrett's Esophagus in the Setting of Endoscopic Therapy.

Early neoplastic progression of Barrett's esophagus (BE) is often treated with endoscopic therapy. Although effective, some patients are refractory to therapy or recur after apparent eradication of the BE. The goal of this study was to determine whether genomic alterations within the treated BE may be associated with persistent or recurrent disease. We performed DNA sequencing on pre-treatment esophageal samples from 45 patients who were successfully treated by endoscopic therapy and did not recur as well as pre-treatment and post-treatment samples from 40 patients who had persistent neoplasia and 21 patients who had recurrent neoplasia. The genomic alterations were compared between groups. The genomic landscape was similar between all groups. Patients with persistent disease were more likely to have pre-treatment alterations involving the receptor tyrosine kinase pathway ( P = 0.01), amplifications of oncogenes ( P = 0.01), and deletions of tumor suppressor genes ( P = 0.02). These associations were no longer significant after adjusting for patient age and BE length. More than half of patients with persistent (52.5%) or recurrent (57.2%) disease showed pre-treatment and post-treatment samples that shared at least 50% of their driver mutations. Pre-treatment samples were genomically similar between those who responded to endoscopic therapy and those who had persistent or recurrent disease, suggesting there is not a strong genomic component to treatment response. Although it was expected to find shared driver mutations in pre-treatment and post-treatment samples in patients with persistent disease, the finding that an equal number of patients with recurrent disease also showed this relation suggests that many recurrences represent undetected minimal residual disease.

Patients with cardinal symptoms of eosinophilic esophagitis. Prejudice affects clinical practice….

Dysphagia and bolus impaction are the cardinal manifestations of eosinophilic esophagitis (EoE). Esophageal biopsy sampling is mandatory for EoE diagnosis, data though suggest that clinician do not always obtain biopsies from patients with cardinal EoE symptoms during upper gastrointestinal endoscopy even if no other entity than EoE can explain patients symptoms. We aimed to search for the esophageal biopsy procurement rate as also for factors that drive clinicians to obtain esophageal biopsies among patients with cardinal EoE symptoms. We retrospectively searched for patients with cardinal EoE symptoms submitted to upper gastrointestinal endoscopy between 1/2018 and 12/2023 in our department. Epidemiologic, clinical, endoscopic, and histological data were analyzed. In total 163 patients with cardinal EoE symptoms (dysphagia: 63 and bolus impaction: 100) were included in the study (M/F: 100/63, mean age: 54 ± 22 years). Biopsy sampling was obtained in 77/163 (47.2%) patients and sampling rates did not differ between patients with bolus impaction or dysphagia (47/100, 47% vs 30/63, 47.6%, P = 0.553). Higher rates of sampling were observed in males ( P = 0.045), those younger than 65 years old ( P < 0.001) and patients with endoscopic EoE signs ( P = 0.004). Age and endoscopic findings compatible to EoE were independently correlated to biopsy sampling. EoE was diagnosed in 35/74 patients (47.3%); the majority of patients were male, with a bolus impaction episode, compatible endoscopic findings and all were younger than 65 years old. Clinicians take esophageal biopsies in half of patients with cardinal EoE. Age and supportive endoscopic evidence drive clinicians' decision to obtain esophageal biopsies.

System transferability of Raman-based oesophageal tissue classification using modern machine learning to support multi-centre clinical diagnostics

BackgroundThe clinical potential of Raman spectroscopy is well established but has yet to become established in routine oncology workflows. One barrier slowing clinical adoption is a lack of evidence demonstrating that data taken on one spectrometer transfers across to data taken on another spectrometer to provide consistent diagnoses.MethodsWe investigated multi-centre transferability using human oesophageal tissue. Raman spectra were taken across three different centres with different spectrometers of the same make and model. By using a common protocol, we aimed to minimise the difference in machine learning performance between centres.Results61 oesophageal samples from 51 patients were interrogated by Raman spectroscopy at each centre and classified into one of five pathologies. The overall accuracy and log-loss did not significantly vary when a model trained upon data from any one centre was applied to data taken at the other centres. Computational methods to correct for the data during pre-processing were not needed.ConclusionWe have found that when using the same make and model of spectrometer, together with a common protocol, across different centres it is possible to achieve system transferability without the need for additional computational instrument correction.

