A single biopsy is valid for genetic diagnosis of eosinophilic esophagitis regardless of tissue preservation or location in the esophagus.
ABSTRACT: A new gene expression profile test may distinguish eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD), but the optimal tissue preparation and biopsy location are unknown. We aimed to determine if formalin-fixed paraffin-embedded (FFPE) and RNA-later (RNAL) preserved specimens from newly diagnosed EoE patients have equivalent gene expression scores and whether scores vary by esophageal biopsy location.We analyzed prospectively collected and banked esophageal biopsies from EoE patients and GERD controls. Paired FFPE and RNAL samples from the distal, mid, and proximal esophagus were used. RNA was extracted, and gene expression for a previously constructed 96 gene panel was quantified with a summary expression score. Scores were compared between EoE and GERD patients, between FFPE and RNAL samples, and between the different esophageal locations.A total of 72 samples, representing paired FFPE and RNAL specimens from 9 EoE cases and 3 GERD controls, were analyzed. Overall median gene expression scores were similar between FFPE and RNAL (238 vs 227; p=0.64), correlation was excellent between FFPE and RNAL (Spearman's rho=0.90; p<0.001), and there were no differences by biopsy level. Median gene scores distinguished EoE from controls (134 vs 402; p=0.02), and overall agreement between preservation methods and EoE case status was perfect (kappa=1.0; p<0.001).Gene expression scores were equivalent in FFPE and RNAL, and were also similar across three esophageal locations. This implies that a single biopsy in either FFPE or RNAL from anywhere in the esophagus may have the potential for genetic diagnosis of EoE.
Project description:The Pediatric Eosinophilic Esophagitis Symptom Score (PEESS v2.0) measures patient-relevant outcomes. However, whether patient-identified domains (dysphagia, gastroesophageal reflux disease [GERD], nausea/vomiting, and pain) align with clinical symptomology and histopathologic and molecular features of eosinophilic esophagitis (EoE) is unclear.The purpose of this study was to determine whether clinical features of EoE, measured through PEESS v2.0, associate with histopathologic and molecular features of EoE. This represents a novel approach for analysis of allergic diseases, given the availability of allergic tissue biopsy specimens.We systematically recruited treated and untreated pediatric patients with EoE (aged 2-18 years) and examined parent proxy-reported symptoms using the PEESS v2.0. Clinical symptomology was collected by questionnaire. Esophageal biopsy samples were quantified for levels of eosinophils, eosinophil peroxidase (EPX) immunohistochemical staining, and mast cells. Molecular features were assessed by using the EoE Diagnostic Panel (94 EoE-related gene transcripts). Associations between domain scores and clinical symptoms and biological features were analyzed with Wilcoxon rank sum and Spearman correlation.The PEESS v2.0 domains correlated to specific parent-reported symptoms: dysphagia (P = .0012), GERD (P = .0001), and nausea/vomiting (P < .0001). Pain correlated with multiple symptoms (P < .0005). Dysphagia correlated most strongly with overall histopathology, particularly in the proximal esophagus (P ? .0049). Markers of esophageal activity (EPX) were significantly associated with dysphagia (strongest r = 0.37, P = .02). Eosinophil levels were more associated with pain (r = 0.27, P = .06) than dysphagia (r = 0.24, P = .13). The dysphagia domain correlated most with esophageal gene transcript levels, predominantly with mast cell-specific genes.We have (1) established a validated, parent proxy-reported measure for pediatric EoE, the PEESS v2.0; (2) verified that the parent proxy effectively captures symptoms; (3) determined that the dysphagia domain most closely aligns with symptoms and tissue-based molecular biomarkers; (4) established that symptoms correlate with EPX staining; and (5) observed association between mast cells and dysphagia.
