RNAseq analyses of Esophageal biopsies from proton pump inhibitor (PPI)-responsive and PPI-unresponsive Pediatric patients with Eosinophilic Esophagitis.

Clinical trials have identified two distinct eosinophilic esophagitis (EoE) treatment phenotypes, proton pump inhibitor (PPI)-responsive (PPI-R) and PPI-unresponsive (PPI-UR). Herein, we performed clinical, endoscopic, and histologic evaluation of esophageal biopsies from pediatric PPI-R and PPI-UR EoE individuals prior to PPI therapy (diagnosis) and following PPI trial. RNAseq analyses of esophageal biopsy samples revealed common immune and inflammatory transcriptional signatures in both PPI-R EoE and PPI-UR EoE at diagnosis and distinct signatures enriched for processes related to neuropeptide signaling and cell cycle and division. PPI therapy induced histologic, endoscopic, and transcriptional remission in PPI-R EoE, but not in PPI-UR EoE. Persistent disease in PPI-UR EoE was associated with the presence of Th2 inflammatory and dedifferentiated esophageal epithelial transcriptomic signatures, while PPI-R EoE revealed genes enriched in cellular responses to LPS, host defense to viruses and type-I interferon signaling. In silico analyses identified common and unique EoE disease gene drivers in PPI-R and PPI-UR EoE. These studies indicate that the two EoE phenotypes have unique transcriptomic elements that underlie the molecular nature of PPI-R and PPI-UR EoE disease. Experimental Design Description: This study employed RNA sequencing to investigate transcriptomic differences in esophageal tissue from pediatric patients with eosinophilic esophagitis (EoE) and their response to proton pump inhibitor (PPI) therapy. Esophageal biopsies were collected from pediatric patients presenting with EoE symptoms who underwent esophagogastroduodenoscopy (EGD) with biopsies at the Mount Sinai Center for Eosinophilic Disorders. Study Groups: PPI-Responsive (PPI-R): EoE patients with symptom resolution and eosinophil counts reduced to <15/HPF following high-dose PPI treatment (n=5). PPI-Unresponsive (PPI-UR): EoE patients who did not meet response criteria after PPI treatment (n=5) Controls: Patients with 0 esophageal eosinophils/HPF, normal endoscopy, and no esophageal or gastrointestinal pathology (n=5). Sample Size: The study included 15 pediatric patients across three groups, with 5 biological replicates per condition to ensure adequate statistical power for differential gene expression analysis. Inclusion/Exclusion Criteria: EoE diagnosis required symptoms of esophageal dysfunction and ≥15 eosinophils per high-power field (HPF) per consensus guidelines. All EoE patients received standardized high-dose omeprazole or lansoprazole treatment (1-2 mg/kg/day up to adult dose twice daily) according to current pediatric and adult guidelines. Patients with concurrent gastrointestinal diseases, additional therapies, or dietary changes during the study period were excluded. Sample Collection and Processing: Esophageal biopsies were immediately preserved in RNALater solution for transcriptomic analysis. RNA sequencing was performed using Illumina platform with stringent quality control measures (RNA Integrity Number >8). Bioinformatics Pipeline: Raw sequencing reads underwent quality assessment using FASTQC and were processed with Trimmomatic for adapter removal and quality trimming. Processed reads were aligned to the human reference genome (GRCh38) using HiSAT2 aligner, followed by gene quantification using feature-counts. Duplicate sample validation confirmed >99% genome alignment identity and >98% sequencing depth consistency across replicates. Statistical Analysis: Differential gene expression analysis was conducted using DESeq2 with statistical significance defined as adjusted p-value ≤0.05 and absolute log2 fold change >1. Study Population: The cohort included both male and female pediatric patients. Reflecting the known male predominance in EoE, males comprised 60% of the study population.