Spencer Rusin

Markers of Eosinophilic Inflammation for Diagnosis of Eosinophilic Esophagitis and Proton Pump Inhibitor–Responsive Esophageal Eosinophilia: A Prospective Study

BACKGROUND & AIMS Distinguishing between eosinophilic esophagitis (EoE), gastroesophageal reflux disease, and proton pump inhibitor-responsive esophageal eosinophilia (PPI-REE) is challenging. We assessed whether immunohistochemical analysis of esophageal tissues for major basic protein (MBP), eotaxin-3, and tryptase can be used for diagnosis of EoE and to differentiate EoE from PPI-REE. METHODS We conducted a prospective study of 196 consecutive adults who underwent outpatient endoscopy at the University of North Carolina from 2009 through 2012. Incident cases of EoE were diagnosed per consensus guidelines. Patients with gastroesophageal reflux disease or dysphagia served as controls. PPI-REE was defined as a symptomatic and histologic response to a PPI. Immunohistochemistry was performed to quantify MBP, eotaxin-3, and tryptase. The maximum density of epithelial staining was determined for each assay; levels were compared between EoE and control groups and then EoE and PPI-REE groups, and receiver operating characteristic curves were constructed. RESULTS Esophageal tissues from patients with EoE (n = 50) had a median 951 MBP-positive cells/mm(2), whereas those from controls (n = 123) had a median 2 MBP-positive cells/mm(2) (P < .001). Samples from patients with EoE had a median 155 eotaxin-3-positive cells/mm(2), and those from controls (n = 123) had 18 eotaxin-3-positive cells/mm(2) (P < .001). Samples from patients with EoE had a median 249 tryptase-positive cells/mm(2), and those from controls (n = 123) had 11 tryptase-positive cells/mm(2) (P < .001). Levels of MBP, eotaxin-3, tryptase, and the combination of all 3 identified patients with EoE with area under the receiver operating characteristic curve values of 0.99, 0.94, 0.99, and 1.00. Analyses of only samples with eosinophil counts of 10-100 eosinophils per high-power field produced similar results. No marker distinguished EoE from PPI-REE. Esophageal tissues from patients with PPI-REE (n = 23) had 987 MBP-positive cells/mm(2) (P = .18, compared with EoE), 160 eotaxin-3-positive cells/mm(2) (P = .33), and 243 tryptase-positive cells/mm(2) (P = .28). CONCLUSIONS Esophageal tissues from patients with EoE have substantially higher levels of MBP, eotaxin-3, and tryptase than controls on the basis of immunohistochemical analysis. Assays for the 3 markers identify patients with EoE with 100% accuracy but cannot distinguish EoE from PPI-REE.

Utility of a Noninvasive Serum Biomarker Panel for Diagnosis and Monitoring of Eosinophilic Esophagitis: A Prospective Study.

OBJECTIVES:Noninvasive biomarkers would be valuable for diagnosis and monitoring of eosinophilic esophagitis (EoE). The aim of this study was to determine the utility of a panel of serum biomarkers for the diagnosis and management of EoE.METHODS:We conducted a prospective cohort study of consecutive adults undergoing outpatient esophagogastroduodenoscopy. Incident cases of EoE were diagnosed per consensus guidelines; controls had gastroesophageal reflux disease (GERD) or dysphagia and did not meet the EoE criteria. EoE cases were treated with topical steroids and had repeat endoscopy. Pre- and post-treatment serum samples were analyzed in a blinded manner for interleukin (IL)-4, IL-5, IL-6, IL-9, IL-13, transforming growth factor (TGF)-α, TGF-β, tumor necrosis factor-α, eotaxin-1, -2, and -3, thymic stromal lymphopoietin (TSLP), major basic protein, and eosinophil-derived neurotoxin. Cases and controls were compared at baseline, and pre- and post-treatment assays were compared in cases.RESULTS:A total of 61 incident EoE cases and 87 controls were enrolled; 51 EoE cases had post-treatment serum analyzed. There were no significant differences in any of the biomarkers between EoE cases and controls at baseline. IL-13 and eotaxin-3 for cases and controls were 85±160 vs. 43±161 pg/ml (P=0.12) and 41±159 vs. 21±73 (P=0.30). There were no significant differences in assay values among cases before and after treatment. There were also no differences after stratification by atopic status or treatment response.CONCLUSIONS:A panel of inflammatory factors known to be associated with EoE pathogenesis were not increased in the serum, nor were they responsive to therapy. None of these biomarkers are likely candidates for a serum test for EoE. Histologic analysis for diagnosis and management of EoE continues to be necessary, and novel, less invasive, biomarkers are needed.

