Human Pathology (2014) xx, xxx–xxx
www.elsevier.com/locate/humpath
Original contribution
Correlation of ALOX15 expression with eosinophilic or reflux esophagitis in a cohort of pediatric patients with esophageal eosinophilia☆,☆☆ Andres Matoso MD a,1 , Danisha Allen MD a,1 , Michael Herzlinger MD b , Jason Ferreira MD c , Sonja Chen MD a , Shaolei Lu MD, PhD a , Valeria Fabre MD d , Renee Monahan BSc a , Dongfang Yang MSc a , Lelia Noble a , Shamlal Mangray MD a , Murray B. Resnick MD, PhD a,⁎ a
Department of Pathology and Laboratory Medicine, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI 02903 b Department of Pediatrics, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI 02903 c Department of Medicine, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI 02903 d Department of Medicine, Johns Hopkins Hospital, Baltimore, MD 21287 Received 18 November 2013; revised 13 January 2014; accepted 17 January 2014
Keywords: Eosinophilic esophagitis; Biomarker; ALOX15
Summary The differential diagnosis between eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) is often challenging. We recently showed that the ALOX15 protein is expressed in 95% of esophageal biopsies from patients with a definitive diagnosis of EoE. Here we correlated ALOX15 expression with the clinical classification of EoE or GERD in a cohort of consecutive pediatric patients (n = 62) with at least 1 esophageal biopsy containing at least 15 eosinophils per high-power field (eos/HPF). The patients were categorized into the following groups: (1) at least 15 eos/HPF in the distal esophagus only (n = 24), (2) at least 15 eos/HPF in the proximal esophagus only (n = 5), and (3) at least 15 eos/HPF in the distal and proximal biopsies (n = 33). Control groups included patients with GERD with biopsies containing 6 to 15 eos/HPF (n = 9), patients with GERD with 5 eos/HPF or less (n = 15), patients with candida esophagitis (n = 15), and patients with normal biopsies (n = 15). ALOX15 was positive in 90.5% of patients with EoE (13/16 in group 1, 4/4 in group 2, 31/33 in group 3) versus 44% of patients with GERD (4/8 in group 1, 0/1 in group 2, and 0/0 in group 3), 2 of 9 (22%) of patients with 6 to 15 eos/HPF, and was negative in all patients with GERD with biopsies containing 5 eos/HPF or less, all patients with candida esophagitis, and all normal controls. In conclusion, ALOX15 is a sensitive marker of EoE; however, subpopulations of patients with GERD with N5 eos/HPF also express ALOX15. Positive ALOX15 expression is more prevalent in EoE than in GERD and may prove to be a useful diagnostic marker in patients with discrepant biopsy findings between the proximal and distal esophagus. © 2014 Elsevier Inc. All rights reserved.
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Disclosures: This study was supported by the Molecular Pathology Core of the COBRE Center for Cancer Research Development, funded by the National Institute of General Medical Sciences of the National Institute of Health (P20GM103421). ☆☆ Conflict of interest: The authors declare no competing interest. ⁎ Corresponding author. Department of Pathology, 593 Eddy St, Providence, RI 02903. E-mail address: [email protected] (M. B. Resnick). 1 These authors contributed equally to this work. 0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2014.01.021
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1. Introduction The clinical diagnosis of eosinophilic esophagitis (EoE) is based on the correlation of clinical, endoscopic, and histopathologic findings. According to the most recent consensus recommendations in EoE [1], 1 or more biopsy specimens must show eosinophil-predominant inflammation, with a minimum threshold of 15 eosinophils per high-power field (eos/HPF). The histopathologic findings that support the diagnosis of EoE, including superficial clustering of intraepithelial eosinophils, basal cell hyperplasia, and elongation of papillae, are not entirely specific of EoE, and there is a considerable overlap with features of gastroesophageal reflux disease (GERD) [2-7]. For instance, some patients with strong clinical evidence of EoE may have biopsies with many of the features mentioned, but with a peak eosinophil count of less than 15/HPF [1]. In addition, a subgroup of patients with typical symptoms and biopsy findings of EoE who have had