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Efficacy, Dose Reduction, and Resistance to High-Dose Fluticasone in Patients With Eosinophilic Esophagitis

      Background & Aims

      We evaluated the efficacy and safety of high-dose swallowed fluticasone propionate (FP) and dose reduction in patients with eosinophilic esophagitis (EoE) and analyzed esophageal transcriptomes to identify mechanisms.

      Methods

      We conducted a randomized, multisite, double-blind, placebo-controlled trial of daily 1760 mcg FP in participants age 3–30 years with active EoE. Twenty-eight participants received FP, and 14 participants received placebo. After 3 months, participants given FP who were in complete remission (CR) received 880 mcg FP daily, and participants in the FP or placebo groups who were not in CR continued or started, respectively, 1760 mcg FP daily for 3 additional months. The primary end point was histologic evidence for CR. Secondary end points were partial remission (PR), symptoms, compliance, esophageal gene expression, esophageal eosinophil count, and the relationship between clinical features and FP responsiveness.

      Results

      After 3 months, 65% of subjects given FP and no subjects given placebo were in CR (P = .0001); 12% of those given FP and 8% of those given placebo were in PR. In the FP group, 73% of subjects remained in CR, and 20% were in PR after the daily dose was reduced by 50%. Extending FP therapy in FP-resistant participants did not induce remission. FP decreased heartburn severity (P = .041). Compliance, age, sex, atopic status, or anthropomorphic features were not associated with response to FP. Gene expression patterns in esophageal tissues of FP responders were similar to those of patients without EoE; there was evidence for heterogeneous steroid signaling in subjects who did not respond to FP and preliminary evidence for transcripts predictive of FP responsiveness.

      Conclusions

      Daily administration of a high dose of FP induces histologic remission in 65%–77% of patients with EoE after 3 months. A 50% dose reduction remained effective in 73%–93% of patients who initially responded to FP. Nonresponders had evidence of steroid resistance; histologic and molecular markers may predict resistance. Clinicaltrials.gov number: NCT00426283.

      Keywords

      Abbreviations used in this paper:

      AE (adverse event), BMI (body mass index), CCHMC (Cincinnati Children's Hospital Medical Center), CFR (Code of Federal Regulations), CR (complete remission), EDP (Eosinophilic Esophagitis Diagnostic Panel), EoE (eosinophilic esophagitis), FP (fluticasone propionate), HPF (high-power field), PPI (proton-pump inhibitor), PR (partial remission)
      See related article, Wolf WA et al, on page 1272 in CGH.
      Eosinophilic esophagitis (EoE) is an emerging immune-mediated disease characterized by intense eosinophil infiltration of the esophageal mucosal epithelium that is refractory to acid-suppressive therapy and often is associated with significant tissue remodeling.
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      Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis.
      The disease has a strong hereditary component with a large sibling risk ratio (∼80 fold),
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      and early genetic analyses have identified susceptibility loci in regions containing candidate genes that are expressed in epithelial cells and strongly implicated in regulating antigen recognition (thymic stromal lymphopoietin) and inflammatory cell recruitment and activation (CCL26, eotaxin-3).
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      Common variants at 5q22 associate with pediatric eosinophilic esophagitis.
      The most effective medicine for the treatment of EoE is off-label use of topical steroids such as fluticasone propionate (FP) and budesonide.
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      Long-term budesonide maintenance treatment is partially effective for patients with eosinophilic esophagitis.
      Faubion et al
      • Faubion Jr., W.A.
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      • et al.
      Treatment of eosinophilic esophagitis with inhaled corticosteroids.
      showed that swallowed FP was effective and safe for treating EoE in 4 pediatric patients. Konikoff et al
      • Konikoff M.R.
      • Noel R.J.
      • Blanchard C.
      • et al.
      A randomized, double-blind, placebo-controlled trial of fluticasone propionate for pediatric eosinophilic esophagitis.
      showed complete remission in 50% of patients with EoE after a 3-month treatment course of 880 mcg daily swallowed FP compared with 9% in the placebo group. Potential theories for this apparent lack of response to FP in half of patients include poor compliance, the existence of a steroid-resistant phenotype, and inadequate FP dosing. It is notable that polymorphisms in the transforming growth factor-β gene are associated with steroid resistance in EoE.
      • Aceves S.S.
      • Newbury R.O.
      • Chen D.
      • et al.
      Resolution of remodeling in eosinophilic esophagitis correlates with epithelial response to topical corticosteroids.
      In addition, responsiveness to FP has been reported to correlate negatively with younger patient age, shorter height, and lower weight.
      • Konikoff M.R.
      • Noel R.J.
      • Blanchard C.
      • et al.
      A randomized, double-blind, placebo-controlled trial of fluticasone propionate for pediatric eosinophilic esophagitis.
      • Noel R.J.
      • Putnam P.E.
      • Collins M.H.
      • et al.
      Clinical and immunopathologic effects of swallowed fluticasone for eosinophilic esophagitis.
      In vitro, cardinal components of the EoE transcriptome including interleukin 13-induced eotaxin-3 in esophageal epithelial cells are inhibited by glucocorticoids in a dose-dependent manner.
      • Blanchard C.
      • Mingler M.K.
      • Vicario M.
      • et al.
      IL-13 involvement in eosinophilic esophagitis: transcriptome analysis and reversibility with glucocorticoids.
      Notably, asthma has parallels with the type of inflammation seen in EoE, and higher dosages of glucocorticoids are more effective than lower dosages in patients with refractory asthma. In addition, we have noted in clinical practice that several patients who did not respond to 880 mcg/day FP responded to a higher dose of 1760 mcg/day FP. Therefore, in this study, we aimed to determine the efficacy and safety of a high FP dosage (1760 mcg/day) in inducing remission, the effect of prolonging high-dose FP therapy in FP-resistant EoE, and the ability to maintain disease remission while reducing FP dosage (880 mcg/day) in FP-responsive EoE. Furthermore, we analyzed esophageal transcriptomes to identify mechanisms involved in remission status.

