Esophageal Samples Research Articles

Carcinogen 4-Nitroquinoline Oxide (4-NQO) Induces Oncostatin-M (OSM) in Esophageal Cells.

The earliest cellular and molecular biologic changes in the esophagus that lead to esophageal cancer were evaluated in a mouse model. We correlated numbers of senescent cells with the levels of expression of potentially carcinogenic genes in sorted side population (SP) cells containing esophageal stem cells and non-stem cells in the non-side population cells in the 4-nitroquinolone oxide (NQO)-treated esophagus. We compared stem cells with non-stem cells from the esophagus of mice treated with the chemical carcinogen 4-NQO (100 μg/ml) in drinking water. We also compared gene expression in human esophagus samples treated with 4-NQO (100 μg/ml media) to non-treated samples. We separated and quantitated the relative levels of expression of RNA using RNAseq analysis. We identified senescent cells by luciferase imaging of p16+/LUC mice and senescent cells in excised esophagus from tdTOMp16+ mice. A significant increase in the levels of RNA for oncostatin-M was found in senescent cells of the esophagus from 4-NQO-treated mice and human esophagus in vitro. Induction of OSM in chemically-induced esophageal cancer in mice correlates with the appearance of senescent cells.

Human papillomavirus is an important risk factor for esophageal carcinoma in a Chinese population

PurposeDifferent types of HPV have been associated with cancer in humans, but the role of HPV in esophageal cancer (EC) is controversial. The purpose of this study was to evaluate the correlation between HPV infection and EC in the Chinese population and to provide the scientific basis for the future prevention, control, early diagnosis, and treatment strategies of EC in China.MethodsPCR detected HPV infection in 1112 esophageal cancer tissue samples, and 89 HPV-positive samples were detected by genotyping. Proximity ligation assays (PLAs) and immunohistochemistry were used to detect the expression of HPV E6 and E7 proteins. Real-time fluorescent quantitative PCR was used to detect the integration of HPV16 E6. The level of HPV-specific antibody IgG in serum was detected by ELISA and PLA.ResultsThe positive rates of HPV L1, HPV16, HPV18, hpv16 + 18 E6 and hpv16/18 E6 in 1,112 EC tissue samples were 77.6%, 41.4%, 27.2%, 14.2% and 55.4% respectively. Multiple HPV subtypes were detected in HPV-positive EC samples. PLA showed that E6 and E7 were expressed in EC109 and formed complexes with p53 and pRb, respectively. Immunohistochemistry showed that the positive rates of hpv16 + 18 E6 and E7 in HPV-positive EC samples were 56.4% and 37.0%, respectively. HPV-DNA integration rate in HPV-positive EC tissues (88.79%) was higher than that in adjacent tissues (54.17%). HPV antibody was found in the serum of EC patients by a serological test.ConclusionThe study suggests that HPV, especially HPV16 and HPV18, the infection may be a risk factor for EC in the Chinese population and that the E6 protein may play a key role in HPV-associated malignancies. These results may be important for the prevention and treatment of HPV-positive EC in China.

First Molecular Identification of Sarcocystis Buffalonis in Goats Worldwide in Misan Governorate, Iraq

The current study was conducted in Misan Governorate to detect the parasite Sarcocystis spp. by both conventional and molecular methods and to identify the species of this genus in goats as intermediate hosts for multiple species of this parasite. Regarding to the traditional study, the rates of infection with the macroscopic type of Sarcocystis spp. were low and recorded 2% in esophageal samples only in goats. The sizes of macroscopic parasite cysts ranged between 1-10 mm in length and 2-6 mm in width. Infection with the microscopic type of parasite recorded high rates. When comparing the efficiency of the traditional methods used in our study to diagnose the infection of the microscopic type in all of the examined organs isolated from goats, which included the methods of digestion with pepsin, squeezing, trichinoscopy. It was found that the method of digestion with pepsin was the most efficient traditional method for diagnosing the microscopic type of parasite, followed by the trichinoscopy. While the squeezing method recorded the least efficient traditional method for diagnosing Sarcocystis spp. microscopically. The molecular study of Sarcocystis spp. included molecular diagnosis of the parasite's identity at the species level through gene amplification using polymerase chain reaction (PCR) of the target segment of DNA extracted from the parasite samples of goats for 18S rRNA gene by 120 microscopic samples and 4 macroscopic samples. 87.9% of goat samples, showed positive infection with the 18S rRNA gene; In which 100% of the macroscopic and microscopic samples of the esophagus were recorded, and both animals had a positive infection, while the infection rates for the diaphragm, tongue and heart samples were recorded as 90%, 86.7 and 80%, respectively. The results obtained from sequence analysis of 18S rRNA gene for six samples showed that four microscopic samples of goats (OP474017.1, OP474018.1, OP476699.1 and OP474036.1) were closely related to the Malaysian species; S. capracanis with accession number (KR155191.1) with identity score percentages as 99.50%, 99.01%, 99.76% and 99.13%, respectively. Finally, the two macroscopic samples of goats (OP474061.1 and OP474062.1) were identical with the Egyptian species; S. buffalonis, with accession numbers (KU247913.1) and (KU247913.1) respectively, with a matching percentage of 84.39% and 85.64% respectively. S. buffalonis has identified in goats at the first time globally in present study. Keywords: Sarcocystis buffalonis, Goats, OP474061.1, OP474062.1.