Nonendoscopic Screening for Barrett's Esophagus and Esophageal Adenocarcinoma in At-Risk Veterans.

Although rates of esophageal adenocarcinoma (EAC) in the United States continue to rise, many patients at risk of disease are not screened. EsoCheck (EC), a nonendoscopic esophageal balloon sampling device coupled with EsoGuard (EG), a DNA-based screening assay, is an US Food and Drug Administration-approved minimally invasive alternative to the traditional screening method of upper endoscopy. The objective of this study was to prospectively determine the diagnostic accuracy, tolerance, and acceptability of the EC/EG test in a screening population. We recruited veterans who met the American College of Gastroenterology Guideline criteria for endoscopic Barrett's esophagus (BE) and EAC screening at the Louis Stokes Cleveland Veterans Affairs Medical Center. All study participants completed unsedated EC-guided distal esophageal sampling followed by a sedated esophagogastroduodenoscopy (EGD). Diagnostic yield of the EG assay and EGD was recorded and used in calculation of sensitivity and specificity of EC/EG in prospective screening. The abbreviated Spielberger State-Trait Anxiety Inventory questionnaire was administered before and after completion of EC. Overall tolerance of EC sampling was evaluated on a 10-point Likert scale. Esophageal cancer screening was accepted by 130 of 782 eligible veterans (16.6%), and we analyzed results of those who completed both screening tests (N = 124). Prevalence of BE/EAC among studied veterans was 12.9% (16/124), based on EGD. Sensitivity and specificity of EC/EG for EGD-detected BE/EAC were 92.9% (95% confidence interval [CI] 66.1-99.8) and 72.2% (95% CI 62.1-80.8), respectively. Positive and negative predictive values were 32.5% (95% CI 18.6-49.1) and 98.6% (95% CI 92.4-100), respectively. Baseline Spielberger State-Trait Anxiety Inventory-6 scores were reflective of notable levels of anxiety among veterans in the periprocedural setting. The mean postprocedure acceptability score for the EC test was 7.23 (SD 2.45). Our data suggest excellent sensitivity and negative predictive value of EC/EG in a screening population of veterans, making this modality a powerful screening tool for BE and EAC.

Esophageal viral and bacterial microbiome unbalances characterize achalasia

Achalasia is a rare neuromuscular disorder of the esophagus whose pathogenesis is poorly understood. It is characterized by the loss of nerve cells in the esophagus, failure of the lower esophageal sphincter to relax, and impaired esophageal peristalsis. In this study, we re-analyzed transcriptomics data from blood and esophageal tissue samples of achalasia patients publicly available. Blood analysis showed hyperactivation of the immune system and dysfunction of the nervous system, consistent with the hypothesis of immune-mediated pathogenesis leading to neuronal loss. Mucosal tissue analysis e showed a remarkable unbalance in virome and bacteriome compositions in achalasia. In particular, bacterial diversity was reduced across the esophagus in achalasia compared to healthy controls, and in the distal esophagus bacteria associated with the healthy state, such as Firmicutes, Tenericutes, and Bacteroidetes, were less represented if not completely absent. Importantly, the only other component of the microbiota that varied significantly between achalasia and controls was the virome. Bacteriophages that parasite Firmicutes were strongly enriched in the esophagus supporting a predator-prey interaction. In conclusion, our study opens new horizons on a circulating signature of achalasia and the role of viral-bacterial interactions in orchestrating mucosal homeostasis and possibly disease pathogenesis. Key summaryAchalasia is a rare swallowing disorder with unknown causes. The role of gut microbiota, especially viruses, in achalasia is unclear. This study investigated the differences in viral and bacterial communities between achalasia patients and healthy people, and how they affect the esophageal mucosa. The findings may help develop new diagnostic tools and therapies based on novel hypotheses about the pathogenesis of achalasia.