Project description:RNA sequencing analysis of proximal and distal esophageal biopsies from a small cohort of EoE, GERD, and control patients was performed to identify differentially expressed genes. Overall design: Proximal and distal esophageal biopsies from three patients with EoE, GERD, and control esophagus, 18 samples total
Project description:BACKGROUND & AIMS:Diagnostic testing for chronic esophageal disorders relies on histopathology analysis of biopsies or uncomfortable transnasal catheters or wireless pH monitoring, which capture abnormal intraluminal refluxate. We therefore developed a balloon mucosal impedance (MI) catheter system that instantly detects changes in esophageal mucosal integrity during endoscopy over a long segment of the esophagus. We performed a prospective study to evaluate the ability of a balloon-incorporated MI catheter to detect and evaluate esophageal disorders, including gastroesophageal reflux disease (GERD) and eosinophilic esophagitis (EoE). METHODS:We performed a prospective study of 69 patients undergoing esophagogastroduodenoscopy with or without wireless pH monitoring. Patients were classified as having GERD (erosive esophagitis or abnormal pH; n = 24), EoE (confirmed with pathology analysis of tissues from both distal and proximal esophagus; n = 21), or non-GERD (normal results from esophagogastroduodenoscopy and pH tests; n = 24). Receiver operating characteristic curves and area under the operating characteristic curve (AUC) were used to compare the accuracy of balloon MI in diagnosis. Probabilities of assignment to each group (GERD, non-GERD, or EoE) were estimated using multinomial logistic regression. Association between MI patterns and diagnoses were validated using data from patients seen at 3 separate institutions. RESULTS:MI pattern along the esophageal axis differed significantly (P < .01) among patients with GERD, EoE, and non-GERD. Patients with non-GERD had higher MI values along all measured segments. The MI pattern for GERD was easily distinguished from that of EoE: in patients with GERD, MI values were low in the distal esophagus and normalized along the proximal esophagus, whereas in patients with EoE, measurements were low in all segments of the esophagus. Intercept and rate of rise of MI value (slope) as distance increased from the squamocolumnar junction identified patients with GERD with an AUC = 0.67, patients with EoE with an AUC = 0.84, and patients with non-GERD with an AUC = 0.83 in the development cohort. One patient had an adverse event (reported mild chest pain after the procedure) and was discharged from the hospital without further events. CONCLUSIONS:We developed a balloon MI catheter system that instantly detects changes in esophageal mucosal integrity during endoscopy and found it to be safe and able to identify patients with GERD, EoE, or non-GERD. We validated our findings in a separate cohort for patients. ClinicalTrials.gov ID NCT03103789.
Project description:Eosinophilic esophagitis (EoE) is an allergy-mediated disease culminating in severe eosinophilic inflammation and dysfunction of the esophagus. This chronic disorder of the esophagus causes significant morbidity, poor quality of life, and complications involving fibrosis and esophageal remodeling. Overlapping features between EoE and gastroesophageal reflux disease (GERD) pose great challenges to differentiating the two conditions, although the two disorders are not mutually exclusive. Recent findings suggest that the confounding condition proton pump inhibitor - responsive esophageal eosinophilia (PPI-REE) is likely a subset of EoE. Since PPIs have therapeutic properties that can benefit EoE, PPIs should be considered as a therapeutic option for EoE rather than a diagnostic screen to differentiate GERD, PPI-REE, and EoE. Other current treatments include dietary therapy, corticosteroids, and dilation. Immunomodulators and biologic agents might have therapeutic value, and larger trials are needed to assess efficacy and safety. Understanding the pathophysiology of EoE is critical to the development of novel therapeutics.