Tumor immune microenvironment in therapy-naive esophageal adenocarcinoma could predict the nodal status.

Currently, preoperative staging of esophageal adenocarcinoma (EAC) has modest reliability and accuracy for pT and pN stages prediction, which heavily affects overall survival. The interplay among immune checkpoints, oncogenes, and intratumoral and peritumoral immune infiltrating cells could be used to predict loco-regional metastatic disease in early EAC. We prospectively evaluated immune markers expression and oncogenes status as well as intratumoral and peritumoral immune infiltrating cells populations in esophageal mucosa samples obtained from neoadjuvant therapy-naïve patients who had esophagectomy for EAC. Vascular invasion and high infiltration of lamina propria mononuclear cells resulted associated with nodal metastasis. Low infiltration of activated CD8+ CD28+ T cells was observed in both intratumoral and peritumoral mucosa of patients with nodal metastasis. Low levels of CD69, MYD88, and TLR4 transcripts were detected in the intratumoral specimen of patients with lymph node involvement. Receiver operating characteristic curve analysis showed good accuracy for detecting nodal metastasis for all the markers tested. Significant lower infiltration of CD8 T cells and M1 macrophages and a lower expression of CD8A, CD8B, and TBX21 were found also in Esophageal Adenocarcinoma TCGA panCancer Atlas in the normal tissue of patients with nodal metastasis. Our data suggest that immune surveillance failure is the main driver of nodal metastasis onset. Moreover, nodal metastasis containment also involves the immune microenvironment of the peritumoral healthy tissue.

Clinical and Genomic Characterization of Esophageal Cancer Brain Metastases

<h3>Purpose/Objective(s)</h3> Brain metastases from esophageal cancer are a rare and understudied form of metastatic spread with limited treatment options. We characterized the clinical and genomic features of esophageal cancer brain metastases. <h3>Materials/Methods</h3> We identified 129 patients who were diagnosed with brain metastases secondary to esophageal cancer at a single institution between 2002 and 2020. Survival analysis was performed using multivariable Cox proportional hazards regression. Results of targeted next-generation sequencing (NGS) panels were obtained for 56 patients representing 62 adenocarcinoma samples and compared to a cohort of 1344 esophageal adenocarcinoma samples identified from the American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE) v11.0. <i>ERBB2</i>/<i>HER2</i> amplified/overexpressed (<i>HER2</i>+) status was determined using routine clinical assays and/or NGS results. <h3>Results</h3> Median age at the time of esophageal cancer diagnosis was 63 years (interquartile range 58-68 years). 14% of the cohort (n=18) was female, 93% (n=120) had adenocarcinoma, and 98% (n=126) of primary tumors involved the distal esophagus and/or gastroesophageal junction. 50% (n=64) of patients had distant metastases at presentation, including 18 patients with brain metastases. 51% (n=66) underwent resection for brain metastases, and 84% (n=108) underwent radiation therapy (n=26 for whole brain, n=55 for partial brain or stereotactic, and n=27 for both). With median follow-up of 19 months for the entire cohort, median overall survival following primary cancer diagnosis was 22 months and significantly higher for <i>HER2</i>+ disease (31 vs 19 months, hazard ratio [HR] 0.64, 95% confidence interval [CI] 0.47-0.87, <i>P</i>=0.004). For patients without brain metastases initially, the median time between initial diagnosis and brain metastasis diagnosis was 13 months and significantly higher for patients without distant metastasis initially (14 vs 12 months, HR 0.59, 95% CI 0.39-0.89, <i>P</i>=0.012). Median overall survival after brain metastasis diagnosis was 7 months and significantly higher for <i>HER2</i>+ disease (14 vs 4 months, HR 0.60, 95% CI 0.45-0.82, <i>P</i>=0.001). NGS results were notable for an enrichment of <i>HER2</i> high amplification events (42% vs 19%, <i>P</i><0.0001), <i>HER2</i> mutations (10% vs 4%, <i>P</i>=0.036), and <i>TP53</i> mutations (97% vs 73%, <i>P</i><0.00001; all with Fisher's exact test) compared to the GENIE cohort. <h3>Conclusion</h3> Esophageal cancer brain metastases are associated with a poor prognosis and have a distinct molecular profile. Further efforts are necessary to elucidate the drivers of this unique and aggressive entity in order to develop novel treatment strategies.