Distribution and variability of esophageal eosinophilia in patients undergoing upper endoscopy

The variability of eosinophilic infiltrates in eosinophilic esophagitis is not well described. This study aimed to determine the distribution of esophageal eosinophilia and the utility of histologic cut-points for eosinophilic esophagitis diagnosis in subjects undergoing endoscopy. We performed a prospective study of adults undergoing outpatient endoscopy. Research protocol esophageal biopsies were obtained from all subjects. Incident cases of eosinophilic esophagitis were diagnosed per consensus guidelines. Biopsies were interpreted following a validated protocol, and maximum eosinophil counts (eosinophils per high-power field; eos/hpf) were determined. Histologic analyses were performed on a per-patient, per-biopsy, and per-hpf basis. There were 213 patients, yielding 923 esophageal biopsies with 4588 hpfs. Overall, 48 patients (23%), 165 biopsy fragments (18%), and 449 hpfs (10%) had ≥15 eos/hpf; most subjects had no or low levels of eosinophils. In the eosinophilic esophagitis cases, 119 biopsy fragments (63%) and 332 hpfs (36%) had ≥15 eos/hpf. There was a mean 104-fold difference between the lowest and highest hpf eosinophil count for the eosinophilic esophagitis patients; 85% of the biopsies from eosinophilic esophagitis cases also had at least one hpf with <15 eos/hpf. The cut-point of 15 eos/hpf had a sensitivity of 100% and a specificity of 96% for diagnosis of eosinophilic esophagitis. In conclusion, most patients have little to no esophageal eosinophilia. In patients with eosinophilic esophagitis, there was marked variability in the eosinophil counts by biopsy and by hpf within a given biopsy. Additionally, the 15 eos/hpf cut-point was highly sensitive and specific for eosinophilic esophagitis. Multiple esophageal biopsies from different locations should be obtained to optimize eosinophilic esophagitis diagnosis.

A Clinical Prediction Tool Identifies Cases of Eosinophilic Esophagitis Without Endoscopic Biopsy: A Prospective Study

OBJECTIVES:Eosinophilic esophagitis (EoE) is difficult to distinguish from gastroesophageal reflux (GERD) and other causes of dysphagia. We assessed the utility of a set of clinical and endoscopic features for predicting EoE without obtaining esophageal biopsies.METHODS:We prospectively enrolled consecutive adults undergoing outpatient upper endoscopy at the University of North Carolina from July 2011 through December 2013. Incident cases of EoE were diagnosed per consensus guidelines. Non-EoE controls had either GERD- or dysphagia-predominant symptoms. A predictive model containing clinical and endoscopic, but no histological, data was assessed. Receiver operator characteristic curves were constructed and the area under the curve (AUC) was calculated.RESULTS:A total of 81 EoE cases (mean age 38 years; 60% male; 93% white; 141 eosinophils per high-power field (eos/hpf)) and 144 controls (mean age 52, 38% male; 82% white; 3 eos/hpf) were enrolled. A combination of clinical (age, sex, dysphagia, food allergy) and endoscopic (rings, furrows, plaques, hiatal hernia) features was highly predictive of EoE. The AUC was 0.944, with sensitivity, specificity, and accuracy of 84, 97, and 92%. Similar values were seen after limiting controls to those with only reflux or dysphagia or to those with esophageal eosinophilia not due to EoE.CONCLUSIONS:We validated a set of clinical and endoscopic features to predict EoE with a high degree of accuracy and allow identification of those at very low risk of disease. Use of these predictors at the point-of-care will avoid the effort and expense of low-yield histological examinations for EoE.

Optimal Histologic Cutpoints for Treatment Response in Patients With Eosinophilic Esophagitis: Analysis of Data From a Prospective Cohort Study

BACKGROUND AND AIMS: No prospective studies substantiate 15 eos/hpf as an appropriate endpoint for treatment of eosinophilic esophagitis (EoE). We aimed to determine a histologic cutpoint that identifies successful treatment of EoE by assessing symptomatic and endoscopic improvement. METHODS: We performed a prospective cohort study of 62 consecutive adult patients undergoing outpatient esophagogastroduodenoscopy at the University of North Carolina from 2009 through 2014. At diagnosis of EoE and after 8 weeks of standard treatment, symptom and endoscopic responses were measured using a visual analogue scale and an endoscopic severity score (ESS), and eosinophil counts were assessed. Receiver operator curves and logistic regression models evaluated the histologic threshold that best predicted symptomatic and endoscopic response. For symptoms, analysis was limited to patients without baseline esophageal dilation. RESULTS: The mean eosinophil count at diagnosis was 124 eos/hpf, falling to 35 eos/hpf after treatment. The mean visual analogue scale decreased from 3.4 at baseline to 1.7 after treatment, and the mean ESS decreased from 3 to 1.6. Twenty‐nine patients had symptom responses (47%) and 34 had endoscopic responses (55%). Post‐treatment eosinophil count thresholds of 8, 15, and 5 eos/hpf best predicted symptom, endoscopic and combined responses, respectively. On logistic regression, decreasing eosinophil count was significantly associated with the probability of symptomatic (P = .01) and endoscopic response (P < .001). CONCLUSIONS: In a prospective study of patients with EoE, we found that a cutpoint of <15 eos/hpf identifies most patients with symptom and endoscopic improvements, providing support for the current diagnostic threshold. A lower threshold (<5 eos/hpf) identifies most patients with a combination of symptom and endoscopic responses; this cutpoint might be used in situations that require a stringent histologic threshold.