Project description:A key question in the allergy field is to understand how tissue-specific disease is manifested. Eosinophilic esophagitis (EoE) is an emerging tissue-specific allergic disease with an unclear pathogenesis.Herein we tested the hypothesis that a defect in tissue-specific esophageal genes is an integral part of EoE pathogenesis.We interrogated the pattern of expression of esophagus-specific signature genes derived from the Human Protein Atlas in the EoE transcriptome and in EPC2 esophageal epithelial cells. Western blotting and immunofluorescence were used for evaluating expression of esophageal proteins in biopsy specimens from control subjects and patients with active EoE. Whole-exome sequencing was performed to identify mutations in esophagus-specific genes.We found that approximately 39% of the esophagus-specific transcripts were altered in patients with EoE, with approximately 90% being downregulated. The majority of transcriptional changes observed in esophagus-specific genes were reproduced in vitro in esophageal epithelial cells differentiated in the presence of IL-13. Functional enrichment analysis revealed keratinization and differentiation as the most affected biological processes and identified IL-1 cytokines and serine peptidase inhibitors as the most dysregulated esophagus-specific protein families in patients with EoE. Accordingly, biopsy specimens from patients with EoE evidenced a profound loss of tissue differentiation, decreased expression of keratin 4 (KRT4) and cornulin (CRNN), and increased expression of KRT5 and KRT14. Whole-exome sequencing of 33 unrelated patients with EoE revealed 39 rare mutations in 18 esophagus-specific differentially expressed genes.A tissue-centered analysis has revealed a profound loss of esophageal tissue differentiation (identity) as an integral and specific part of the pathophysiology of EoE and implicated protease- and IL-1-related activities as putative central pathways in disease pathogenesis.
Project description:BACKGROUND AND AIMS:Subepithelial fibrosis in eosinophilic esophagitis (EoE) can be detected only in esophageal biopsy specimens with adequate amounts of lamina propria (LP). We investigated how often pediatric esophageal biopsy specimens contain adequate LP, and whether esophageal eosinophilia influences the acquisition rates. METHODS:We evaluated 284 esophageal biopsy specimens from 39 patients with EoE, and 87 biopsy specimens from 32 patients without esophageal eosinophilia or other esophageal abnormalities for the presence of adequate LP and fibrosis. RESULTS:On a per biopsy specimen basis, there was no significant difference in the rate of procuring adequate amounts of LP between patients with EoE and patients without esophageal eosinophilia (43% vs 31%, P = .14). Eighty-five percent of patients with EoE had fibrosis. Fibrosis in patients with EoE was patchy and more likely to be detected in the middle or distal esophagus (odds ratio, 19.93; 95% confidence interval, 4.12-91.52). Among patients with fibrosis, the probability of its detection reached >95% with 7 middle-distal esophageal biopsy specimens. Most children with newly diagnosed EoE already had subepithelial fibrosis despite exhibiting only inflammatory endoscopic features. CONCLUSIONS:Most individual esophageal biopsy specimens in children are inadequate for assessing subepithelial fibrosis, and the rates of procuring adequate LP per biopsy specimen are similar in patients with and without EoE. To reliably detect fibrosis in patients with EoE, at least 7 biopsy specimens should be taken from the middle-distal esophagus. The finding of fibrosis in children with newly diagnosed EoE and only inflammatory endoscopic features suggests that fibrosis can occur early in this disease.
Project description:<h4>Background and aims</h4>The prospective, observational Swiss Eosinophilic Esophagitis Cohort Study (SEECS) was set up in 2015 with the following goals in mind: (1) to provide up-to-date epidemiologic data; (2) to assess the appropriateness of care; (3) to evaluate the psychosocial impact; and (4) to foster translational research projects. Data capture relies on validated instruments to assess disease activity and focuses on epidemiologic variables and biosamples (esophageal biopsies and blood specimens). An annual inclusion of 70 new patients with eosinophilic esophagitis (EoE) or proton pump inhibitor-responsive esophageal eosinophilia (PPI-REE) is intended. We herein describe the SEECS cohort profile.<h4>Methods</h4>The SEECS includes adult patients (age ?18 years) with EoE or PPI-REE diagnosed according to published criteria. After inclusion, the patients are typically seen once a year for a clinical and endoscopic/histologic follow-up examination. Data are captured using validated questionnaires. Biosamples from patients with gastroesophageal reflux disease (GERD) and controls with a healthy esophagus are collected as well.<h4>Results</h4>From January 2016 to July 2017, a total of 111 patients with EoE and 10 patients with PPI-REE were recruited. In addition, esophageal biopsies and blood samples from 11 patients with GERD and 20 controls with a healthy esophagus were collected. The mean age of the patients with EoE and those with PPI-REE was 39.6 ± 12.9 and 44.6 ± 15.6 years, respectively. A male predominance was found among both the patients with EoE (77.5%) and those with PPI-REE (70%). Concomitant allergic disorders were found in 79.3% of the patients with EoE and 90% of the patients with PPI-REE. At inclusion, the EoE patients were treated with the following therapeutic regimens: no therapy (0.9%), PPI (36%), swallowed topical corticosteroids (82.9%), elimination diets (15.3%), and esophageal dilation (19.8%).<h4>Conclusions</h4>The SEECS is the first national cohort study of patients with EoE or PPI-REE. The SEECS will provide up-to-date epidemiologic data and foster translational research projects.