Analysis of local expression of inflammatory cytokines genes in patients with gastroesophageal reflux disease and its association with types and number of gastroesophageal refluxes

The aim of the study was to evaluate gene expression of proinflammatory cytokines in esophageal mucosa in patients with erosive esophagitis (EE) and non-erosive form (NERD) of gastroesophageal reflux disease (GERD) and perform correlation analysis between the genes expression and types and number of gastroesophageal refluxes. Methods. This was a single-center prospective study. Esophageal mucosa samples were taken from lower part of the esophagus during endoscopy. Expression of IL-1b, IL-10, IL-18, TNFA, TLR4, GATA3, CD68 and beta-2 macroglobulin genes was assessed with ImmunoQuantex assays. Multichannel intraluminal esophageal pH-impedance studies were performed with Ohmega recorder and 2pH-6impedance catheters. Results. Data of 60 patients (females - 34; mean age (M±SD) 54.6 years, 20 - with erosive esophagitis) were available for the analysis. In those with EE, there was higher expression of IL-18 (5.89±0.4 vs 5.28±1.1, p=0.04), GATA3 (2.92±0.86 vs 2.23±0.96, p=0.03) genes, and inflammatory index (92.12±32.41% vs 70.1±30.8%, p=0.02) compared to NERD. Direct correlation was found between acid exposure time and expression of IL-1b (Spearman rank R = 0.29), IL-18 (R=0.31), TNFA (R=0.35), GATA3 (R=0.34), TLR4 (R=0.29), CD68 (R=0.37) genes and inflammatory index (R=0.3). Indirect correlation was revealed between mean рН and expressions of IL-18 (R= -0.28), TNFA (R= -0.33), GATA3 (R= -0.28), TLR4 (R= -0.28), CD68 (R= -0.39) genes and inflammatory index (R= -0.17). No correlation was found between the genes’ expression and number of gastroesophageal refluxes. Conclusions: Expression of proinflammatory cytokines’ genes differ in patients with EE and NERD. There is a correlation between expression of studied genes and esophageal acidity.

Decellularized esophageal tubular scaffold microperforated by quantum molecular resonance technology and seeded with mesenchymal stromal cells for tissue engineering esophageal regeneration.

Current surgical options for patients requiring esophageal replacement suffer from several limitations and do not assure a satisfactory quality of life. Tissue engineering techniques for the creation of customized “self-developing” esophageal substitutes, which are obtained by seeding autologous cells on artificial or natural scaffolds, allow simplifying surgical procedures and achieving good clinical outcomes. In this context, an appealing approach is based on the exploitation of decellularized tissues as biological matrices to be colonized by the appropriate cell types to regenerate the desired organs. With specific regard to the esophagus, the presence of a thick connective texture in the decellularized scaffold hampers an adequate penetration and spatial distribution of cells. In the present work, the Quantum Molecular Resonance® (QMR) technology was used to create a regular microchannel structure inside the connective tissue of full-thickness decellularized tubular porcine esophagi to facilitate a diffuse and uniform spreading of seeded mesenchymal stromal cells within the scaffold. Esophageal samples were thoroughly characterized before and after decellularization and microperforation in terms of residual DNA content, matrix composition, structure and biomechanical features. The scaffold was seeded with mesenchymal stromal cells under dynamic conditions, to assess the ability to be repopulated before its implantation in a large animal model. At the end of the procedure, they resemble the original esophagus, preserving the characteristic multilayer composition and maintaining biomechanical properties adequate for surgery. After the sacrifice we had histological and immunohistochemical evidence of the full-thickness regeneration of the esophageal wall, resembling the native organ. These results suggest the QMR microperforated decellularized esophageal scaffold as a promising device for esophagus regeneration in patients needing esophageal substitution.

S2257 Cirrhosis, Varices, and Barrett’s Esophagus: WATS3D-only Surveillance and Band Ligation in the Management of Nondysplastic Barrett’s Esophagus Progressing to High Grade Dysplasia With Underlying Esophageal Varices