GERD excluded may show favorable clinicopathological response to treatment with proton pump inhibitors (PPI-responsive esophageal eosinophilia) [8-10]. The distinction between EoE and GERD is clinically important because medical treatment differs between these 2 entities; while reflux disease is treated with antacid medication, EoE responds to topical steroids and dietary allergen elimination. Several studies have been performed to better characterize the molecular signature of EoE [11-16]. We previously conducted a validation study to confirm the presence of several novel biomarkers in EoE detectable by immunohistochemistry [17]. In this study, we found that 95% of the biopsies of patients with clinically proven EoE were positive for the protein arachidonate-15 lipoxigenase (ALOX15). More recently, Wen et al [18] introduced an EoE diagnostic panel based on a 96-gene expression profile that included overexpression of ALOX15. In Wen’s study, ALOX15 was expressed more than 1000-fold higher in biopsies of patients with EoE compared with normal controls. Although the overexpression of ALOX15 was highly sensitive and specific for EoE, all of the biopsies included in our previous study were from patients with straightforward diagnoses who presented with all of the histopathologic and clinical features of EoE, including involvement of the proximal esophagus. In the present study, we aimed to evaluate ALOX15 expression in a cohort of consecutive pediatric patients with esophageal eosinophilia and correlated the findings with their clinical classification as EoE versus GERD.
2. Materials and methods
A. Matoso et al. tal. The cohort included consecutive pediatric patients with biopsies from the distal and proximal esophagus obtained from 2009 through 2012 (ages 0-18 years) with at least 1 biopsy with 15 or more intraepithelial eos/HPF. Control groups included patients with GERD with biopsies containing 6 to 15 eos/HPF, patients with GERD with biopsies containing up to 5 eos/HPF, patients with candida esophagitis, and patients with normal squamous mucosa. Clinical diagnosis of EoE was made as defined by the 2011 consensus guidelines [1]. Specifically, patients were required to have symptoms of esophageal dysfunction, a trial with PPIs without remission of symptoms, clinical history of other allergies and/or food impaction, endoscopic findings more consistent with EoE (furrows, rings, and ridging), 1 or more esophageal biopsies with a minimum of 15 eos/HPF, and other causes of esophageal eosinophilia excluded. Clinical diagnosis of GERD included clinical presentation and histopathology consistent with reflux in patients with good symptomatic response to acid suppression. Two pathologists (A. M. and M. R.) reviewed the hematoxylin and eosin slides to confirm the histologic diagnosis. A pediatric gastroenterologist (M. H.) reviewed the patients' medical records to ensure that these patients fit the diagnostic selection criteria for either EoE or GERD.
2.2. Immunohistochemistry Immunohistochemistry was performed using the antibody against ALOX15 (11-K; Santa Cruz Biotechnology, Santa Cruz, CA; 1:100 dilution) as described previously [17]. Known cases of EoE were used as a positive control. Negative controls were achieved by replacing the primary antibody with normal serum. Results were scored using a semiquantitative system, which takes into consideration the extent and intensity of staining. For extent, points were assigned as follows: 0, negative; 1, up to 10% positive cells; 2, 10% to 50% positive cells; and 3, more than 50% positive cells. For intensity, points were assigned as follows: 0, negative (no stain); 1, weakly positive (pale brown cytoplasmic stain); 2, moderately positive (dark-brown cytoplasmic stain in part or all the cytoplasm); and 3, intensely positive (very dark-brown stain covering the entire cytoplasm of all positive cells). A total score of 3 or higher was considered positive and 2 or lower was considered negative. Only staining of squamous cells was considered positive staining. Owing to the discontinuous nature of EoE, “patchy” lesions were graded on areas of histologic changes consistent with EoE. A. M. and M. R. independently scored each section without knowledge of the clinical follow-up. Concordance was high, and discrepant cases were reviewed to reach consensus.