      Materials and Methods

       Participants

      This study was initiated at Cincinnati Children's Hospital Medical Center (CCHMC) on December 12, 2006. Subsequently, the University of Utah, the Colorado Children's Hospital, and the Children's Hospital of Philadelphia were added as study sites. All participants underwent allergy testing and either completed a 3-month elimination diet or refused such diet treatment. Participants were age 3–30 years and were required to have an upper endoscopy (ie, esophagogastroduodenoscopy) that showed active EoE at the time of screening. Active EoE was defined as 24 or more eosinophils/high-power field (HPF) in the proximal or distal esophagus while being treated with a proton-pump inhibitor (PPI) for at least 2 months or having a negative pH probe. Participants were both newly diagnosed and relapsed EoE patients. Potential participants were excluded if there was a history of poor tolerance to FP, an inability to use a metered-dose inhaler, concurrent or recent use of systemic corticosteroids, comorbid eosinophilic disorders, or a diagnosis of or being at risk for diabetes (type I or type II). The study was approved by the institutional review board at each institution, and assent and/or informed consent were obtained. The study was conducted in compliance with the International Conference on Harmonization Good Clinical Practice E6 guidelines, and the applicable regulatory requirements, including the US Code of Federal Regulations (45 CFR 46, 21 CFR 50, 21 CFR 56, 21 CFR 312). All authors had access to the study data and have reviewed and approved the final manuscript. Participants were instructed not to change PPI dosage and/or diet therapy during the study.

       Study Design

      This 6-month study was a double-blind, placebo-controlled, randomized trial to determine the safety and efficacy of daily high-dose (1760 mcg) FP in participants with EoE. Participants who met inclusion criteria were assigned randomly to either high-dose FP, 1760 mcg divided into 2 daily 880-mcg doses, or placebo for 3 months (phase 1). Two thirds of the participants were assigned to the FP group. The placebo generously was provided by GlaxoSmithKline. Randomization was performed by members of the CCHMC investigational pharmacy. The randomization scheme was generated by using the website Randomization.com (available: http://www.randomization.com). Participants were assigned to a single treatment by using a method of randomly permuted blocks. The allocation sequence was known only by the CCHMC pharmacist, and the site pharmacist was made aware of individual assignments only after enrollment/assignment. To maintain blinding, only 1 staff member, in addition to the pharmacy staff, at each site was unblinded. This individual was unblinded to the individual participant randomization after the first 3 months to stratify each participant for the following 3 months. After all participants completed the study and the database was finalized, team members were unblinded for data analysis purposes.
      After 3 months, participants underwent an esophagogastroduodenoscopy. Participants assigned to FP who were in complete remission (CR), 1 or less eosinophil/HPF in the proximal and distal esophagus (FP responders), were assigned to receive half the dosage of FP (1 daily 880-mcg dose) for 3 additional months (phase 2). FP-treated participants who had more than 1 eosinophil/HPF after 3 months (FP nonresponders) were assigned to continue receiving the high dose of FP (1760 mcg, 2 daily 880-mcg doses) for 3 additional months. Placebo-treated participants who had more than 1 eosinophil/HPF at month 3 were assigned to receive high-dose FP (1760 mcg, 2 daily 880-mcg doses) for 3 additional months. Placebo-treated participants who had 1 or fewer eosinophil/HPF after 3 months were to discontinue placebo treatment and the study. Participants completed a semivalidated EoE symptom score,
      • Pentiuk S.
      • Putnam P.E.
      • Collins M.H.
      • et al.
      Dissociation between symptoms and histological severity in pediatric eosinophilic esophagitis.
      which included both frequency and severity questions, at the beginning of the study and after 3 months (phase 1) and 6 months (phase 2) (see the Supplementary Materials and Methods section for more detail).

       Molecular Gene Expression Profiling by EoE Diagnostic Panel

      We chose 94 representative EoE genes as the molecular foundation of the EoE Diagnostic Panel (EDP).
      • Wen T.
      • Stucke E.M.
      • Grotjan T.M.
      • et al.
      Molecular diagnosis of eosinophilic esophagitis by gene expression profiling.
      Among these 94 genes, 77 served as definitive diagnostic and treatment assessment genes. RNA extracted from fresh distal biopsy specimens at CCHMC was reverse-transcribed into complementary DNA, which was subjected to TaqMan quantitative polymerase chain reaction amplification of the 94 genes using a 7900HT quantitative polymerase chain reaction amplification system (Applied Biosciences, Grand Island, NY).

       Statistical Analysis

      Demographic and participant characteristic data were summarized within each treatment group using the frequency and percentage for categoric data and the mean and standard deviation or the median and interquartile range for continuous data. Treatment groups were compared using the Fisher exact test for categoric data and the Wilcoxon rank-sum test for continuous data.

       Analysis of 3-Month Data

      Based on the study by Konikoff et al,
      • Konikoff M.R.
      • Noel R.J.
      • Blanchard C.
      • et al.
      A randomized, double-blind, placebo-controlled trial of fluticasone propionate for pediatric eosinophilic esophagitis.
      it was estimated that the study participants would have a 75% response rate to 1760 mcg/day FP and a 10% response rate to daily placebo. Under these assumptions, a Fisher exact test with a 0.050 two-sided significance level would have 95% power to detect the difference between the FP and placebo group if at least 39 subjects completed the study (26 FP, 13 placebo). The primary end point for this study was remission at 3 months (categoric outcome) and was summarized using frequencies and percentages, which were compared between treatment groups using the Fisher exact test. At the interim analysis, the test was conducted at an α value of .01, and at the final analysis, the test was conducted at an α value of .04, to maintain the overall study type I error at 5%.
      For symptom data, the change from screening score (continuous outcome) was compared between treatment groups using the Wilcoxon rank-sum test. Changes in eosinophil levels were analyzed separately by location: distal, proximal, and maximum of distal and proximal.
      To investigate the effect of demographic and participant characteristics on remission, logistic regression models were conducted with the following covariates separately, including the interaction of the covariate and treatment: age, weight, height, body mass index (BMI) Z-score, atopic status, and compliance. Similar analyses were conducted for the percentage change in eosinophil counts since screening using analysis of covariance models.
      The FP responsiveness analysis included participants randomized to the FP treatment group. To determine whether participant characteristics were predictive of achieving remission, logistic regression models were conducted separately for each of the following predictors: age, weight, height, BMI Z-score, race, ethnicity, atopic status, eosinophil level at the time of screening, and compliance. Analyses of the change in eosinophil counts were conducted using regression analysis.
      P values less than .05 were considered significant for all tests except for the primary end point at the interim analysis and at the end of the study. All statistical tests were completed using SAS 9.3 (Cary, NC).