Introduction: Wide-area transepithelial sampling with 3-dimensional computer-assisted analysis (WATS3D) is an emerging technique used to collect esophageal samples via transepithelial brushing. WATS3D can be a safe and useful tool in Barrett’s esophagus (BE) management in patients with underlying small esophageal varices (EV) where traditional 4 quadrant cold forceps biopsies (CFB) would be contraindicated due to the risk of adverse events such as bleeding. We describe the utility of WATS3D-only surveillance combined with band ligation in the management and eradication of BE which progressed to high grade dysplasia (HGD) in a patient with underlying cirrhosis and EV. Case Description/Methods: We present a 66-year-old male with a history of nondysplastic (ND) BE (C0-M1), small Grade 1 EV and cirrhosis who presented for BE surveillance. Due to underlying EV and associated risk of bleeding, CFB was not performed and sampling with WATS3D-only was undertaken. WATS3D was first performed with surveillance esophagogastroduodenoscopy (EGD) in 2015 and demonstrated ND BE. On further EGD surveillance, BE histology remained ND until early 2021 where WATS3D revealed low grade dysplasia (LGD). Repeat EGD with WATS3D brushing was performed 4 months later, which demonstrated progression to HGD. Interval surveillance EGD with WATS3D brushing was, again, performed 4 months later and demonstrated LGD with rare foci of HGD. Of note, in both instances band ligation of the dysplastic segments was done. EGD with WATS3D 3 months later revealed endoscopic clearance of BE post-banding and pathology report revealed, ND, non-goblet cell metaplasia. The final interval surveillance EGD with WATS3D, 3 months later, showed complete eradication of BE with no evidence of intestinal metaplasia. Table 1 provides a thorough chronological description from first EGD to complete resolution of BE, 8 years later. Discussion: WATS3D has been increasingly shown to be an effective BE surveillance and screening tool. However, given the lack of data regarding appropriate surveillance windows for WATS3D-only BE surveillance, we performed sampling at shorter intervals than what would otherwise be recommended, to confirm the lack of dysplasia. This case presents a unique approach to WATS3D-only surveillance combined with band ligation therapy of dysplasia in the management of BE in a cirrhotic patient with underlying EV. Additionally, this case is unique in clearly documenting progression of a short segment of ND BE to HGD with WATS3D sampling alone. Table 1. - Summary of findings and intervention from first EGD to complete resolution of BE (8 year window) Date Variceal Grade Prague Score Number of BE Islands Sampling done by WATS3D Pathology Findings Banding therapy (# of bands) 04/09/14 2 C0-M1 3 No N/A 0 07/29/15 2 C0-M1 Scattered No N/A 0 12/2315 1 C0-M1 Scattered Yes No dysplasia 0 06/15/16 1 C0-M1 Scattered Yes No dysplasia 0 02/08/17 2 C0-M1 Scattered No N/A 4 01/22/18 1 C0-M1 1 No N/A 0 01/30/19 2 C0-M1 2 No N/A 0 01/29/20 1 C0-M1 2 No N/A 0 02/01/21 1 C0-M1 2 Yes Low-grade dysplasia 0 06/01/21 1 C0-M1 2 Yes High-grade dysplasia 3 10/25/21 1 C0-M1 2 Yes Low-grade with rare foci of high-grade dysplasia 2 01/25/22 1 N/A 3 Yes Non-goblet cell metaplasia, no dysplasia 0 04/25/22 1 N/A 0 Yes Squamocolumnar, no dysplasia or metaplasia 0

Changes to the upper gastrointestinal microbiotas of children with reflux oesophagitis and oesophageal metaplasia.

Little is known of the relationships among paediatric upper gastrointestinal microbiotas, and the impact of medication use and disease on their diversity. Here, we investigated the diversity of three microbiotas in the upper gastrointestinal tract of paediatric patients in relation to each other and to host factors. Oral, oesophageal and gastric microbiotas from a prospective paediatric cohort (n=54) were profiled using the 16S rRNA gene and ITS2 amplicon sequencing. 16S rRNA gene amplicon sequencing of oesophageal biopsies from a retrospective paediatric cohort (n=96) and shotgun metagenomics data from oesophageal brushings (n=88) were employed for genomic signature validation. Bacterial diversity and composition showed substantial differences across oral, oesophageal and gastric fluid samples that were not replicated for fungi, and the presence of reflux led to increased homogeneity in the bacterial component of these three microbiotas. The oral and oesophageal microbiotas were associated with age, sex, history of oesophageal atresia and presence of oesophageal metaplasia, with the latter characterized by Prevotella enrichment. Proton pump inhibitor use was associated with increased oral bacterial richness in the gastric fluid, and this correlated with increased levels of gastric pro-inflammatory cytokines. Profiling of oesophageal biopsies from a retrospective paediatric cohort confirmed an increased Prevotella prevalence in samples with metaplasia. Analysis of metagenome-derived oesophageal Prevotella melaninogenica genomes identified strain-specific features that were significantly increased in prevalence in samples with metaplasia. Prevotella enrichment is a signature associated with paediatric oesophageal metaplasia, and proton pump inhibitor use substantially alters the paediatric gastric microenvironment.

149. LOCAL INFLAMMATORY RESPONSE TO GASTROESOPHAGEAL REFLUX: ASSOCIATION OF GENE EXPRESSION OF INFLAMMATORY CYTOKINES WITH MULTICHANNEL INTRALUMINAL PH-IMPEDANCE DATA