2.1. Patients and tissue samples Archival esophageal biopsies were obtained from the Pathology Department of the Rhode Island Hospital (Providence, RI), following a protocol approved by the institutional review board of Lifespan/Rhode Island Hospi-
2.3. Statistical analysis Statistical analysis was performed using the STATA (version 13) software (Stata, College Station, TX). P values
ALOX15 in eosinophilic esophagitis
3 etc) were excluded from analysis. Control groups included patients with GERD with biopsies containing 6 to 15 eos/HPF (n = 9), patients with GERD with biopsies containing up to 5 eos/HPF (n = 15), patients with candida esophagitis (n = 15), and patients with normal esophageal biopsies (n = 15). Because involvement of the proximal esophagus is characteristic of EoE and involvement of the distal esophagus is more characteristic of GERD, patients were grouped as follows: group 1, more than 15 eos/HPF in the distal esophagus only (n = 24); group 2, more than 15 eos/ HPF in the proximal esophagus only (n = 5); and group 3, more than 15 eos/HPF in both distal and proximal esophagus (n = 33). The clinicopathological characteristics of all patients are summarized in Table 1.
were calculated using the Fisher exact test, t test, Woolf test, and Spearman rank test. Differences were expressed as mean ± SEM and considered significant at a P value of equal or less than .05.
3. Results 3.1. Study population A total of 77 patients were identified in the pathology database. All subjects underwent upper endoscopy for evaluation of gastrointestinal symptoms suggestive of esophageal dysfunction. As per protocol in our institution, biopsies were taken from the proximal and distal esophagus; all of the study patients had at least 1 esophageal biopsy with 15 or more intraepithelial eos/HPF. Only first-time biopsies from patients without history of any EoE-specific therapy were included in the study. Medical records were reviewed for documentation of symptoms, endoscopic findings, and medical management, and patients were classified as having EoE or GERD based on the criteria outlined in Materials and Methods. Instances in which there were insufficient data to determine a clinical diagnosis of either EoE or GERD (n = 8) or in which there was a confounding gastrointestinal disorder (n = 7, celiac, Crohn disease, Helicobacter pylori infection,
Table 1
3.2. ALOX15 immunohistochemistry Expression of ALOX15 was detected in the cytoplasm of squamous epithelial cells and eosinophils as previously described [17]. Although scoring was restricted to epithelial expression in squamous cells, positive staining of eosinophils was used as an internal control in cases of negative squamous staining, as they were consistently positive. Glandular cells at the gastroesophageal junction and subepithelial stromal cells were consistently negative (Fig. 1A and B). There were occasional biopsies with less
Clinicopathological characteristics of patients
Age (y), mean ± SEM Sex (male/female) Symptoms Abdominal pain (%) Vomiting (%) Dysphagia (%) Food impaction (%) Heartburn (%) Failure to thrive (%) Endoscopy Normal (%) Erythema (%) Rings (%) Ridging (%) Furrows (%) White plaques (%) Allergies Food allergy (%) Asthma (%) Rhinitis or dermatitis (%) Eosinophils/HPF, mean ± SEM Distal Proximal
Group 1 (≥15 eos/HPF in distal only; n = 24)
Group 2 (≥15 eos/HPF in proximal only; n = 5)
Group 3 (≥15 eos/HPF in proximal and distal; n = 33)
GERD with 6-15 eos/HPF (n = 9)
10.9 ± 1.13 16:8
9.8 ± 1.8 2:3
12.1 ± 0.8 21:12
10.4 ± 1.6 5:4
7 (29) 10 (41) 15 (63) 6 (25) 6 (25) 10 (41)
0 0 3 4 2 3
(0) (0) (60) (80) (40) (60)
8 (24) 16 (48) 20 (60) 10 (30) 7 (21) 19 (58)
2 4 3 3 2 1
(22) (44) (33) (33) (22) (11)
7 (29) 5 (21) 2 (8) 9 (37) 12 (50) 3 (13)
1 1 1 4 2 3
(20) (20) (20) (80) (40) (60)
12 (36) 3 (9) 12 (36) 21 (64) 15 (45) 2 (6)
2 0 0 1 1 0
(22) (0) (0) (11) (11) (0)
11 (46) 8 (33) 8 (33)
2 (40) 1 (20) 1 (20)
12 (36) 3 (9) 17 (51)
1 (11) 3 (33) 2 (22)
38.2 ± 4.5 4.5 ± 0.9
8.2 ± 2.5 38.6 ± 8.4
43.9 ± 3.9 49.4 ± 5.0
10.6 ± 0.9 1.6 ± 0.6
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Fig. 1 ALOX15 immunohistochemistry in esophageal mucosa. A, Low-power view of a biopsy from the gastroesophageal junction shows diffuse and strong ALOX15 immunostaining in the squamous epithelium (arrow) and negative staining in the glandular (cardia-type) epithelium (arrowhead). B, Strong cytoplasmic staining of the squamous cells, whereas subepithelial stroma is negative (arrowhead). C, Positive staining both the cytoplasm of squamous cells (arrow) and in eosinophils (arrowheads). D, An esophageal biopsy showing negative staining in the squamous cells but positive staining in eosinophils (arrowheads).