       Analysis at 6 Months

      The percentage of participants in remission at the end of the study and the change in eosinophil counts from baseline were summarized for the treatment groups resulting from the initial randomization and subsequent treatment strategy for phase 2. No statistical comparisons across groups were conducted.

      Results

       Participant Characteristics

      Fifty-one participants were screened for the study; 9 were excluded (7 did not meet inclusion criteria and 2 declined to participate), and 42 were randomized (Figure 1). Of the 42 randomized participants, 28 received FP and 14 received placebo. Thirty-six participants completed phase 1 of the study (n = 17, 11, 5, and 3 from CCHMC, the University of Utah, the Colorado Children's Hospital, and the Children's Hospital of Philadelphia, respectively). Five participants withdrew from the FP group during phases 1 and 2 because of prohibited medications, 2 because of loss to follow-up evaluation, and 1 because of an adverse event (AE) (absence seizure that was deemed unlikely to be related to FP). One participant from the placebo group was lost to follow-up evaluation during phase 1. During phase 2, there were 2 participants who withdrew from the study for unknown reasons. Thus, 34 participants completed phases 1 and 2 of the study. Baseline participant characteristics were similar between the FP and placebo groups. The participants of both groups were primarily male, white (race), atopic, and on a PPI at the time of the screening visit (Table 1).
      Figure thumbnail gr1
      Figure 1Flow diagram of the study participants. Fifty-one participants were screened for the study; 9 were excluded and 42 were randomized. Of the 42 randomized participants, 28 received FP and 14 received placebo. Thirty-six participants completed phase 1, and 34 participants completed phases 1 and 2 of the study. NR, no response.
      Table 1Baseline Participant Characteristics
      P > .05 for all comparisons, when data were available.
      VariableFluticasonePlacebo
      Sex, male/female (% male)22/6 (79%)13/1 (93%)
      White race25 (89%)14 (100%)
      Mean age at entry, y (range)12.2 (3.54–26.90)13.5 (4.10–29.80)
      Height, cm (SD)141.3 (29)148.9 (23)
      Weight, kg (SD)43.4 (25)47.5 (24)
      Atopic status21 (78%)14 (100%)
      Proton pump inhibitor at screening20 (71%)10 (71%)
      Peak eosinophils/HPF proximal esophagus (SD, range)53.5 (62, 0–235)87.1 (108, 1–343)
      Peak eosinophils/HPF distal esophagus (SD, (range)56.3 (40, 1–155)97.7 (98, 30–296)
      Mean % blood eosinophils, SD (range)5.5 (3)7.8 (6)
      Mean absolute blood eosinophils, K/mm3 (SD)0.35 (0.2)0.53 (0.4)
      a P > .05 for all comparisons, when data were available.

       Interim Analysis

      Twenty-six participants were included in the interim analysis (17 FP, 9 placebo). The percentage of CR was significantly higher in the FP group (71%) than in the placebo group (0%) (P = .0007). On the basis of these results, we closed recruitment. Participants who already were enrolled in the trial were allowed to complete the trial.

       Efficacy

      In phase 1, the percentage of CR (both proximal and distal peak eosinophil count of ≤1 eosinophil/HPF) was significantly higher in the FP group (15 of 23; 65%) than in the placebo group (0 of 13, 0%; P = .0001). Responsiveness also was evaluated using different threshold values, as reported by Konikoff et al.
      • Konikoff M.R.
      • Noel R.J.
      • Blanchard C.
      • et al.
      A randomized, double-blind, placebo-controlled trial of fluticasone propionate for pediatric eosinophilic esophagitis.
      The values comprised a peak eosinophil count of 6 or fewer or 14 or fewer eosinophils/HPF, a mean (of proximal and distal peak values) eosinophil count of 1 or fewer or 2 or fewer eosinophils/HPF, and decreases of 90% or more or 95% or more of peak eosinophil count values. On the basis of these criteria, 73%, 77%, 63%, 68%, 75%, or 70% remission was observed after FP, respectively (Figure 2A). In the placebo group, 1 participant had partial remission (≤14 eosinophils/HPF). None of the placebo participants met the criteria for remission in any other category (Figure 2A). Responsiveness also was evaluated individually for the proximal or distal esophagus, by peak of 1 or fewer, 6, or 14 eosinophils, and decreases of 90% or more or 95% or more of peak eosinophil count values. In the proximal esophagus (Figure 2B), the FP group had 73%, 77%, 82%, 81%, and 81%, whereas the placebo group had 0%, 15%, 15%, 8%, and 0%, respectively. In the distal esophagus, the FP group had 59%, 73%, 77%, 70%, and 65%. The placebo group had remission rates of 8%, 15%, 23%, 8%, and 8%, respectively. The mean, proximal only, and distal only peak values had similar efficacy (Figure 2B and C).
      Figure thumbnail gr2
      Figure 2Responsiveness in phase 1. Response was defined as a peak count of 1 or fewer eosinophils/HPF in (A) both the proximal and distal esophagus, (B) proximal only, and (C) distal only. Additional definitions were calculated on the basis of peak counts of 6 or fewer and 14 or fewer eosinophils/HPF, mean counts of 1 or fewer and 2 or fewer eosinophils/HPF, and decreases of 90% or greater and 95% or greater in eosinophil levels. *P < .01, **P < .001, ***P < .0001. (A) P = 0 for a peak of 6 or fewer eosinophils/HPF. (B) P = 0 for a peak of 1 or fewer eosinophils/HPF.
      At the end of phase 2, FP phase 1 nonresponders who had continued on high-dose FP in phase 2 had EoE that remained largely FP resistant (Supplementary Figure 1A). One participant who was in partial remission (PR) after phase 1 and continued on high-dose FP in phase 2 was in PR after the 3 additional months of phase 2. Sixty-two percent of the participants from the phase 1 placebo group who initiated high-dose FP in phase 2 were in CR after 3 months, and 69% or more were in remission using different threshold values of response as reported by Konikoff et al
      • Konikoff M.R.
      • Noel R.J.
      • Blanchard C.
      • et al.
      A randomized, double-blind, placebo-controlled trial of fluticasone propionate for pediatric eosinophilic esophagitis.
      (Supplementary Figure 1A). Of the FP phase 1 responders, who had their dose halved in phase 2, there were 73% in CR (Figure 3), and 80% or more were in remission using different threshold values of response (Supplementary Figure 1A).
      • Konikoff M.R.
      • Noel R.J.
      • Blanchard C.
      • et al.
      A randomized, double-blind, placebo-controlled trial of fluticasone propionate for pediatric eosinophilic esophagitis.
      These same thresholds were used to evaluate remission individually in the distal or proximal esophagus (Supplementary Figure 1B and C).
      Figure thumbnail gr3
      Figure 3Effect of dose reduction on phase 1 FP responders. FP responders had their dosage of FP reduced from 1760 to 880 mcg/day in an open-label study and had a repeat esophagogastroduodenoscopy after 3 months. The peak eosinophil value is for the distal and proximal esophagus. Each solid line represents a unique patient. Dashed lines represent the 15 eosinophils/HPF threshold for diagnosing EoE by consensus recommendations and 1 eosinophil/HPF for CR.
      A responder analysis as a function of age, height, weight, BMI Z-score, compliance, atopic status, and screening esophageal eosinophil count showed that none of these variables correlated with response (Supplementary Tables 1 and 2). Participants with a new diagnosis of EoE and participants with long-standing EoE at the time of enrollment had 70% and 61.5% complete response, respectively.

