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Accuracy of digital mRNA profiling of oesophageal biopsies as a novel diagnostic approach to eosinophilic oesophagitis.

ABSTRACT: Quantification of tissue eosinophils remains the golden standard in diagnosing eosinophilic oesophagitis (EoE), but this approach suffers from poor specificity. It has been recognized that histopathological changes that occur in patients with EoE are associated with a disease-specific tissue transcriptome.We hypothesized that digital mRNA profiling targeted at a set of EoE-specific and Th2 inflammatory genes in oesophageal biopsies could help differentiate patients with EoE from those with reflux oesophagitis (RE) or normal tissue histology (NH).The mRNA expression levels of 79 target genes were defined in both proximal and distal biopsies of 196 patients with nCounter® (Nanostring) technology. According to clinicopathological diagnosis, these patients were grouped in a training set (35 EoE, 30 RE, 30 NH) for building of a three-class prediction model using the random forest method, and a blinded predictive set (n = 47) for model validation.A diagnostic model built on ten differentially expressed genes was able to differentiate with 100% sensitivity and specificity between conditions in the training set. In a blinded predictive set, this model was able to correctly predict EoE in 14 of 18 patients in distal (sensitivity 78%, 95% CI 52-93%) and 16 of 18 patients in proximal biopsies (sensitivity 89%, 95% CI 64-98%), without false-positive diagnosis of EoE in RE or NH patients (specificity 100%, 95% CI 85-100%). Sensitivity was increased to 94% (95% CI 71-100%) when either the best predictive distal or proximal biopsy was used.We conclude that mRNA profiling of oesophageal tissue is an accurate diagnostic strategy in detecting EoE.

SUBMITTER: Lexmond WS

PROVIDER: S-EPMC4773201 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Accuracy of digital mRNA profiling of oesophageal biopsies as a novel diagnostic approach to eosinophilic oesophagitis.

Lexmond Willem S WS Hu Lan L Pardo Michael M Heinz Nicole N Rooney Katharine K LaRosa Jessica J Dehlink Eleonora E Fiebiger Edda E Nurko Samuel S

Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 20150801 8

<h4>Background</h4>Quantification of tissue eosinophils remains the golden standard in diagnosing eosinophilic oesophagitis (EoE), but this approach suffers from poor specificity. It has been recognized that histopathological changes that occur in patients with EoE are associated with a disease-specific tissue transcriptome.<h4>Objective</h4>We hypothesized that digital mRNA profiling targeted at a set of EoE-specific and Th2 inflammatory genes in oesophageal biopsies could help differentiate pa ...[more]

PMID: 25728460

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Project description:ObjectiveThe influence of eosinophilic oesophagitis (EoE)-associated inflammation upon oesophageal epithelial biology remains poorly understood. We investigated the functional role of autophagy in oesophageal epithelial cells (keratinocytes) exposed to the inflammatory EoE milieu.DesignFunctional consequences of genetic or pharmacological autophagy inhibition were assessed in endoscopic oesophageal biopsies, human oesophageal keratinocytes, single cell-derived ex vivo murine oesophageal organoids as well as a murine model recapitulating EoE-like inflammation and basal cell hyperplasia. Gene expression, morphological and functional characterisation of autophagy and oxidative stress were performed by transmission electron microscopy, immunostaining, immunoblotting, live cell imaging and flow cytometry.ResultsEoE-relevant inflammatory conditions promoted autophagy and basal cell hyperplasia in three independent murine EoE models and oesophageal organoids. Inhibition of autophagic flux via chloroquine treatment augmented basal cell hyperplasia in these model systems. Oesophageal keratinocytes stimulated with EoE-relevant cytokines, including tumour necrosis factor-α and interleukin-13 exhibited activation of autophagic flux in a reactive oxygen species-dependent manner. Autophagy inhibition via chloroquine treatment or depletion of Beclin-1 or ATG-7, augmented oxidative stress induced by EoE-relevant stimuli in murine EoE, oesophageal organoids and human oesophageal keratinocytes. Oesophageal epithelia of paediatric EoE patients with active inflammation displayed increased autophagic vesicle content compared with normal and EoE remission subjects. Functional flow cytometric analysis revealed autophagic flux in human oesophageal biopsies.ConclusionsOur findings reveal for the first time that autophagy may function as a cytoprotective mechanism to maintain epithelial redox balance and homeostasis under EoE inflammation-associated stress, providing mechanistic insights into the role of autophagy in EoE pathogenesis.

Project description:BackgroundThe characterization of miRNAs and their target mRNAs involved in regulation of the immune process is an area of intense research and relatively little is known governing these processes in allergic inflammation. Here we present novel findings defining the miRNA and mRNA transcriptome in eosinophilic esophagitis (EoE), an increasing recognized allergic disorder.MethodsEsophageal epithelial miRNA and mRNA from five paired biopsies pre- and post-treatment with glucocorticosteroids were profiled using Taqman and Affymetrix arrays. Validation was performed on additional paired biopsies, untreated EoE specimens and normal controls. Differentially regulated miRNAs and mRNAs were generated, within which miRNA-mRNA target pairs with high predicted confidence were identified.ResultsCompared to the post-glucocorticoid treated esophageal mucosa, of all the 377 miRNA sequences examined, 32 miRNAs were significantly upregulated and four downregulated in the pre-treated biopsies. MiR-214 was the most upregulated (150 fold) and miR-146b-5b, 146a, 145, 142-3p and 21 were upregulated by at least 10 fold. Out of 12 miRNAs chosen for validation by qRT-PCR, five (miR-214, 146b-5p, 146a, 142-3p and 21) were confirmed and 11 shared the same trend. When the expression of the 12 miRNAs in the EoE mucosa was compared to unrelated normal mucosa, six (miR-214, 146b-5p, 146a, 21, 203, and 489) showed similar significant changes as in the paired samples and 10 of them shared the same trend. In the same five pairs of samples used to profile miRNA, 311 mRNAs were down-regulated and 35 were up-regulated in pre-treated EoE mucosa. Among them, 164 mRNAs were identified as potential targets of differentially regulated miRNAs. Further analysis revealed that immune-related genes, targeted and non-targeted by miRNAs, were among the most important genes involved in the pathogenesis of EoE.ConclusionsOur findings add to the accumulating body of data defining a regulatory role for miRNA in immune and allergic processes.

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Accuracy of digital mRNA profiling of oesophageal biopsies as a novel diagnostic approach to eosinophilic oesophagitis.

Publications

Accuracy of digital mRNA profiling of oesophageal biopsies as a novel diagnostic approach to eosinophilic oesophagitis.

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