The oesophageal string test: a novel, minimally invasive method measures mucosal inflammation in eosinophilic oesophagitis

Abstract

Objective: Eosinophil predominant inflammation characterises histological features of eosinophilic oesophagitis (EoE). Endoscopy with biopsy is currently the only method to assess oesophageal mucosal inflammation in EoE. We hypothesised that measurements of luminal eosinophil-derived proteins would correlate with oesophageal mucosal inflammation in children with EoE.

Design: The Enterotest diagnostic device was used to develop an oesophageal string test (EST) as a minimally invasive clinical device. EST samples and oesophageal mucosal biopsies were obtained from children undergoing upper endoscopy for clinically defined indications. Eosinophil-derived proteins including eosinophil secondary granule proteins (major basic protein-1, eosinophil-derived neurotoxin, eosinophil cationic protein, eosinophil peroxidase) and Charcot-Leyden crystal protein/galectin-10 were measured by ELISA in luminal effluents eluted from ESTs and extracts of mucosal biopsies.

Results: ESTs were performed in 41 children with active EoE (n=14), EoE in remission (n=8), gastro-oesophageal reflux disease (n=4) and controls with normal oesophagus (n=15). EST measurement of eosinophil-derived protein biomarkers significantly distinguished between children with active EoE, treated EoE in remission, gastro-oesophageal reflux disease and normal oesophagus. Levels of luminal eosinophil-derived proteins in EST samples significantly correlated with peak and mean oesophageal eosinophils/high power field (HPF), eosinophil peroxidase indices and levels of the same eosinophil-derived proteins in extracts of oesophageal biopsies.

Conclusions: The presence of eosinophil-derived proteins in luminal secretions is reflective of mucosal inflammation in children with EoE. The EST is a novel, minimally invasive device for measuring oesophageal eosinophilic inflammation in children with EoE.

Keywords: Eosinophil; allergy; breast milk; childhood nutrition; enterotest string test; epithelial barrier; gastrointestinal tract; inflammation; oesophageal disease; oesophageal disorders; oesophageal lesions; oesophageal reflux; oesophageal strictures; oesophagitis; pediatric.

Figure 1

Eosinophil-derived protein levels in oesophageal biopsy and oesophageal string test (EST) samples. Oesophageal mucosal biopsies concentrations of (A1) major basic protein-1 (MBP1), (A2) eosinophil-derived neurotoxin (EDN), (A3) eosinophil cationic protein (ECP), (A4) eosinophil peroxidase (EPX) and (A5) Charcot–Leyden crystal protein/galectin-10 (CLC/Gal-10). Eosinophil secondary granule proteins and CLC/Gal-10 as measured by ELISA are shown in samples from subject groups (eosinophilic oesophagitis (EoE) active—black bars, EoE treated—light gray bars, gastro-oesophageal reflux disease (GORD)—black bars, normal—white bars). Results are reported as ng of eosinophil protein per mg of total protein in the biopsy extract. EST concentrations of (B1) MBP1, (B2) EDN, (B3) ECP (B4) EPX and (B5) CLC/Gal-10 from the same subject groups are reported as ng of eosinophil protein per ml of EST supernatant. Biomarker levels were compared across groups using ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS, not significant.

Figure 2

Correlation of eosinophil-derived proteins in oesophageal string test samples with eosinophil counts in oesophageal mucosal biopsy samples. Spearman analyses correlating oesophageal string test sample eosinophil-derived protein levels with peak (panel A, 1–5) and mean (panel B, 1–5) eosinophil counts were performed. Spearman rank correlation coefficients (r) and associated p values are shown. EoE-no Rx (untreated—active disease) (solid circles), EoE-Rx (treated—in remission) (open circles), GORD (open triangles) and normal oesophagus (open squares). CLC/Gal-10, Charcot–Leyden crystal protein/galectin-10; ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; EoE, eosinophilic oesophagitis; EPX, eosinophil peroxidase; GORD, gastro-oesophageal reflux disease; MBP1, major basic protein-1.

Figure 3

Correlation of eosinophil-derived protein concentrations in luminal oesophageal string test (EST) samples with eosinophil-derived proteins in oesophageal mucosal biopsy samples. (A) major basic protein-1 (MBP1), (B) eosinophil-derived neurotoxin (EDN), (C) eosinophil cationic protein (ECP), (D) eosinophil peroxidase (EPX) and (E) Charcot–Leyden crystal protein/galectin-10 (CLC/Gal-10) concentrations in EST and mucosal biopsy extracts were correlated using Spearman analyses (rank correlation coefficients (r) and associated p values). Eosinophilic oesophagitis (EoE)-no Rx (untreated—active disease) (solid circles), EoE-Rx (treated—in remission) (open circles), gastro-oesophageal reflux disease (GORD) (open triangles) and normal oesophagus (open squares).

Figure 4

Receiver operating characteristic (ROC) curves for the diagnosis of eosinophilic oesophagitis with biopsies and oesophageal string test (EST) sampling of eosinophil-derived proteins. ROC sensitivity versus specificity curves (A) are shown for biopsies (left panels) and ESTs (right panels) for measurements of major basic protein-1 (MBP1) and Charcot–Leyden crystal protein/galectin-10 (CLC/Gal-10) in biopsy extracts and EST supernatants. C-statistics (the area under the ROC curve) are indicated as a measure of the discriminating ability of each biomarker. (B) C-values for the ROC curves for biopsies and EST samples for other eosinophil secondary granule proteins are presented here and graphically in online supplementary figure 6. ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; EPX, eosinophil peroxidase.