       Safety

      Vital signs and laboratory results were stable throughout the study (data not shown). AEs were documented from the time of consent until a 30-day follow-up interview. AEs were reported according to the Medical Dictionary for Regulatory Activities System Organ Class (Table 2). One participant reported oral thrush at the 3-month study visit, which spontaneously resolved by the follow-up telephone call at the end of phase 2. This participant was in the high-dose FP group during phase 1 and remained on high-dose FP during phase 2. No statistically significant differences in AEs were observed between the high-dose FP and placebo groups during phase 1. Instances of decreased cortisol (n = 8) (Supplementary Table 3), increased cortisol (n = 1), and increased glucose (n = 1) levels were reported as AEs. For decreased cortisol level, 7 of 8 events occurred after a participant was on high-dose FP, and 1 of 8 events occurred at screening before randomization. Of the 7 events that occurred after randomization, 5 of 7 occurred initially during phase 1 while the participant was on high-dose FP, and 2 of 7 occurred during phase 2 in participants who went from placebo to high-dose FP. For the 5 events of decreased cortisol occurring during phase 1 of the study, 1 resolved before the 3-month visit, 3 resolved during phase 2 after FP dose reduction, and 1 did not resolve after dose reduction. The decreased cortisol events in phase 2 did not resolve before the end of the study. Although the decreased cortisol events between the FP and placebo groups during phase 1 were not significant (P = .15), there was a trend toward decreased cortisol in participants on high-dose FP. All decreased cortisol AEs were from 1 study site only (CCHMC), and most were measured via saliva (71%; 5 of 7). CCHMC was the only study site to collect cortisol via saliva. No participant was reported to show clinical signs of adrenal insufficiency or glucocorticoid toxicity.
      Table 2Number of Participants Experiencing Adverse Events in Phase 1
      Medical Dictionary for Regulatory Activities System Organ ClassControl (n = 14)Fluticasone (n = 28)Fisher exact P value
      Eye disorders2 (14.3%)0 (0.0%).11
      Gastrointestinal disorders2 (14.3%)7 (25.0%).69
      General disorders and administration site conditions: chest pain1 (7.1%)1 (3.6%)1.00
      Immune system disorders0 (0.0%)2 (7.1%).54
      Infections and infestations3 (21.4%)3 (10.7%).38
      Injury, poisoning, and procedural complications: scrapes and cuts0 (0.0%)1 (3.6%)1.00
      Investigations: abnormal laboratory values1 (7.1%)5 (17.9%).64
      Nervous system disorders0 (0.0%)4 (14.3%).28
      Respiratory, thoracic, and mediastinal disorders3 (21.4%)3 (10.7%).38
      Skin and subcutaneous tissue disorders1 (7.1%)0 (0.0%).33

       Symptom Severity and Frequency

      No statistically significant differences were found between the FP and placebo groups for total frequency or total severity of symptoms from the time of screening to month 3, the end of phase 1. Early satiety and abdominal pain were the most frequent symptom at screening in the FP group (71% and 71%, respectively), whereas dysphagia was the most frequent symptom in the placebo group (69%). At month 3, abdominal pain was the most frequent symptom in the FP group (61%), and heartburn was the most frequent in the placebo group (58%). The change in heartburn severity from screening to month 3 was statistically significant between the FP and placebo groups (P = .041) owing to a decrease in heartburn severity in 8 participants in the FP group compared with 0 participants in the placebo group. Of note, 50% of placebo participants and 41% of FP participants reported “none” relative to heartburn severity at screening. No other significant differences were found between the treatment groups in screening frequency or severity changes for individual symptoms in phase 1. This study was not designed or powered to statistically compare age groups, but additional conclusions are summarized in Supplementary Table 4.

