Monitoring Treatment Response in Eosinophilic Esophagitis
Monitoring Treatment Response in Eosinophilic Esophagitis
This study was performed as a secondary analysis of a randomised, placebo-controlled, multicenter trial, which investigated budesonide in different formulations and dosages for short-term treatment of EoE. This trial was conducted in accordance with the ICH Guideline for Good Clinical Practice and was approved by the Ethics Committee of the Physician's Chamber Hamburg, Germany, as well as by the national ethics committees in the participating countries and was registered at ClinicalTrials.gov (Identifier: NCT02280616). Patients between 18 and 75 years of age with a confirmed clinicopathological diagnosis of active EoE were eligible and randomly received treatment with either budesonide effervescent tablets for orodispersible use 2 × 1 mg/day or 2 × 2 mg/day or oral viscous budesonide suspension 2 × 5 mL [0.4 mg/mL]/day or placebo for 14 days. The primary endpoint was the rate of histological remission defined as mean number of <16 eos/mm hpf at end-of-treatment.
Upper endoscopy was performed by board-certified gastroenterologists at baseline (day 0) and at the end-of-treatment (day 14). For assessment of an endoscopic intensity score, characteristic abnormalities such as white exudates, furrows, oedema, fixed rings, crepe paper sign, short-segment stenosis, long-distance stenosis were recorded and classified as either absent (0 points), mild (1 point), moderate (2 points) or severe (3 points). Thus, total scores ranged from 0 to 21 points. During endoscopies, two biopsies each from the distal, mid and proximal oesophagus preferably from visible lesions were obtained. The biopsy specimen were placed into separate tubes with neutral-pH-buffered 4% paraformaldehyde solution and sent to the central pathologist (MV or CB) for further analysis.
From the paraffin-embedded oesophageal biopsies 10 sections were cut for haematoxylin & eosin (HE) staining. Biopsies were assessed either by MV using an Olympus AX70 microscope (Olympus, Hamburg, Germany) with Olympus SWH 10x-H/26.5 ocular (field number: 26.5) and plan apochromat 40× objective, resulting in an area of microscopic field of 0.345 mm or by CB using a Zeiss-Axio Imager A1 microscope (Carl Zeiss, Jena, Germany) with Carl Zeiss Pl 10x/23ocular (field number: 23) and a Zeiss EC Plan-Neofluar 40× objective, resulting in an area of microscopic field of 0.260 mm. On each oesophageal biopsy specimen all levels were surveyed and the eosinophils in the most densely infiltrated area were counted in 5 hpf and the mean and peak number of eos/hpf and eos/mm hpf were calculated.
Symptoms were assessed by a dysphagia score, which has been previously described. This score assessed frequency of dysphagia ranging from none (0 points) to several times per day (4 points) as well as intensity of dysphagia ranging from unhindered swallowing (0 points) to long-lasting complete obstruction requiring endoscopic intervention (5 points). Thus, total scores ranged from 0 to 9 points.
Venous blood was drawn from the study subjects at the respective recruiting study site, immediately processed, and sent to the central laboratory (Spranger Lab, Ingolstadt, Germany). All samples were blinded to the treatment group status as well as to all other assessed parameters. Samples for peripheral blood count were collected in tubes containing potassium-ethylenediaminetetraacetic-acid (S-Monovette EDTA-K, Sarstedt, Nürnbrecht, Germany) and an automated haematology analyser at the central laboratory determined absolute eosinophil count. Samples for serum marker analysis were collected in tubes containing silicate-coated granules (S-Monovette Serum, Sarstedt). Serum was obtained by centrifugation at 1000 g for 10 min at 4 °C 30 min after blood collection. All serum samples were stored frozen at −20 °C until used for further analyses, which were carried out at the laboratories of the '2nd Medical Department, Klinikum rechts der Isar der TU München' and at the 'Department of Dermatology and Allergy Biederstein, TU München' in Munich, Germany respectively. Prior to analyses the serum samples were checked with respect to amount and quality and aliquots were taken for further measurement of each marker. Insufficient samples were excluded from further analyses. Serum-values of CCL-17, CCL-18 and CCL-26 were measured using a commercially available enzyme-linked immunosorbent assay (Quantikine kit; R&D Systems Europe, Bad Nauheim, Germany) per the manufacturer's instructions. Serum ECP- and MCT-values were measured using the ImmunoCap System (Phadia, Uppsala, Sweden). All samples were measured in duplicates.
For quantitative data mean and standard deviation (SD) are presented, qualitative data are summarised by absolute and relative frequencies. Comparisons of data were conducted using Student's t-test for quantitative data or Fisher's exact test for qualitative data. The association between quantitative measures was assessed by Spearman's rank correlation coefficient. ROC-AUC-analyses were used to investigate the capabilities of pre- and post-treatment values of each assessed marker to predict the primary end point of histological remission. Maximum sum of sensitivity and specificity were calculated by the Youden-index. A two-sided level of significance of 5% was used for each test. All analyses were performed using the software package sas V.9.3 (SAS Institute, Cary, NC, USA) Subjects without a measurement at baseline or at the end-of-treatment of a particular marker were excluded from all of the analyses of that marker.
