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The current paradigm for diagnosing eosinophilic esophagitis (EoE) requires the presence of symptoms of esophageal dysfunction, an esophageal biopsy with at least 15 eosinophils per high-power field (eos/hpf) after a course of a proton pump inhibitor (PPI), and exclusion of other potential causes of esophageal eosinophilia.1, 2 Although this definition has helped to provide more consistency in the field,3, 4 diagnosis of EoE remains challenging. Symptoms of esophageal dysfunction can be seen in multiple conditions, including gastroesophageal reflux disease and esophageal motility disorders, and esophageal eosinophilia, even at very high levels, is not specific for EoE.5, 6 Moreover, histological assessment of eosinophilia in clinical practice is fraught with problems related to tissue sampling, section thickness, field selection, degranulation, and even microscope used.3, 7 Finally, although endoscopic signs and biopsy findings can be highly suggestive, there are no pathognomonic signs of EoE and there is significant clinical overlap between EoE, gastroesophageal reflux disease, and other causes of dysphagia.8 Because of these issues, there has been significant research interest in optimizing the diagnosis of EoE, including clinical symptom scores,9, 10, 11, 12 immunohistochemical staining of esophageal biopsies,13, 14, 15, 16, 17 endoscopic severity scores,18, 19, 20 biomarkers,21, 22, 23, 24 and gene expression.25, 26, 27 Recently, analysis of the EoE transcriptome identified a panel of 94 differentially expressed genes that held promise for diagnosis of EoE.28 Genes were selected based on the degree to which they were upregulated or downregulated, their relation to EoE pathogenesis, and their involvement in pathways related to EoE inflammation, and the panel contains pro-inflammatory genes, epithelial/barrier function genes, and mast cell–related genes, among others.28, 29 The study by Wen et al.28 showed that a gene expression summary score was highly sensitive and specific for distinguishing EoE cases from non-EoE controls, but the score was primarily derived and validated in a pediatric population, with relatively few adults included in the study. In addition, although the score appeared to normalize in EoE cases after successful treatment, assessment of treatment response was not a major focus of that study, and the utility of this test has not been validated in an independent external population. The aim of this study was to evaluate whether a gene expression score could accurately differentiate adult EoE cases from non-EoE controls and to determine whether scores were affected by treatment for EoE. We used consensus guidelines as the gold standard to clinically diagnose EoE cases.1, 2 Cases were required to have at least one symptom of esophageal dysfunction, ≥15 eos/hpf on esophageal biopsy after an 8-week proton pump inhibitor trial (20–40 mg twice daily of any of the available agents, prescribed at the discretion of the clinician), and exclusion of other causes of esophageal eosinophilia. Controls were subjects who did not meet clinical or histological criteria for EoE after endoscopy and biopsy. Subjects with proton pump inhibitor–responsive esophageal eosinophilia were not included in this study based on prior data that gene expression profiling could not distinguish them from EoE cases.32 In addition, we included a set of patients who were “clinically challenging” from a diagnostic standpoint: they had ≥15 eos/hpf on esophageal biopsy but could not be readily classified as a case or control based on initial clinical presentation alone. In addition to biopsies obtained for histological assessment, we also collected additional biopsy samples that were labeled with a de-identified study number, masked as to case/control status, and stored at −80 °C RNA-later (Life Technologies/Thermo-Fisher Scientific, Grand Island, NY) for future use. This study utilized a single RNA-later-preserved biopsy from the mid-esophagus (10 cm above the gastroesophageal junction) for gene expression determination. The decision to use a single mid-esophageal biopsy was based on our prior work showing that gene expression in EoE and controls was similar throughout the esophagus.31 Patients diagnosed with EoE were treated as clinically indicated by their gastroenterologist. They could receive treatment with topical corticosteroids (either oval viscous budesonide 1 mg twice daily or fluticasone from a multi-dose inhaler, 880 mcg twice daily) for 8 weeks36, 37, 38 or dietary therapy with the six-food elimination diet for 6 weeks39, 40 based on personal preference. When the initial course of therapy was completed, patients had a repeat upper endoscopy with biopsy during which time a repeat set of esophageal biopsies was obtained using identical protocols as the baseline endoscopy. Using