       Molecular Gene Expression Profiling

      A concurrent distal esophageal biopsy was subjected to molecular expression signature analysis based on representative EoE genes.
      • Blanchard C.
      • Wang N.
      • Stringer K.F.
      • et al.
      Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis.
      • Saito K.
      • Katsuno T.
      • Nakagawa T.
      • et al.
      Predictive factors of response to intravenous cyclosporin in severe ulcerative colitis: the development of a novel prediction formula.
      The EDP has been shown to possess high sensitivity and specificity with only a single biopsy specimen; in contrast, histology requires 5–6 biopsy specimens to reach more than 95% sensitivity.
      • Gozansky W.S.
      • Lynn J.S.
      • Laudenslage M.L.
      • et al.
      Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic-pituitary-adrenal axis activity.
      Thus, in addition to providing key molecular insight into the action of steroids in the esophagus, this study provided an opportunity to examine the value of the EDP in a prospective controlled trial. In the format of a heat diagram, the 77 definitive diagnostic gene expression profiles from each group at screening and the end of each of the 2 trial phases were juxtaposed (Figure 4A) with known EoE and control cases shown as references. Bidirectional gene signature changes readily can be observed from the heat diagram. Phase 1 participants receiving the placebo did not have signature reversal because bidirectional genes still were dysregulated, similarly to at the time of initial screening (P = .16, 2-tailed Student t test). In contrast, a large portion of the participants receiving FP in phase 1 showed a normalized signature compared with the dysregulated screening and placebo signatures (P < .0001 for both, 2-tailed Student t tests), albeit there was still modestly dysregulated gene expression. Notably, the 6 FP participants with histologic PR or no remission also had a partial reversal with a signature different from the placebo group. After the placebo group received FP in phase 2, their transcriptomes were normalized dramatically. However, there were still a few molecular nonresponders whose signatures failed to normalize upon FP treatment, consistent with the histologic findings. These findings likely are attributable to individual FP sensitivity or the suboptimal effect of the FP dose.
      Figure thumbnail gr4
      Figure 4Esophageal transcriptome analysis. Total RNA from separately acquired biopsy specimens was subjected to EDP signature analysis. (A) The heat diagram depicts the changes after FP exposure in the bidirectional gene dysregulation in each group with reference historical EoE and normal (NL) cohorts (n = 15 and 14, respectively), and the samples at screening (no FP exposure) on the left. At the end of phases 1 and 2, expression signatures from FP nonresponders (NRs), FP responders with partial remission (PRs), and FP responders with complete remission (CRs) are shown in the heat diagram (yellow, increased expression; blue, decreased expression). (B) On the basis of the expression levels of the 77 diagnostic genes and a dimensionality reduction algorithm, an EoE score reflecting the EoE disease activity was calculated at the end of phase 1 (**P < .001, ***P < .0001, 2-tailed Student t test). (C) To assess the prediction value of the EDP on FP responsiveness, samples at screening (ie, pre-FP exposure) and the placebo samples at the end of phase 1 (ie, pre-FP exposure) were screened by bioinformatics in the context of their subsequent FP responsiveness on the basis of the CR criteria of fewer than 2 eosinophils/HPF in the distal esophagus, where the biopsy specimens were acquired. The 15-gene cluster listed shows a potential for FP efficacy prediction with a P value of less than .05 and fold change greater than 2.0.
      The EDP is associated with an EoE score algorithm reflecting disease status and severity in a quantifiable number—the EoE score, based on a core set of 77 diagnostic genes (Dx-77). The EoE score was calculated for each subgroup (Figure 4B) and showed that FP responders had quantifiable improvement in the transcriptome that was not observed to the same degree in participants with FP-resistant EoE. Notably, although the EoE score normalized in FP complete responders, it still was significantly different from the EoE score of healthy controls (normal vs FP-CR) (Figure 4B; Supplementary Table 5). Receiver operating characteristic analysis indicated a sensitivity of 92% and a specificity of 92% for the EDP to distinguish patients with active disease vs complete remission. By using the reported Dx-77 scoring, an analysis of active EoE samples (placebo treated and initial screening) vs samples from patients in complete remission (phase 1 and 2), showed a sensitivity of 96% and a specificity of 80%. Finally, the EDP score inversely correlated with the esophageal eosinophil count (Spearman R, -0.82; P < .0001).
      We also aimed to identify esophageal gene expressions that might predict FP responsiveness by statistically screening the pre–FP treatment distal biopsy samples of the FP responder and nonresponder cohorts. A total of 15 genes (Figure 4C) (P < .05; fold-change, >2.0) on the EDP were identified that had a tendency to predict subsequent FP efficacy on the basis of CR criteria (≤1 eosinophil/HPF in the distal esophagus).

