MicroRNAs in plasma-derived extracellular vesicles as non-invasive biomarkers for eosinophilic esophagitis

Background: The lack of non-invasive biomarkers imposes the dependence on endoscopy with biopsies for the diagnosis and monitoring of eosinophilic esophagitis (EoE), a prevalent chronic inflammation of the esophagus mediated by a type 2 immune response. We aimed to identify potential non-invasive biomarkers using microRNAs (miRNAs) in plasma-derived extracellular vesicles (pEVs). Methods: This is a prospective single-center observational study including a discovery cohort of EoE patients (n=26) with active disease (EoE.Basal) and after anti-inflammatory treatment (EoE.Post.tx), and control subjects (n=16). Small-RNA-seq of RNA-pEVs was performed to identify differentially regulated small-RNAs (sRNAs) in EoE.Basal vs. controls and EoE.Basal vs. EoE.Post.tx comparisons. Candidate miRNAs were validated in an independent cohort (EoE patients, n=33; controls, n=14), and the diagnostic potential of miRNAs-pairs was assessed. Results: the pEVs-sRNA cargo differed among controls, EoE.Basal and EoE.Post.tx conditions. Compared with controls, Ser_Comb_22, Leu_Comb_5, miR-10b-5p and miR-125a-5 were upregulated in EoE.Basal, and miR-224-5p, miR-221-3p, let-7d-5p and miR-191-5p were downregulated. Combination of miR-221-3p and miR-10b-5p showed the best diagnostic performance in discovery (AUC=0.87) and validation (AUC=0.70) cohorts. Comparing EoE.Basal and EoE.Post.tx paired samples, miR-374a-5p and miR-30a-3p were upregulated in EoE.Basal, while miR-15a-5p and let-7d-5p were downregulated. Here, combination of mi-30a-3p and miR-15a-5p showed AUC values of 0.90 and 0.71 in discovery and validation cohorts respectively. MiR-30a-3p remained highly upregulated in EoE.Basal when compared to EoE.Post.tx in the validation cohort (p=0.001). Conclusions: The sRNA cargo of pEVs is related to the inflammatory activity in EoE. This study pioneers the use of pEVs as a non-invasive biomarker for EoE. To investigate the potential of extracellular vesicle (EVs) dervied small-RNA as non-invasive biomarker for EoE we obtained plasma samples from control subjects, and paired samples from patients with EoE with active inflammation (EoE.Basal) and after succesful anti-inflammatory treatment (EoE.Post.tx). Total RNA from plasma EVs of participants was subjected to small-RNAseq to identify differentially regulated smallRNAs, and performed two comparisons: 1) unpaired analysis for comparing controls with EoE.Basal samples to identify potential diagnostic biomarkers, and 2) paired analysis for EoE.Basal and EoE.Post.tx. Identified small-RNAs, to identify potential monitoring biomarkers. Data from sRNA-seq was analyzed to identfy microRNA and transference fragment RNAs (tRFs). Finally, Finally, was used to best combinations of two miRNAs were selected to discriminate between the groups compared.