Project description:It is not clear why only a minority of patients with gastroesophageal reflux disease (GERD) develop Barrett's esophagus. We hypothesized that differences among individuals in molecular pathways activated when esophageal squamous epithelium is exposed to reflux underlie the development of Barrett's metaplasia.We used esophageal squamous cell lines from patients who had GERD with Barrett's esophagus (normal esophageal squamous [NES]-B3T and NES-B10T) and without Barrett's esophagus (NES-G2T and NES-G4T) to study effects of acid and bile salts on expression of the CDX2 gene. Bay 11-705, Ad5 inhibitor kappaB(IkappaB)alpha-SR, and site-directed mutagenesis were used to explore effects of nuclear factor-kappaB (NF-kappaB) inhibition on CDX2 promoter activity; DNA binding of the NF-kappaB subunits p50 and p65 was assessed by chromatin immune-precipitation.Acid and bile salts increased CDX2 messenger RNA (mRNA), protein, and promoter activity in NES-B3T and NES-B10T cells, but not in NES-G2T or NES-G4T cells. Inhibition of NF-kappaB abolished the increase in CDX2 promoter activity. Increased CDX2 promoter activity was associated with nuclear translocation of p50, which bound to the promoter. We found CDX2 mRNA in 7 of 10 esophageal squamous biopsy specimens from patients with Barrett's esophagus, but in only 1 of 10 such specimens from patients who had GERD without Barrett's esophagus.Acid and bile salts induce CDX2 mRNA and protein expression in esophageal squamous cells from patients with Barrett's esophagus, but not from GERD patients without Barrett's esophagus. We speculate that these differences in acid- and bile salt-induced activation of molecular pathways may underlie the development of Barrett's metaplasia.
Project description: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.
Project description:<h4>Background & aims</h4>Gastroesophageal reflux causes inflammation and intestinal metaplasia and its downstream sequelum adenocarcinoma in the distal esophagus. The incidence of esophageal adenocarcinoma has increased approximately 6-fold in the United States since the 1970s, accompanied with a significant increase in the prevalence of gastroesophageal reflux disease (GERD). Despite extensive epidemiologic study, the cause for GERD and the unexpected increases remain unexplainable. Microbes are among the environmental factors that may contribute to the etiology of GERD, but very little research has been done on the esophageal microbiome, particularly in its relation to GERD. This is the first comprehensive reported correlation between a change in the esophageal microbiome and esophageal diseases.<h4>Methods</h4>Biopsy samples of the distal esophagus were collected from 34 patients. Host phenotypes were histologically defined as normal, esophagitis, or Barrett's esophagus (intestinal metaplasia). Microbiomes from the biopsy samples were analyzed by bacterial 16S ribosomal RNA gene survey and classified into types using unsupervised cluster analysis and phenotype-guided analyses. Independence between host phenotypes and microbiome types were analyzed by Fisher exact test.<h4>Results</h4>Esophageal microbiomes can be classified into 2 types. The type I microbiome was dominated by the genus Streptococcus and concentrated in the phenotypically normal esophagus. Conversely, the type II microbiome contained a greater proportion of gram-negative anaerobes/microaerophiles and primarily correlated with esophagitis (odds ratio, 15.4) and Barrett's esophagus (odds ratio, 16.5).<h4>Conclusions</h4>In the human distal esophagus, inflammation and intestinal metaplasia are associated with global alteration of the microbiome. These findings raise the issue of a possible role for dysbiosis in the pathogenesis of reflux-related disorders.