Abstract Expression of inflammatory cytokines’ genes may take part in pathophysiology of different forms of gastroesophageal reflux disease (GERD). Aim: to explore the expression of inflammatory cytokines’ genes in esophageal mucosa in patients with erosive esophagitis and non-erosive forms of gastroesophageal reflux disease and its association with data of esophageal multichannel intraluminal pH-impedance measurements. This was a single-center prospective study. Esophageal mucosa samples were taken from lower part of the esophagus during endoscopy. Expression of IL-1β, IL-10, IL-18, TNFA, TLR4, GATA3, CD68, beta-2 macroglobulin genes in esophageal mucosa was assessed with ImmunoQuantex assays. Multichannel intraluminal esophageal pH-impedance (MII-pH) studies performed to all the participants. Diagnosis of GERD was confirmed by the results of MII-pH data. Patients were allocated to erosive esophagitis (EE) or non-erosive GERD groups (NERD). Non-symptomatic subjects with normal endoscopy and MII-pH results served as a control. Spearman rank correlation was used to asses association of genes expression and results of MII-pH. Data of 60 patients with GERD and 10 subjects of the control group were available for the analysis. Higher expression of IL-18 (5.89 ± 0.4 vs 5.28 ± 1.1, p = 0.04) and GATA3 (2.92 ± 0.86 vs 2.23 ± 0.96, p = 0.03) genes was found in EE group compared to NERD. Expression of IL-1β, IL-18, TNFA, and TLR4 genes was lower (P &amp;lt; 0.05) in the control group compared to EE and NERD. Esophageal acid exposure correlated with the expression of IL-1β (R = 0.29), IL-18 (R = 0.31), TNFA (R = 0.35), GATA3 (R = 0.34), TLR4 (R = 0.29), CD68 (R = 0.37) genes. Mean esophageal рН correlated inversely with the expressions of IL-18, TNFA, GATA3, TLR4, CD68 genes. In patients with erosive esophagitis, local expression of interleukin 18 and GATA3 transcription factor genes was higher compared to subjects with non-erosive form of gastroesophageal reflux disease. Esophageal acid exposure correlated directly with expression of IL-1β, IL-18, TNFA, TLR4, CD68 and B2M genes. Inverse correlation revealed between expression of IL-18, TNFA, GATA3, TLR4 CD68 genes and mean esophageal pH.

Comparison of tumor-associated and nontumor-associated esophageal mucosa microbiota in patients with esophageal squamous cell carcinoma.

Esophageal microbiota plays important roles in esophageal squamous cell carcinoma (ESCC). The aims of this study were to clarify the changes in the bacterial community during ESCC development and identify latent pathogenic bacteria which may contribute to esophageal carcinogenesis and progression. Fresh tumor and nontumor esophageal mucosal samples were collected from 31 men with ESCC. High-throughput 16s rRNA sequencing was performed, and the operational taxonomic unit data and bacterial classification annotation were obtained and analyzed. The Ace, Chao, Shannon, Simpson indexes, and operational taxonomic unit numbers were higher in nontumor tissues than in tumor tissues, although without statistical significance. There were 4 phyla and 28 genera found to show significant differences between tumor and nontumor samples. The general probiotic Lactobacillus was 1.98-fold higher in nontumor tissues, while the general pathogenic genera Fusobacterium was 4.35-fold higher in tumor tissues. For tumor tissue samples, the genera Treponema and Brevibacillus were significantly higher in N1 and N2 stages, respectively, and Acinetobacter was significantly higher in T3 stage. For nontumor tissues, the genus Fusicatenibacter was significantly higher in T2 stage, and Corynebacterium, Aggregatibacter, Saccharimonadaceae-TM7x, and Cupriavidus were significantly higher in T4 stage. Additionally, bacteria related to nitrotoluene degradation were enriched in nontumor tissues, while bacteria related to base excision repair were enriched in tumor tissues. The relative abundance of several phyla and genera are different between tumor and nontumor tissue samples. The altered bacterial microbiota is correlated with different tumor stages and some microbes may take part in the carcinogenesis and development of ESCC.

Local inflammatory response to gastroesophageal reflux: Association of gene expression of inflammatory cytokines with esophageal multichannel intraluminal impedance-pH data.

BACKGROUNDGene expression of inflammatory cytokines may take part in the pathophysiology of different forms of gastroesophageal reflux disease (GERD).AIMTo explore gene expression of inflammatory cytokines in esophageal mucosa in patients with erosive esophagitis (EE) and non-erosive forms of GERD (NERD) and its association with data of esophageal multichannel intraluminal impedance-pH (MII-pH) measurements.METHODSThis was a single-center prospective study. Esophageal mucosa samples were taken from the lower part of the esophagus during endoscopy. Expression of interleukin (IL)-1β, IL-10, IL-18, tumor necrosis factor α (TNFA), toll-like receptor 4 (TLR4), GATA binding protein 3 (GATA3), differentiation cluster 68 (CD68) and β-2 microglobulin genes in esophageal mucosa was assessed with ImmunoQuantex assays. MII-pH measurements were performed on all the participants. Diagnosis of GERD was confirmed by the results of the MII-pH data. Based on the endoscopy, patients were allocated to the groups of EE and NERD. The control group consisted of non-symptomatic subjects with normal endoscopy and MII-pH results. We used nonparametric statistics to compare the differences between the groups. Association of expression of the mentioned genes with the results of the MII-pH data was assessed with Spearman’s rank method.RESULTSData from 60 patients with GERD and 10 subjects of the control group were available for the analysis. Higher expression of IL-18 (5.89 ± 0.4 vs 5.28 ± 1.1, P = 0.04) and GATA3 (2.92 ± 0.86 vs 2.23 ± 0.96, P = 0.03) was found in the EE group compared to NERD. Expression of IL-1β, IL-18, TNFA, and TLR4 was lower (P < 0.05) in the control group compared to EE and NERD. Esophageal acid exposure correlated with the expression of IL-1β (Spearman’s rank r = 0.29), IL-18 (r = 0.31), TNFA (r = 0.35), GATA3 (r = 0.34), TLR4 (r = 0.29), and CD68 (r = 0.37). Mean esophageal рН correlated inversely with the expression of IL-18, TNFA, GATA3, TLR4, and CD68. No association of gene expression with the number of gastroesophageal refluxes was found.CONCLUSIONIn patients with EE, local expression of IL-18 and GATA3 was higher compared to subjects with NERD. Esophageal acid exposure correlated directly with expression of IL-1β, IL-18, TNFA, TLR4, CD68, and β-2 microglobulin genes. Inverse correlation was revealed between expression of IL-18, TNFA, GATA3, TLR4, and CD68 and mean esophageal pH.