this expression data, a summary score was calculated using a previously established algorithm.28 Specifically, the Ct value of the housekeeping gene was subtracted from the Ct value of each gene of interest to acquire the ΔCT, and then the absolute values of the normalized gene Ct values were summed for each gene in the gene expression panel. Because the clinical, endoscopic, and histological features of EoE are non-specific, the diagnosis of EoE remains challenging. The description of a characteristic gene expression pattern in EoE patients, the EoE transcriptome,25, 26 was the foundation for the eventual development of a molecular strategy for diagnosis of EoE. A summary score based on a panel of 94 genes with differential expression in EoE compared with non-EoE controls showed great promise for diagnosis of EoE.28 Our study focused on the clinical utility of this gene expression summary score in adults and several results were notable. First, the score discriminated EoE cases from non-EoE controls with a high degree of accuracy, though not perfectly. Second, we were able to adapt a scoring system to provide clinically relevant probabilistic score ranges, rather than a single dichotomous threshold for diagnosis. Third, we showed that a positive gene score reliably normalizes after histological treatment response, suggesting that it may be used to assess treatment response in place of a set of biopsies obtained for histological analysis. Finally, there appeared to be some utility in applying the gene score to cases that were clinically indeterminate. There have been several studies that have examined gene expression in EoE, showing differences in individual genes or microRNAs between EoE cases and controls and that these individual markers could normalize with treatment.25, 26, 27, 28, 42, 43, 44, 45 However, the landmark paper by Wen et al.28 was the first to develop an overall gene score and then to go further by validating its use for EoE diagnosis. Although the score was primarily evaluated in a pediatric population with relatively few adults included, and while treatment response was not the main focus, the performance of this summary score was impressive. A cutoff of 333 perfectly distinguished EoE cases from non-EoE controls in the initial study phase (AUC=1.00) and was nearly perfect in the follow-up validation phase (AUC=0.97). Our results also showed an outstanding AUC (0.93). The main difference was that the threshold of 333, though good, was not perfect (κ=0.71) and some patients were misclassified. This discrepancy may be explained by methodology. Because this is the first large-scale external validation of the gene expression panel, it is not surprising that a cut point developed in a different population does not have identical performance characteristics. In addition, the prior study processed samples individually in a research laboratory, while the present study utilized a more automated process that could be used in the clinical setting. To address this, we defined ranges, based on the clinical probability of EoE, that maximized PPV and NPV and minimized indeterminate results. In addition, the test had promise in cases that had high levels of eosinophilia on biopsy but had a clinically indeterminate presentation, something that no study to date has examined. In addition to the test’s diagnostic utility, we were able to show that it might have an even stronger role in monitoring of therapy. Currently, there are few studies that systematically assess or define the “best” histologicalal threshold in EoE,41 clinical trials have substantial heterogeneity in the end points that have been used,5, 46 and guidelines do not recommend a specific cut point to monitor response.2 In practice, multiple biopsies are obtained from multiple locations throughout the esophagus, and the posttreatment peak eosinophil count is determined from review of these samples. Because eosinophilia is patchy,34, 47 there could be sampling error during this evaluation. In contrast, it appears that gene expression may be more consistent throughout the esophagus,31 and our results demonstrate that a single biopsy from a patient who had histological response also had normalization of a gene expression. It is therefore intriguing to speculate whether gene expression normalization might provide a more efficient and accurate way to define tissue response to treatment in EoE. However, future studies will need to assess other measures of response (symptoms, endoscopic findings, histological findings besides eosinophil count alone) to fully explore the use of this gene panel as a treatment outcome measure. Additionally, even with the data presented here, the role and availability of this test in clinical diagnostic and treatment monitoring algorithms must still be defined. As of now, there may not be a need for use in clear-cut cases, but