      Discussion

      This study shows that high-dose FP (1760 mcg/day) induces histologic CR in 65% of EoE participants and at least partial remission in 77% with barely any placebo effect. Furthermore, after achieving remission, reducing the dose of FP from 1760 to 880 mcg/day results in 93% of EoE participants maintaining in CR or PR. This finding supports dose reduction after CR is achieved with high-dose FP. Of note, the total dose is halved for the 1760 to 880 mcg group, as well as administration of 4 puffs twice daily to 2 puffs twice daily. For EoE that does not respond to high-dose FP in 3 months, extending the timeframe for high-dose FP to 6 months does not increase remission status. Therefore, 3 months is an adequate timeframe to evaluate histologic response after introduction of FP therapy, and we suggest that the 3-month evaluation be considered in clinical practice after initiation of FP. Our study provides definitive evidence for substantial rates of steroid resistance in EoE (approximately 25%), even at the relatively high doses and prolonged exposure durations associated with our study. High-dose FP responders were not found to be different from nonresponders in age, height, weight, BMI z-score, compliance, or atopic status. Notably, at the higher dose (1760 mcg) administered in this study, there was no effect of these phenotypic markers on responsiveness, strongly suggesting steroid resistance rather than inadequate dosage or delivery format. It is important to point out that we cannot directly conclude that there is a 15% difference in the response rate previously reported in our prior study with FP at 880 mcg
      • Konikoff M.R.
      • Noel R.J.
      • Blanchard C.
      • et al.
      A randomized, double-blind, placebo-controlled trial of fluticasone propionate for pediatric eosinophilic esophagitis.
      because the 880 vs 1760 mcg doses were not directly compared in the same study; it remains possible that at least some of these patients would have responded to the lower dosage. Nevertheless, the observed response rate to 1760 mcg of fluticasone suggests that starting at this higher dosage and then stepping down is warranted, but further clinical trials are required before this can become a recommendation.
      Molecular gene expression profiling represents the next generation of EoE diagnosis, and the representative EoE gene array, the EDP, was developed for this purpose.
      • Wen T.
      • Stucke E.M.
      • Grotjan T.M.
      • et al.
      Molecular diagnosis of eosinophilic esophagitis by gene expression profiling.
      As a parallel study, we used the EDP to monitor the FP intervention efficiency by assessing the EoE signature and activity and steroid exposure, all of which can be quantified into a readily comprehended score by associated EDP algorithms.
      • Wen T.
      • Stucke E.M.
      • Grotjan T.M.
      • et al.
      Molecular diagnosis of eosinophilic esophagitis by gene expression profiling.
      We showed that in those cases in which FP achieved histologic CR, the molecular signature also normalized. However, the EDP EoE score from FP complete responders still was significantly different from the EoE score of healthy controls (Figure 4B), providing evidence for the persistence of molecular abnormalities even in patients with histologic CR and a potential molecular explanation for disease relapse.
      Meanwhile, those cases in which FP failed to elicit histologic CR were associated with a partial EoE-like intermediate transcriptome. Therefore, FP was effective in reversing the pathogenic gene expression signatures to a certain extent but failed to elicit CR. These findings indicate that these FP nonresponders were indeed exposed to FP. The partial EoE-like intermediate signature is consistent with some participants having steroid resistance rather than noncompliance, which is in accord with the responder analysis (Supplementary Table 1). Indeed, participant compliance with medication intake did not correlate with the participant transcriptome changes on the basis of the data available (data not shown). The signature normalization in most of the participants who received high-dose FP in phase 1 and high-dose FP in phase 2 highlights the bona fide efficacy of FP intervention in EoE. Notably, this report shows the value of the EDP in measuring disease activity in a controlled, prospective clinical trial.
      Predicative medicine aims to select the optimal form of treatment on a patient-by-patient basis, which has been shown to be successful in some other gastrointestinal disorders, such as ulcerative colitis.
      • Saito K.
      • Katsuno T.
      • Nakagawa T.
      • et al.
      Predictive factors of response to intravenous cyclosporin in severe ulcerative colitis: the development of a novel prediction formula.
      Therefore, we also screened the pre–FP treatment samples for their subsequent FP responsiveness and identified 15 genes showing a tendency for FP efficacy predictions. Of note, we were limited by the sample size and did not have a power high enough to apply a false-positive correction filter on this result; therefore, these findings should be considered preliminary. Further validating studies on these candidate genes would be of significant interest.
      Although AE occurrences were not significantly different between high-dose FP and placebo, the small sample size results in low power for detecting signals. CCHMC was the only site with low cortisol values, and most, but not all, were measured in saliva samples. CCHMC was the only site that included salivary cortisol measurements. Salivary cortisol measurements have limitations because the reference ranges have not been agreed upon and the cortisol may not be stable during the period before participants deliver the samples. At the same time, salivary measures free cortisol levels whereas serum measures free and bound cortisol; because free is the active form, it remains possible that salivary cortisol is a more sensitive measurement under these conditions.
      • Gozansky W.S.
      • Lynn J.S.
      • Laudenslage M.L.
      • et al.
      Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic-pituitary-adrenal axis activity.
      In addition, salivary levels are more stable compared with the diurnal variation seen in the blood (which peaks in the morning); thus, it may be easier to discern lower levels in the saliva.
      • Inder W.J.
      • Dimeski G.
      • Russell A.
      Measurement of salivary cortisol in 2012-laboratory techniques and clinical indications.
      Long-term topical steroid therapy (0.5 mg/day of budesonide in adults) has been reported to be well tolerated and effective in maintaining disease remission, although an increase in eosinophil levels during the 50-week observation period has been reported to occur.
      • Straumann A.
      • Conus S.
      • Degen L.
      • et al.
      Long-term budesonide maintenance treatment is partially effective for patients with eosinophilic esophagitis.
      Further assessment of the effect of FP on the adrenal corticoid axis is warranted in future studies, especially because follow-up evaluation of the low cortisol values were not examined in this study.
      The high-dose FP group had improved heartburn severity at the 3-month visit, whereas the placebo group remained the same. The frequency and severity for all other symptoms was similar between the groups. At the time of study initiation, a validated EoE symptom score measure was not available, which was a limitation to this component of our study. Although we observed no significant differences in changes in symptom frequency and severity, with the exception of heartburn severity, between treatment groups, participants were not required to have symptoms at study entry, and the study was not powered to detect differences in symptom frequency or severity. Nevertheless, we observed an improvement in heartburn severity in the FP group compared with the placebo group in phase 1. All participants had PPI-confirmed EoE and were instructed not to alter any medicine or diet during the trial. Therefore, it is unlikely that the reduction in heartburn severity in the FP group was caused by PPI use. It is notable that symptom reduction is often less impressive than histologic response. There are several explanations for this finding, including the possibility of insufficient duration of therapy, fixed fibrosis (although in our study this was not present), and the use of nonvalidated and/or insufficient patient-reported outcome metrics, as recently discussed.
      • Rothenberg M.E.
      • Aceves S.
      • Bonis P.A.
      • et al.
      Working with the US Food and Drug Administration: progress and timelines in understanding and treating patients with eosinophilic esophagitis.
      Besides FP, budesonide has been shown to improve dysphagia
      • Dohil R.
      • Newbury R.
      • Fox L.
      • et al.
      Oral viscous budesonide is effective in children with eosinophilic esophagitis in a randomized, placebo-controlled trial.
      • Straumann A.
      • Conus S.
      • Degen L.
      • et al.
      Long-term budesonide maintenance treatment is partially effective for patients with eosinophilic esophagitis.
      • Aceves S.S.
      • Newbury R.O.
      • Chen D.
      • et al.
      Resolution of remodeling in eosinophilic esophagitis correlates with epithelial response to topical corticosteroids.
      and to reduce esophageal eosinophils significantly,
      • Straumann A.
      • Conus S.
      • Degen L.
      • et al.
      Budesonide is effective in adolescent and adult patients with active eosinophilic esophagitis.
      • Straumann A.
      • Conus S.
      • Degen L.
      • et al.
      Long-term budesonide maintenance treatment is partially effective for patients with eosinophilic esophagitis.
      as compared with placebo, with CR of 35%-72% or 0%–11% after budesonide or placebo, respectively. Dellon et al
      • Dellon E.S.
      • Sheikh A.
      • Speck O.
      • et al.
      Viscous topical is more effective than nebulized steroid therapy for patients with eosinophilic esophagitis.
      conducted a randomized trial comparing nebulized budesonide and viscous topical budesonide (as an oral slurry) in EoE and showed that symptoms improved in all groups; however, the esophageal eosinophil reduction was greater with viscous topical administration, supporting the view that a topical mechanism was operational.
      • Dellon E.S.
      • Sheikh A.
      • Speck O.
      • et al.
      Viscous topical is more effective than nebulized steroid therapy for patients with eosinophilic esophagitis.
      Indeed, swallowed glucocorticoids induce de novo gene expression in the esophagus, suggesting a local effect, particularly on the epithelium.
      • Caldwell J.M.
      • Blanchard C.
      • Collins M.H.
      • et al.
      Glucocorticoid-regulated genes in eosinophilic esophagitis: a role for FKBP51.
      Although FP and budesonide are the most highly studied glucocorticoids for the treatment of EoE, a number of other steroids have been examined recently. Swallowed ciclesonide also has been shown to improve symptoms and histology but only in a small set of patients in an open-label study (n = 8).
      • Schroeder S.
      • Fleischer D.M.
      • Masterson J.C.
      • et al.
      Successful treatment of eosinophilic esophagitis with ciclesonide.
      • Lee J.J.
      • Fried A.J.
      • Hait E.
      • et al.
      Topical inhaled ciclesonide for treatment of eosinophilic esophagitis.
      Bergquist et al
      • Bergquist H.
      • Larsson H.
      • Johansson L.
      • et al.
      Dysphagia and quality of life may improve with mometasone treatment in patients with eosinophilic esophagitis: a pilot study.
      conducted a pilot study with topical mometasone furoate in 31 patients with EoE. Esophageal eosinophil levels were not assessed, but dysphagia and health-related quality of life improved after treatment.
      In conclusion, high-dose FP is effective in inducing histologic remission in EoE in 65%–77% of participants after 3 months of therapy, with the range dependent on the degree of histologic improvement. In FP responders, reducing the dose in half results in CR and PR in 73% and 20% of participants, respectively. In FP nonresponders, extending high-dose treatment for an additional 3 months provided no benefit. Evidence for bona fide steroid resistance is present at both histologic and molecular levels, further substantiating the need for the development of additional therapeutic interventions for this emerging medical disorder.