Low-cost and clinically applicable copy number profiling using repeat DNA

BackgroundSomatic copy number alterations (SCNAs) are an important class of genomic alteration in cancer. They are frequently observed in cancer samples, with studies showing that, on average, SCNAs affect 34% of a cancer cell’s genome. Furthermore, SCNAs have been shown to be major drivers of tumour development and have been associated with response to therapy and prognosis. Large-scale cancer genome studies suggest that tumours are driven by somatic copy number alterations (SCNAs) or single-nucleotide variants (SNVs). Despite the frequency of SCNAs and their clinical relevance, the use of genomics assays in the clinic is biased towards targeted gene panels, which identify SNVs but provide limited scope to detect SCNAs throughout the genome. There is a need for a comparably low-cost and simple method for high-resolution SCNA profiling.ResultsWe present conliga, a fully probabilistic method that infers SCNA profiles from a low-cost, simple, and clinically-relevant assay (FAST-SeqS). When applied to 11 high-purity oesophageal adenocarcinoma samples, we obtain good agreement (Spearman’s rank correlation coefficient, rs=0.94) between conliga’s inferred SCNA profiles using FAST-SeqS data (approximately £14 per sample) and those inferred by ASCAT using high-coverage WGS (gold-standard). We find that conliga outperforms CNVkit (rs=0.89), also applied to FAST-SeqS data, and is comparable to QDNAseq (rs=0.96) applied to low-coverage WGS, which is approximately four-fold more expensive, more laborious and less clinically-relevant. By performing an in silico dilution series experiment, we find that conliga is particularly suited to detecting SCNAs in low tumour purity samples. At two million reads per sample, conliga is able to detect SCNAs in all nine samples at 3% tumour purity and as low as 0.5% purity in one sample. Crucially, we show that conliga’s hidden state information can be used to decide when a sample is abnormal or normal, whereas CNVkit and QDNAseq cannot provide this critical information.ConclusionsWe show that conliga provides high-resolution SCNA profiles using a convenient, low-cost assay. We believe conliga makes FAST-SeqS a more clinically valuable assay as well as a useful research tool, enabling inexpensive and fast copy number profiling of pre-malignant and cancer samples.

Molecular Identification of Sarcocystis Species in Sheep from Lithuania.

Simple SummaryMembers of the genus Sarcocystis (Apicomplexa: Sarcocystidae) are protozoans that have a two-host, prey–predator life cycle. These parasites are most thoroughly examined in economically important animals. Several Sarcocystis species are known to form macroscopic and microscopic sarcocysts in the muscle tissues of sheep. A previous study carried out in Lithuania has shown a maximum Sarcocystis infection prevalence in sheep and a high parasite burden; however, species were not identified. In the present study, diaphragm, oesophagus, and heart samples of 69 sheep raised in Lithuania were examined. No macrocysts were detected in the sheep muscles, while the microcysts found corresponded to two morphological types. By molecular methods, two Sarcocystis species, S. arieticanis and S. tenella, using canids as their definitive hosts, were identified for the first time in sheep from Lithuania. Both species were detected in all the studied animals. In summary, very high detection rates of S. arieticanis and S. tenella ranging from 82.61% to 100% were established in the examined diaphragm, oesophagus, and heart muscle samples of sheep.Data on the distribution of different Sarcocystis species in various muscles of sheep are scarce. In the present study, 190 diaphragm, oesophagus, and heart muscle samples of 69 sheep raised in Lithuania were examined for the presence of Sarcocystis spp. Under a light microscope, two morphological types of microcysts corresponding to S. arieticanis and S. tenella were detected. Eight and 12 sarcocysts of S. arieticanis and S. tenella, respectively, were isolated and characterised by the sequencing of a portion of cox1. The sequence comparisons revealed the highest similarity between European and Asian isolates of S. arieticanis and S. tenella obtained from domestic sheep and other wild Caprinae hosts. Based on peptic digestion, nested PCR targeting cox1, and sequencing, a 100% infection prevalence of S. arieticanis and S. tenella was observed in the 69 studied animals. The occurrence of S. tenella was significantly higher in the diaphragm than in the oesophagus (χ2 = 13.14, p < 0.001), whereas differences in the prevalence of S. arieticanis in the studied muscle types were insignificant (χ2 = 1.28, p > 0.05). Further molecularly based epidemiological studies are needed to compare the prevalence of Sarcocystis species in various muscles of sheep raised in different geographic regions.