because diagnostic features of EoE are not specific, the test may have the most value in settings of clinical uncertainty or when a baseline value is needed for subsequent treatment monitoring. This paper has some limitations to acknowledge. This study was conducted in adults at a single tertiary care referral center, so it is possible that results are not generalizable. However, the study design enrolled all patients undergoing endoscopy for symptoms of esophageal dysfunction, a population that is likely to be similar to that in many other endoscopic suites. In addition, the biopsy samples that we used were preserved in RNA-later. Were a gene panel to be employed in clinical practice, formalin-fixed paraffin-embedded (FFPE) samples would most likely be used. The same gene expression panel has been shown to have good discriminative ability in FFPE samples, but we did not perform external validation, and this additional validation as well as confirmation of the proposed score ranges from this paper must still be explored for RNA derived from FFPE. Moreover, external validation of the cut points proposed in this study would also be required at different centers and in different populations. Although we included a patient population with high eosinophil counts and clinically indeterminate presentations, the clinical care and follow-up testing of these patients were not standardized, so those results should be interpreted with caution. The indeterminate patients also represented a relatively small sample, and in a number of cases a careful clinical evaluation could yield a correct diagnosis without using a gene expression test. In addition, our study cannot address the issue of whether gene expression can replace standard histological analysis in EoE diagnostic algorithms, and future research will need to elucidate the best way to utilize a test that could “rule in EoE”, in contrast to an eosinophil count that is non-specific. The strengths of the study include the rigorous prospective design, meticulous sample handling that was identical for all subjects, and obtaining samples before case/control status was assigned. Moreover, this large case/control population was independent from the population used to develop and initially validate the test, the laboratory was masked to case/control status when gene scores were determined, and samples were processed and run with the same set of equipment that would be used in a clinical setting. In conclusion, this prospective cohort study showed that a gene expression profile run on a single esophageal biopsy had an excellent ability to discriminate EoE cases from non-EoE controls, and we were able to generate probabilistic score ranges with high PPV and NPVs while minimizing samples with intermediate scores. In addition, patients with marked esophageal eosinophilia but with an indeterminate initial clinical presentation were able to be categorized as unlikely to have EoE based on higher gene scores. Although this test could be used in clinical algorithms, its exact place in these algorithms and utility above standard histological analysis for diagnosis is still exploratory and yet to be determined. Finally, because the gene scores were responsive to treatment and normalized in patients who also had histological response, it is possible that in the future a single esophageal biopsy posttreatment could be used to define tissue response in EoE.  Guarantor of the article: Evan S. Dellon, MD, MPH. Specific author contributions: Evan S. Dellon: project conception/design, obtained funding, data analysis/interpretation, drafting of the article, critical revision, approved final draft; Ranjitha Veerappan: processed and analyzed specimens, data analysis/interpretation, critical revision, approved final draft; Sara R. Selitsky and Joel S. Parker: data analysis/interpretation, critical revision, approved final draft; RoseMary Beitia and Leana L. Higgins: patient recruitment, data collection, critical revision, approved final draft; Robert M. Genta: project conception, data interpretation, critical revision, approved final draft; Richard H. Lash: project conception, data interpretation, critical revision, approved final draft. Financial support: This work was supported, in part, by NIH Awards K23DK090073 (E.S.D) and an investigator-initiated research grant from Miraca Life Sciences (E.S.D.) and uses resources from the UNC Center for GI Biology and Disease (P30DK34987) and the UNC Translational Pathology laboratory (P30CA016086). Potential competing interests: Dr Veerappan, Dr Genta, and Dr Lash are employees of Miraca Life Sciences. The other authors declare no conflict of interest. Supplementary Information accompanies this paper on the Clinical and Translational Gastroenterology website

A Gene Expression Panel is Accurate for Diagnosis and Monitoring Treatment of Eosinophilic Esophagitis in Adults