Prospective assessment of serum periostin as a biomarker for diagnosis and monitoring of eosinophilic oesophagitis

Periostin is highly expressed in eosinophilic oesophagitis (EoE), but has not been extensively studied as a non‐invasive biomarker.

Determination of esophageal eosinophil counts and other histologic features of eosinophilic esophagitis by pathology trainees is highly accurate

Many studies of eosinophilic esophagitis (EoE) use expert pathology review, but it is unknown whether less experienced pathologists can reliably assess EoE histology. We aimed to determine whether trainee pathologists can accurately quantify esophageal eosinophil counts and identify associated histologic features of EoE, as compared with expert pathologists. We used a set of 40 digitized slides from patients with varying degrees of esophageal eosinophilia. Each of 6 trainee pathologists underwent a teaching session and used our validated protocol to determine eosinophil counts and associated EoE findings. The same slides had previously been evaluated by expert pathologists, and these results comprised the criterion standard. Eosinophil counts were correlated, and agreement was calculated for the diagnostic threshold of 15 eosinophils per high-power field as well as for associated EoE findings. Peak eosinophil counts were highly correlated between the trainees and the criterion standard (ρ ranged from 0.87 to 0.92; P<.001 for all). Peak counts were also highly correlated between trainees (0.75-0.91; P<.001), and results were similar for mean counts. Agreement was excellent for determining if a count exceeded the diagnostic threshold (κ ranged from 0.83 to 0.89; P<.001). Agreement was very good for eosinophil degranulation (κ = 0.54-0.83; P<.01) and spongiosis (κ = 0.44-0.87; P<.01) but was lower for eosinophil microabscesses (κ = 0.37-0.64; P<.01). In conclusion, using a teaching session, digitized slide set, and validated protocol, the agreement between pathology trainees and expert pathologists for determining eosinophil counts was excellent. Agreement was very good for eosinophil degranulation and spongiosis but less so for microabscesses.

Clinical and Molecular Factors Associated With Histologic Response to Topical Steroid Treatment in Patients With Eosinophilic Esophagitis

BACKGROUND & AIMS Few factors have been identified that can be used to predict response of patients with eosinophilic esophagitis (EoE) to topical steroid treatment. We aimed to determine whether baseline clinical, endoscopic, histologic, and molecular features of EoE can be used to predict histologic response. METHODS We collected data from 97 patients with EoE, from 2009 through 2015, treated with a topical steroid for 8 weeks; 59 patients had a histologic response to treatment. Baseline clinicopathologic features and gene expression patterns were compared between patients with a histologic response to treatment (<15 eos/hpf) and non‐responders (≥15 eos/hpf). We performed sensitivity analyses for alternative histologic response definitions. Multivariate logistic regression was performed to identify predictive factors associated with response to therapy, which were assessed with area under the receiver operator characteristic (AUROC) curves. RESULTS Baseline dilation was the only independent predictor of non‐response (odds ratio [OR], 0.30; 95% CI, 0.10–0.89). When an alternate response (<1 eos/hpf) and non‐response (<50% decrease in baseline eos/hpf) definition was used, independent predictors of response status were age (OR, 1.08; 95% CI, 1.02–1.14), food allergies (OR, 12.95; 95% CI, 2.20–76.15), baseline dilation (OR, 0.17; 95% CI, 0.03–0.88), edema or decreased vascularity (OR, 0.20; 95% CI, 0.04–1.03), and hiatal hernia (OR, 0.07; 95% CI, 0.01–0.66). Using these 5 factors, we developed a predictive model that discriminated complete responders from non‐responders with an AUROC of 0.88. Baseline gene expression patterns were not associated with treatment response and did not change with different histologic response thresholds. CONCLUSIONS In an analysis of 97 patients with EoE, we found dilation to be the only baseline factor associated with non‐response to steroid treatment (<15 eos/hpf). However, a model comprising 5 clinical, endoscopic, and histologic factors identified patients with a complete response (<1 eos/hpf). A baseline gene expression panel was not predictive of treatment response at any threshold.