Inhibition of APLN suppresses cell proliferation and migration and promotes cell apoptosis in esophageal cancer cells in vitro, through activating PI3K/mTOR signaling pathway.

Esophageal cancer is the sixth leading cause of cancer mortalities globally with a high incidence rate. Apelin (APLN) plays regulatory roles in different organs. However, its role in esophageal cancer remains unknown. Therefore, our study aims to explore the effect of APLN on esophageal cancer. One hundred and eighty-four (184) esophageal tumor tissues samples from patients with esophageal cancer, and 11 esophageal tissues samples from healthy volunteers were analyzed for the expression of APLN. APLN was highly expressed in the tumor of patients with esophageal cancer and esophageal cancer cells. Patients with high expressions of APLN had a lower survival rate than the ones with low to medium expressions of APLN. Human esophageal carcinoma cell lines, TE-1 and ECA-109 cells were transfected with APLN siRNA to knockdown APLN, or transfected with pcDNA-APLN to overexpress APLN. Inhibition of APLN by siRNA-APLN reduced proliferative, migrative, and invasive abilities of esophageal cancer cells and promoted cell apoptosis, which could be all restored by pcDNA-APLN. Moreover, knocking down APLN by siRNA-APLN suppressed the PI3K/mTOR signaling pathway. These findings identify that APLN inhibition might ameliorate esophageal cancer through activating the PI3K/mTOR signaling pathway, thus APLN could be a potential target for esophageal cancer.

Eosinophilic esophagitis auxiliary diagnosis based on a peptide ligand to eosinophil cationic protein in esophageal mucus of pediatric patients

Eosinophilic esophagitis (EoE) is a chronic inflammatory condition of the esophagus characterized by increased number of eosinophils. Currently, EoE diagnosis is based on endoscopic procedures for histopathological examination, eosinophils’ counting and, often, in clinical practice, the challenge is the differentiation between EoE and gastroesophageal reflux disease (GERD). Our aim was to develop novel peptide ligand to Eosinophil cationic protein (ECP) present in EoE biopsies of patients with potential to be used for detection. We performed a comparative proteomic analysis using liquid chromatography-tandem mass spectrometry (LC–MS/MS) of esophageal biopsies from pediatric patients with eosinophilic esophagitis, gastroesophageal reflux disease and control individuals. Then, phage display technology was used to select peptides against specific up-regulated protein from EoE patients. Twelve phage clones were selected after three biopanning rounds, and the best phage clone reactivity was evaluated by phage-ELISA assay using esophageal mucus samples from 94 pediatric patients. Mass spectrometry showed that eosinophil cationic protein (ECP) was one of the most up-regulated proteins in EoE patients, which is an eosinophil granule protein usually deposited on tissues to mediate remodeling, but in excess may cause fibrosis and hypertrophy, especially in allergic responses. A highly reactive ECP-ligand peptide (E5) was able to distinguish reactive mucus of EoE patients from GERD and the control individuals by Phage-ELISA, achieving a sensitivity of 84.62%, and a specificity of 82.72%. This is the first study that successfully demonstrated an antibody-like peptide targeting ECP at the esophagus mucus as a useful auxilliary tool for EoE diagnosis with a significant association with atopic disorders and dysphagia.ClinicalTrials.gov no.: NCT03069573.

Multi-Omics Integration Reveals the Crucial Role of Fusobacterium in the Inflammatory Immune Microenvironment in Head and Neck Squamous Cell Carcinoma.

ABSTRACTThe tumor microbiome is believed to have a profound impact on tumor progression owing to its local colonization in the tumor microenvironment (TME). Using the Cancer Microbiome Atlas (TCMA), a database of curated, decontaminated microbial profiles for 3,689 oropharyngeal, esophageal, gastrointestinal, and colorectal tissue samples from 1,772 patients, we conducted a comprehensive multi-omics analysis to reveal microbial signatures among various cancers and the potential mechanisms involved in tumor progression of head and neck squamous cell carcinoma (HNSC). We found that compared with other cancer types, the tumor-resident microbiome of HNSC accounted for the highest bacterial abundance and strongest association with host TME signatures. Fusobacterium was found to be enriched in HNSC tissues, which was associated with an increased inflammatory effect and inferior prognosis. Moreover, we revealed that the microbiota-associated inflammatory TME was attributed to the competing endogenouse RNA (ceRNA) network and chromatin accessibility.IMPORTANCE Studies on revealing the composition and potential mechanisms of the tumor microbiome are still at an initial stage. We uncovered the potential contribution of the tumor-resident microbiota on the immunosuppressive microenvironment in HNSC, which will provide a new perspective for tumor microbiome research and yield valuable insights into the clinical management of HNSC.