Combining non-invasive liquid biopsy and a methylation analysis to assess surgical risk for early esophageal cancer.

While the widespread use of endoscopic submucosal dissection (ESD) has significantly reduced the incidence of early esophageal cancer (ESCA), the limited ability of ESD to strip deep infiltrating esophageal lesions results in a considerable risk of intraoperative perforation. Circulating-free DNA (cfDNA) is widely used in modern tumor screening due to its non-invasive detection capabilities. A methylation analysis offers vital insights into the condition and advancement of malignancies due to its unique positioning, such as a marker of cancer. This study investigated the potential of combining a non-invasive liquid biopsy technique, along with a methylation analysis, to assess the surgical perforation risk of ESCA patients. In this study, we conducted an analysis of gene expression differences between stage I esophageal squamous carcinoma samples and healthy tissue samples using data from The Cancer Genome Atlas (TCGA) database. We also identified the genes associated with progression-free survival (PFS) in esophageal squamous carcinoma. Integrating the framework of the methylation analysis, we explored the methylated sites of these distinct genes. To refine this process, we used the Shiny Methylation Analysis Resource Tool (SMART) to conduct a comprehensive analysis of these sites. We then confirmed the stability of the methylation sites in different lesion conditions using methylation-specific quantitative polymerase chain reaction (MS-qPCR) with paraffin tissue samples collected after ESD. We analyzed RNA-sequencing data from 42 early stage ESCA patients and 17 controls, identifying 1,263 up-regulated and 460 down-regulated genes. Functional analyses revealed involvement in key pathways such as cell cycle regulation and immune responses. Furthermore, we identified 38 differentially expressed genes associated with PFS. Using SMART analysis, we found 217 hyper-methylated regions in 38 genes, suggesting potential early markers for ESCA. Validation experiments confirmed the reliability of 29 hyper-methylated regions in FFPE tissue samples and 6 regions in cfDNA. A LunaCAM model showed high accuracy [area under the curve (AUC) =0.89] in discriminating early ESCA. Integrated assessment of six highly methylated regions significantly improved predictive performance, with 90.56% sensitivity, highlighting the importance of combinatorial biomarker evaluation for early cancer detection. This study established a novel approach that integrates non-invasive testing with a methylation analysis to assess the surgical risk of early ESCA patients. The significance of changes in methylation sites in relation to lesion status should not be underestimated, as they have the potential to offer vital insights for proactive risk assessments before surgery.

Ethanol exposure exacerbates 4-nitroquinoline-1-oxide induced esophageal carcinogenesis and induces invasive carcinoma with muscularis propria infiltration in a mouse model

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal cancers worldwide. Most ESCC patients are diagnosed at an advanced stage; however, current research on in vivo animal models accurately reflecting their clinical presentation is lacking. Alcohol consumption is a major risk factor for ESCC and has been used in several disease models for disease induction. In this study, we used 4-nitroquinoline-1-oxide in combination with ethanol to induce an in vivo ESCC mouse model. Esophageal tissues were stained with hematoxylin and eosin for histopathological examination and lesion scoring. In cellular experiments, cell adhesion and migration invasion ability were observed using phalloidin staining, cell scratch and transwell assays, respectively, and the expression of epithelial-mesenchymal transition-related markers was detected using quantitative reverse transcription polymerase chain reaction and western blotting. The results showed that ethanol-exposed mice lost more weight and had an increased number of esophageal nodules. Histological examination revealed that the lesion scores of the ethanol-exposed esophageal samples were significantly higher than those of the unexposed esophageal samples. Furthermore, ethanol-exposed esophageal cancer samples had more severe lesions with infiltration of tumor cells into the muscularis propria. In vitro cellular experiments showed that ethanol exposure induced cytoskeletal microfilament formation, promoted cell migration invasion elevated the expression of N-cadherin and Snail, and decreased the expression of E-cadherin. In conclusion, ethanol exposure exacerbates ESCC, promotes tumor cell infiltration into the muscularis propria, and could be an effective agent for establishing innovative models of invasive carcinoma.