Su1112 A Set of Clinical and Endoscopic Features Predicts the Presence of Eosinophilic Esophagitis With High Accuracy, Allowing Omission of Low-Yield Biopsies: Results From a Prospective Study

increasing in the population especially in pediatrics and reach 1:1000 in diseases such as EoE (Eosinophilic Esophagitis) (Lee 2013). CD24 is a small, mucin-like glycoprotein that functions both in signal transduction and as an adhesion molecule (Sagiv, 2008). CD24 is expressed on many hematopoietic cells, non-hematopoietic cells and malignancies (Fang, 2010). Previous studies in eosinophils have suggested CD24 as an IL-5-upregulated gene that is involved in eosinophil apoptosis and survival (Temple, 2011). The role evoked for CD24 in IL-5-induced survival of eosinophils remains to be investigated. We hypothesize that CD24 is a key molecular checkpoint, regulating eosinophil functions in chronic mucosal GI diseases, all of which are characterized by significant eosinophil infiltration. Aim: 1) To assess the expression of CD24 in eosinophils in the GI tract in wild-type (WT) mice; 2) To assess the level of eosinophils in the GI tract in CD24 knockout (-/-) and to compare it to WT mice.Methods: We have previously established a colony of CD24-/mice, on a C57BL/ 6J genetic background, and their WT littermates at our local animal facility. The levels of eosinophils were assessed in WT and CD24-/mice. Bone marrow (BM) cells were collected, and grown in medium containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5). On day 13 the cells were counted and levels of eosinophils were assessed by FACS. The levels of eosinophil and CD24 expression in different tissues were also evaluated. Results: Eosinophil counts in BM extracted from mice were significantly (p-value=0.0436) higher in CD24-/compared to WT mice, (Fig. 1). Fluorescense intensity was stronger in CD24-/compared to WT mice even though there were no differences in the eosinophil population of mature cells (Fig 2). Eosinophil count was higher in BM from CD24-/compared to WT mice, and lower in blood and addipose tissue (Fig. 3). CD24 expression was higher in peripheral tissues compared to the blood and BM (Fig 4). Conclusion: Based on our preliminary results, it is suggested that CD24 has an important role in negatively regulating eosinophil proliferation/maturation/chemotaxis. Understanding the role of eosinophil in GI disorders may have a large imapct on our understanding of the EGIDs. Targeting specific proteins that play an important role in these processes may lead to novel therapeutic approaches

Su1113 In Search of a Non-Invasive Blood Test for Diagnosis and Monitoring of EoE: A Prospective Study of a Serum Biomarker Panel

increasing in the population especially in pediatrics and reach 1:1000 in diseases such as EoE (Eosinophilic Esophagitis) (Lee 2013). CD24 is a small, mucin-like glycoprotein that functions both in signal transduction and as an adhesion molecule (Sagiv, 2008). CD24 is expressed on many hematopoietic cells, non-hematopoietic cells and malignancies (Fang, 2010). Previous studies in eosinophils have suggested CD24 as an IL-5-upregulated gene that is involved in eosinophil apoptosis and survival (Temple, 2011). The role evoked for CD24 in IL-5-induced survival of eosinophils remains to be investigated. We hypothesize that CD24 is a key molecular checkpoint, regulating eosinophil functions in chronic mucosal GI diseases, all of which are characterized by significant eosinophil infiltration. Aim: 1) To assess the expression of CD24 in eosinophils in the GI tract in wild-type (WT) mice; 2) To assess the level of eosinophils in the GI tract in CD24 knockout (-/-) and to compare it to WT mice.Methods: We have previously established a colony of CD24-/mice, on a C57BL/ 6J genetic background, and their WT littermates at our local animal facility. The levels of eosinophils were assessed in WT and CD24-/mice. Bone marrow (BM) cells were collected, and grown in medium containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5). On day 13 the cells were counted and levels of eosinophils were assessed by FACS. The levels of eosinophil and CD24 expression in different tissues were also evaluated. Results: Eosinophil counts in BM extracted from mice were significantly (p-value=0.0436) higher in CD24-/compared to WT mice, (Fig. 1). Fluorescense intensity was stronger in CD24-/compared to WT mice even though there were no differences in the eosinophil population of mature cells (Fig 2). Eosinophil count was higher in BM from CD24-/compared to WT mice, and lower in blood and addipose tissue (Fig. 3). CD24 expression was higher in peripheral tissues compared to the blood and BM (Fig 4). Conclusion: Based on our preliminary results, it is suggested that CD24 has an important role in negatively regulating eosinophil proliferation/maturation/chemotaxis. Understanding the role of eosinophil in GI disorders may have a large imapct on our understanding of the EGIDs. Targeting specific proteins that play an important role in these processes may lead to novel therapeutic approaches