Sarcocystis sinensis in slaughtered cattle from Central of Iran

Sarcocystis is a food borne parasite as an intracellular parasite with two-host life cycle. Humans serve as either intermediate or definitive hosts among different species. Sarcocystosis is a very important disease because of its widespread prevalence with an impact on production and animal health. We aimed to assess S. sinensis in cattle from the desert area of Iran, using conventional PCR, targeting 18S rRNA. Also, the phylogenic analysis was done to mention isolate and the related ones in Genbank.From the 108 beef samples of heart, esophagus, intercostal muscle, diaphragm, and tongue harboring Sarcocystis spp. stored in the BioBank of the Research Center for Health and Food Safety, Shahid Sadoughi University of Medical Sciences, Yazd, Iran, DNA extraction was done. Amplification was done using the primer pair of SAR-F: 5'-TGGCTAATACATGCGCAAATA-3' and SAR-R: 5'-AACTTGAATGATCTATCGCCA-3'For the target of 18S rRNA gene. Sequencing was done. The interesting sequence was edited and then analyzed with BLAST. Then, multiple alignments were conducted using T-coffee. The mentioned sequence was submitted to GenBank in NCBI. Evolutionary analyses were conducted in MEGA7. In order to statistical analysis of the presence of Sarcocystis sinensis significantly with sex, age, and location, the Fisher’s Exact test was done using SPSS 16.0 (SPSS Inc. Chicago, USA). The p-value &lt; 0.05 was considered significant.Out of 108 samples, five isolates were identified as S. sinensis. The distribution of S. sinensis was related to age (p&lt;0.05) but had no significant difference with gender (p&gt;0.05). Phylogenic analysis showed a similarity of S. sinensis with S. hominis. From a food safety viewpoint, this study employed a molecular approach to identify S. sinensisin in cattle meat. To the best of our knowledge, this is the first report of S. sinensis in the desert area from Iran.

Pilot Study Showing Feasibility of Phosphoproteomic Profiling of Pathway-Level Molecular Alterations in Barrett's Esophagus.

(1) Background: Barrett’s esophagus is a major risk factor for esophageal adenocarcinoma. In this pilot study, we employed precision mass spectrometry to map global (phospho)protein perturbations in Barrett’s esophagus lesions and adjacent normal tissue to glean insights into disease progression. (2) Methods: Biopsies were collected from two small but independent cohorts. Comparative analyses were performed between Barrett’s esophagus samples and adjacent matched (normal) tissues from patients with known pathology, while specimens from healthy patients served as additional controls. (3) Results: We identified and quantified 6810 proteins and 6395 phosphosites in the discovery cohort, revealing hundreds of statistically significant differences in protein abundances and phosphorylation states. We identified a robust proteomic signature that accurately classified the disease status of samples from the independent patient cohorts. Pathway-level analysis of the phosphoproteomic profiles revealed the dysregulation of specific cellular processes, including DNA repair, in Barrett’s esophagus relative to paired controls. Comparative analysis with previously published transcriptomic profiles provided independent evidence in support of these preliminary findings. (4) Conclusions: This pilot study establishes the feasibility of using unbiased quantitative phosphoproteomics to identify molecular perturbations associated with disease progression in Barrett’s esophagus to define potentially clinically actionable targets warranting further assessment.

Do the Nonlinear Dynamic Acoustic Measurements, Nonlinear Energy Difference Ratio and Spectrum Convergence Ratio, Correlate with Perceptual Evaluation of Esophageal Voice Speakers?

The acoustic assessment of phonation after total laryngectomy is challenged by signal aperiodicity which makes frequency-based acoustic measures less reliable. This is important for patients who use esophageal voice since voice samples mostly include type III (highly aperiodic) and 4 (chaotic) signals. As such, using non-linear measures, which are better suited for aperiodic phonation, may be useful to investigate the relationship between acoustic signal characteristics and perception of esophageal voice quality. This study aimed to investigate whether nonlinear dynamic acoustic methods, nonlinear energy difference Ratio (NEDR) and spectrum convergence ratio (SCR), were correlated with perceptual measures in subjects who used esophageal phonation. Thirty-one subjects who had undergone total laryngectomy and use esophageal voice as a rehabilitation method were included in this study. Expert and non-expert raters listened to the esophageal voice samples from the subjects and rated vowels and connected speech samples on a scale from 1 to 7 on dysphonia severity and intelligibility. In addition, non-linear acoustic analysis was performed to calculate NEDR and SCR. Analysis from the raters was compared to the non-linear acoustic analysis to find the correlation between the variables. There were no significant correlations between any of the non-linear acoustic measures NEDR and SCR and the perceptual ratings at the significance level of 0.05. Correlations were calculated for each acoustic measure among the expert raters and among the non-expert raters in both connected speech samples and sustained vowel fragments. In conclusion, the nonlinear dynamic acoustic analyses of spectrum convergence ratio and nonlinear energy difference ratio do not have a significant correlation with perceptual measures of esophageal voice.