      Acknowledgements

      The authors would like to thank Marshall Plaut, MD, and Joy Laurienzo, RN, BSN, for their oversight of the study progress, and Dr Daniel Rotrosen for his support of the study and review of the manuscript.
      Marc Rothenberg obtained funding and supervised the study; Bridget Butz, Gerald Gleich, Glenn Furuta, Jonathan Spergel, Robert Kramer, W. Daniel Jackson, and Molly O'Gorman facilitated recruitment; Ting Wen performed the Eosinophilic Esophagitis Diagnostic Panel experiments; Emily Stucke processed study samples; Margaret Collins provided pathologic insight regarding the biopsy sections involved and reviewed specimens; Colleen Mangeot and Eileen King analyzed and interpreted the data; and Bridget Butz, Gerald Gleich, Glenn Furuta, Jonathan Spergel, Margaret Collins, J. Pablo Abonia, Scott Pentiuk, Philip Putnam, and Marc Rothenberg were involved in the study concept and design. All authors were involved in writing the manuscript.
      Transcript Profiling: The microarray data were not of sufficient scope that they should be included in a repository (96 genes).

      Supplementary Materials and Methods

       Interim Analysis

      An interim analysis was added to the study after initiation. The interim analysis was conducted using the 3-month remission status for 26 participants (60% of the sample size). The primary end point for the interim analysis was the percentage of participants who attained CR (defined as a peak eosinophil count of 1 or fewer eosinophils/HPF at the 3-month esophagogastroduodenoscopy). The 3-month remission status used was recorded as either remission (≤1 eosinophil/HPF), active (≥24 eosinophils/HPF), or other (>1 and <24 eosinophils/HPF).

       Assessing Efficacy and Safety

      The primary objective was histologic remission defined by a peak eosinophil count of 1 or fewer eosinophils/HPF (400× field) in both the proximal and distal esophagus at 3-month esophagogastroduodenoscopy.
      The secondary objectives were to measure safety via cortisol, glucose, and adverse reaction data and the relationship between FP responsiveness and participant age, height, weight, BMI Z-score, race, ethnicity, atopic status, compliance, and screening eosinophil level. Atopic status was defined by a personal history of allergic disease (allergic rhinitis, hay fever, atopic dermatitis, eczema, food anaphylaxis, asthma, or positive skin prick tests).

       Histology and Blood

      Esophagogastroduodenoscopies with biopsies were performed at the screening visits and after 3 and 6 months. Biopsy specimens were reviewed by the pathologist at each site to assess disease state. The site pathologist generated quantitative peak eosinophil counts. For cases in which the site pathologist did not provide a peak eosinophil count, the slides were sent to CCHMC for review by the CCHMC site pathologist.
      Participants had baseline blood work (complete blood count, glucose, AM cortisol level) that was repeated at the 3- and 6-month visits. Glucose and AM cortisol levels also were measured at 4 and 8 weeks. The complete blood count, glucose, and AM cortisol level evaluations were performed at Clinical Laboratory Improvement Amendments–certified laboratories. For convenience, some participants (38%) had cortisol measured via saliva, which was performed at CCHMC using the salivary cortisol enzyme immunoassay kit from Salimetrics, LLC (State College, PA).

       Adverse Event

      AE data were coded per the Medical Dictionary for Regulatory Activities by System Organ Class by an independent coder at CCHMC. Laboratory values were considered to be AEs if they were outside of the reference ranges provided by the laboratory and deemed clinically significant by an investigator at each site. The safety of FP was evaluated by glucose and cortisol results and AE data. Decreased cortisol values in consecutive measurements required referral to an endocrinologist and potential removal from the study.