Methods
Study Design and Study Population
This study was performed as a secondary analysis of a randomised, placebo-controlled, multicenter trial, which investigated budesonide in different formulations and dosages for short-term treatment of EoE. This trial was conducted in accordance with the ICH Guideline for Good Clinical Practice and was approved by the Ethics Committee of the Physician's Chamber Hamburg, Germany, as well as by the national ethics committees in the participating countries and was registered at ClinicalTrials.gov (Identifier: NCT02280616). Patients between 18 and 75 years of age with a confirmed clinicopathological diagnosis of active EoE were eligible and randomly received treatment with either budesonide effervescent tablets for orodispersible use 2 × 1 mg/day or 2 × 2 mg/day or oral viscous budesonide suspension 2 × 5 mL [0.4 mg/mL]/day or placebo for 14 days. The primary endpoint was the rate of histological remission defined as mean number of <16 eos/mm hpf at end-of-treatment.
Endoscopy
Upper endoscopy was performed by board-certified gastroenterologists at baseline (day 0) and at the end-of-treatment (day 14). For assessment of an endoscopic intensity score, characteristic abnormalities such as white exudates, furrows, oedema, fixed rings, crepe paper sign, short-segment stenosis, long-distance stenosis were recorded and classified as either absent (0 points), mild (1 point), moderate (2 points) or severe (3 points). Thus, total scores ranged from 0 to 21 points. During endoscopies, two biopsies each from the distal, mid and proximal oesophagus preferably from visible lesions were obtained. The biopsy specimen were placed into separate tubes with neutral-pH-buffered 4% paraformaldehyde solution and sent to the central pathologist (MV or CB) for further analysis.
Histology
From the paraffin-embedded oesophageal biopsies 10 sections were cut for haematoxylin & eosin (HE) staining. Biopsies were assessed either by MV using an Olympus AX70 microscope (Olympus, Hamburg, Germany) with Olympus SWH 10x-H/26.5 ocular (field number: 26.5) and plan apochromat 40× objective, resulting in an area of microscopic field of 0.345 mm or by CB using a Zeiss-Axio Imager A1 microscope (Carl Zeiss, Jena, Germany) with Carl Zeiss Pl 10x/23ocular (field number: 23) and a Zeiss EC Plan-Neofluar 40× objective, resulting in an area of microscopic field of 0.260 mm. On each oesophageal biopsy specimen all levels were surveyed and the eosinophils in the most densely infiltrated area were counted in 5 hpf and the mean and peak number of eos/hpf and eos/mm hpf were calculated.
Symptom Evaluation
Symptoms were assessed by a dysphagia score, which has been previously described. This score assessed frequency of dysphagia ranging from none (0 points) to several times per day (4 points) as well as intensity of dysphagia ranging from unhindered swallowing (0 points) to long-lasting complete obstruction requiring endoscopic intervention (5 points). Thus, total scores ranged from 0 to 9 points.
Biomarker Analyses
Venous blood was drawn from the study subjects at the respective recruiting study site, immediately processed, and sent to the central laboratory (Spranger Lab, Ingolstadt, Germany). All samples were blinded to the treatment group status as well as to all other assessed parameters. Samples for peripheral blood count were collected in tubes containing potassium-ethylenediaminetetraacetic-acid (S-Monovette EDTA-K, Sarstedt, Nürnbrecht, Germany) and an automated haematology analyser at the central laboratory determined absolute eosinophil count. Samples for serum marker analysis were collected in tubes containing silicate-coated granules (S-Monovette Serum, Sarstedt). Serum was obtained by centrifugation at 1000 g for 10 min at 4 °C 30 min after blood collection. All serum samples were stored frozen at −20 °C until used for further analyses, which were carried out at the laboratories of the '2nd Medical Department, Klinikum rechts der Isar der TU München' and at the 'Department of Dermatology and Allergy Biederstein, TU München' in Munich, Germany respectively. Prior to analyses the serum samples were checked with respect to amount and quality and aliquots were taken for further measurement of each marker. Insufficient samples were excluded from further analyses. Serum-values of CCL-17, CCL-18 and CCL-26 were measured using a commercially available enzyme-linked immunosorbent assay (Quantikine kit; R&D Systems Europe, Bad Nauheim, Germany) per the manufacturer's instructions. Serum ECP- and MCT-values were measured using the ImmunoCap System (Phadia, Uppsala, Sweden). All samples were measured in duplicates.
Statistical Analyses
For quantitative data mean and standard deviation (SD) are presented, qualitative data are summarised by absolute and relative frequencies. Comparisons of data were conducted using Student's t-test for quantitative data or Fisher's exact test for qualitative data. The association between quantitative measures was assessed by Spearman's rank correlation coefficient. ROC-AUC-analyses were used to investigate the capabilities of pre- and post-treatment values of each assessed marker to predict the primary end point of histological remission. Maximum sum of sensitivity and specificity were calculated by the Youden-index. A two-sided level of significance of 5% was used for each test. All analyses were performed using the software package sas V.9.3 (SAS Institute, Cary, NC, USA) Subjects without a measurement at baseline or at the end-of-treatment of a particular marker were excluded from all of the analyses of that marker.