Neoadjuvant sintilimab, albumin-bound paclitaxel, and carboplatin for locally advanced resectable esophageal squamous cell carcinoma: A prospective phase 2 study.

e16097 Background: Whether combined immunotherapy on the basis of neoadjuvant chemotherapy is beneficial for locally advanced esophageal squamous cell carcinoma (ESCC) remain to be inconclusive. This phase 2 study (ChineseClinical Trial Registry: ChiCTR2000041081) evaluate the efficacy and safety of neoadjuvant sintilimab, albumin-bound paclitaxel, and carboplatin in locally advanced ESCC. Methods: Patients with locally advanced ESCC (T2N+M0, T3N0-2M0, T4aN0-2M0, or no distant metastases, or viable or potentially viable) were enrolled and received 3 cycles of neoadjuvant sintilimab, albumin-bound paclitaxel, and carboplatin. Surgery was scheduled within four to six weeks of completing the neoadjuvant treatment. Three additional cycles of adjuvant sintilimab monotherapy or sintilimab plus chemotherapy were scheduled after a multidisciplinary discussion. The primary endpoint was the MPR rate. Results: From November 2020 to November 2021, twenty-four patients (median age: 64.5 years, men: 21/24,lower segment tumor location12/24,stage IV disease 15/24) were included in this study. All patients completed three cycles of neoadjuvant treatment and surgery. The median dose intensities for sintilimab, carboplatin and albumin-bound paclitaxel were 100% (range, 66.7–105.0%), 98.4% (range, 64.1–104.0%), and 100% (range, 38.1–105.8%), respectively. The R0 resection rate was 100%. The pCR rate and MPR rate was 33.3% and 41.7%. Among the 10 patients with PD-L1 CPS of ≥1, the pCR and MPR rates were 40% and 50%. 20 (83.1%) had TNM downstaging. The median DFS and median OS had not been reached yet, 500 days DFS and OS rate was 85.3% (95%CI 85.4%-97.2%) and 94.1% (95%CI 94.8%-100.0%). PD-L1 protein expression showed a higher life expectancy trend in the post-neoadjuvant therapy esophageal tissue samples than in the pre-therapy samples. The most common grade 3-4 treatment-related adverse events were anemia (29.2%), increased gamma-glutamyl transferase level (4.2%) and hyponatremia (4.2%). Emergency reoperation and intensive care were not required, and there were no postoperative mortalities. Conclusions: Neoadjuvant sintilimab, albumin-bound paclitaxel, and carboplatin preliminarily demonstrate encouraging clinical benefits and acceptable safety. Preoperative immunotherapy combined with chemotherapy alters the immune microenvironment of locally advanced resectable ESCC. Clinical trial information: ChiCTR2000041081.

ZNF717 or PABPC1 Gene Variants Contribute to Congenital Esophageal Atresia by Interfering with Normal Esophageal Growth

Congenital esophageal atresia (EA) is an abnormality induced by the incomplete differentiation of the foregut in infants, and is frequently accompanied by tracheoesophageal fistula (TEF). Our understanding of the pathogenesis of EA-TEF is limited, additionally, there is still a lack of standard animal or cell models for in vitro EA-TEF investigation. Therefore, we analyzed esophageal tissue samples from 10 children with EA-TEF via Exome sequencing (ES) to identify gene variants. And esophageal organoid units (EOUs) were established as an in vitro model of EA by culturing esophageal tissues from Adriamycin-challenged rats. The ES results indicated 11 mutated genes, including the frameshift variants of ZNF717 and PABPC1. The EA organoids expressed the esophageal marker proteins CK13 and CK4 and showed a significantly slower rate of growth and dysplasia of cell development. In EA organoids, the transcription of SOX2, ZNF717, and PABPC1 was downregulated at varying levels, while NOGGIN transcription was markedly upregulated. Furthermore, when siRNA-ZNF717 or siRNA-PABPC1 was transfected into normal esophageal organoids, the proliferation of esophageal cells was significantly decreased. In conclusion, we found that normal ZNF717 and PABPC1 expressions are essential to the esophageal development, whereas the variant or deficiency of these genes might lead to EA-TEF.