       Compliance

      Compliance was measured by the use of the Doser by MediTrack (North Easton, MA), which attached to the metered-dose inhaler to measure daily inhalations for 30-day intervals. The use of participant diaries to record daily metered-dose inhaler use was incorporated after 18 participants were enrolled.

       Molecular Gene Expression Profiling

      Figure thumbnail fx1
      Supplementary Figure 1Response was defined as a peak count of ≤ eosinophil/HPF in (A) both the proximal and distal esophagus, (B) proximal only, and (C) distal only. Additional definitiions were calculated on the basis of peak counts ≤6 and ≤14 eosinophil/HPF, mean counts of ≤1 and ≤2 eosinophil/HPF, and decreases of ≥90% and ≥95% in eosinophil levels. FP, fluticasone propionate.
      Supplementary Table 1Responder Analysis
      VariableOdds ratio estimateLower confidence limitUpper confidence limitP value
      Age0.9070.7791.055.2058
      Height0.9660.9251.008.1126
      Weight0.9650.9271.005.0864
      BMI Z-score0.5410.2051.433.2167
      Proximal esophageal eosinophils1.0070.9881.027.4488
      Distal esophageal eosinophils1.0030.9801.026.8093
      Peak esophageal eosinophils1.0080.9891.028.3869
      Compliance1.0370.9881.089.1366
      Atopic status0.5720.0496.607.6543
      Supplementary Table 2Efficacy Results Summarized by Age Group: Younger Than Age 18 Versus Age 18 Years and Older
      Complete remissionPartial remissionNo remission
      <18 y (n = 29)
       Control (n = 11)0 (0%)1 (9%)10 (91%)
       Fluticasone (n = 18)12 (67%)2 (11%)4 (22%)
      ≥18 y (n = 7)
       Control (n = 2)0 (0%)0 (0%)2 (100%)
       Fluticasone (n = 5)3 (60%)1 (20%)1 (20%)
      NOTE. The remission rates were consistent across age groups. This study was not designed or powered to statistically compare the age groups. However, summary statistics support the conclusion of consistency across age groups.
      Supplementary Table 3Decreased Cortisol Information
      ParticipantValue, μg/dLSample typeReference range, μg/dLVisit, drug
      10.035Saliva0.084–0.839Month 3, FP 1760
      10.043Saliva0.084–0.839Month 6, FP 880
      20.038Saliva0.112–0.743Month 6, FP 1760
      3<0.012Saliva0.021–0.883Month 6, FP 1760
      45.8Blood6.7–22.6Screening
      50.7Blood3–21Week 8, FP 1760
      5<0.012Saliva0.084–0.839Month 3, FP 1760
      60.061Saliva0.084–0.839Month 3, FP 1760
      70.233Saliva0.272–1.348Month 3, FP 1760
      80.4Blood2.3–19.4Week 8, FP 1760
      Supplementary Table 4Symptoms Divided by Age Group
      <18≥18
      ScreenMonth 3ScreenMonth 3
      Overall (N = 33)Control (N = 11)Fluticasone (N = 22)Control (N = 10)Fluticasone (N = 18)Overall (N = 8)Control (N = 2)Fluticasone (N = 6)Control (N = 2)Fluticasone (N = 5)
      Abdominal pain21 (64%)7 (64%)14 (67%)5 (50%)11 (61%)6 (75%)15 (83%)13 (60%)
      Chest pain17 (52%)5 (50%)12 (55%)3 (30%)8 (44%)4 (50%)13 (50%)12 (40%)
      Dysphagia19 (58%)7 (63%)12 (55%)3 (30%)8 (44%)7 (88%)25 (83%)23 (60%)
      Early satiety22 (67%)6 (55%)16 (73%)4 (40%)11 (61%)4 (50%)04 (67%)11 (20%)
      Food impaction10 (30%)2 (18%)8 (36%)1 (10%)3 (17%)3 (38%)12 (33%)12 (40%)
      Heartburn16 (48%)6 (55%)10 (45%)6 (60%)6 (33%)5 (63%)14 (67%)13 (60%)
      Nausea16 (48%)4 (36%)12 (55%)4 (40%)8 (44%)7 (88%)25 (83%)15 (100%)
      Poor appetite
      Data are missing for 1 respondent in the control group.
      18 (56%)3 (27%)15 (68%)2 (22%)8 (47%)5 (63%)14 (67%)11 (20%)
      Regurgitation20 (61%)5 (45%)15 (68%)2 (20%)7 (39%)4 (50%)04 (67%)23 (60%)
      Vomiting14 (42%)3 (27%)11 (50%)2 (20%)4 (22%)2 (25%)11 (17%)12 (40%)
      NOTE. This table shows the number (%) of subjects responding that they experienced the symptom (ie, they did not respond “never”). Responses were dichotomized into “never” and “sometimes.” This study was not designed or powered to statistically compare age groups. However, on the basis of the summary statistics, the following conclusions were made on the basis of differences in rates of at least 20%: at screening, youth had directionally lower rates of dysphagia and nausea than adults. At month 3, comparisons were made between age groups only for the fluticasone group because only 2 adult patients were in the control group. Youth had directionally higher rates of early satiety and poor appetite than adults. Youth had directionally lower rates of food impaction, heartburn, nausea, regurgitation, and vomiting than adults.
      a Data are missing for 1 respondent in the control group.
      Supplementary Table 5Genes That Do Not Completely Normalize After Complete Remission
      Gene symbolt test
      GLDC4.71E-07
      APOBEC3A2.38E-06
      CFI1.41E-05
      CD200R13.14E-05
      SAMSN19.9E-05
      RUNX20.000103
      MUC40.000126
      CHL10.00043
      PHLDB20.00053
      CXCL60.000834
      IL80.000843
      GCNT30.00087
      EPPK10.000958
      POSTN0.001883
      UPK1B0.002525
      HRH10.002773
      FCGR3A;FCGR3B0.003258
      TPSB2;TPSAB10.004412
      SLC16A60.00616
      CTSC0.007837
      CDH260.012913
      UPK1A0.016188
      ALOX150.028083
      TNFAIP60.029846
      CITED20.030902
      CA20.035152
      CXCL10.037734
      SLC26A40.043581

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