Fig. 2 Relationship between ALOX15 immunostaining and eosinophil count. Peak number of intraepithelial eosinophils in biopsies of patients of ALOX15-positive versus ALOX15-negative cases. A, Group 1, more than 15 eos/HPF in the distal esophagus only. B, Group 2, more than 15 eos/HPF in the proximal esophagus only. C, Group 3, more than 15 eos/HPF in the distal and proximal esophagus. D, Total, groups 1 to 3.
ALOX15 in eosinophilic esophagitis than 15 eos/HPF and positive ALOX15 immunostaining in squamous cells (Fig. 1C), and a subset of biopsies with more than 15 eos/HPF that were negative for ALOX15 immunostaining in squamous cells but exhibiting positive staining of eosinophils (Fig. 1D).
3.3. Relationship between ALOX15 immunohistochemistry results and clinical diagnosis A total of 53 (85%) patients were classified clinically as having EoE, and 9 (15%) patients were classified as having GERD. ALOX15 immunostaining was positive in biopsies of 48 (90.5%) patients classified as having EoE versus 4 (44.4%) biopsies of patients classified as having GERD (P = .003). ALOX15-positive biopsies had a higher peak number of eos/HPF when compared with ALOX15-negative samples (50.5 ± 3.8 versus 28.6 ± 4.8; P b .01; Fig. 2). Similarly, the average peak number of eos/HPF was higher in patients clinically classified as having EoE than in patients classified as having GERD (49.9 ± 3.7 versus 29.4 ± 6.6; P = .03; Fig. 3). Within the patient group classified as having GERD, ALOX15-positive biopsies showed a tendency to having a higher average number of peak eosinophils/HPF (38.0 ± 14.3 versus 22.60 ± 3.0; P = .2). However, within the group of patients classified as having EoE, ALOX15-positive and
5 ALOX15-negative biopsies had similar average numbers of peak eosinophils/HPF (42.8 ± 4.9 versus 38.8 ± 10.1; P = .7). Overall, there was a correlation between the peak number of eos/HPF and ALOX15 immunostaining regardless of the clinical diagnosis in both the proximal (r = 0.23, P b .01) and distal esophagus (r = 0.55, P b .01; Fig. 4). The minimum number of eos/HPF in which there was ALOX15-positive immunostaining was 7 eos/HPF. 3.3.1. Group 1 (N15 eos/HPF in the distal biopsy only) There were 24 patients with more than 15 eos/HPF in the distal esophagus and less than 15 eos/HPF in the proximal esophagus. Following the diagnostic criteria specified above, 16 (67%) were classified as having EoE and 8 (33%) as having GERD. Of the 16 patients classified as having EoE, 13 were positive for ALOX15 (81%) versus 4 of 8 (50%) patients classified as having GERD (Table 2). ALOX15 expression was detected in the distal biopsy (N15 eos/HPF) in all positive cases and in the distal and proximal in 2 cases. Both cases with ALOX15-positive proximal esophageal biopsies (b15 eos/HPF) were classified clinically as EoE. Although this group of patients was characterized by more than 15 eos/HPF in the distal esophagus only, the proximal esophagus frequently showed intraepithelial eosinophils, with an average peak number of eos/HPF of 8.5 ± 1.5 in the ALOX15-positive cases and 4.1 ± 1.0 in the ALOX15negative ones (P = .20).
Fig. 3 Relationship between clinical diagnosis and eosinophil count. Peak number of intraepithelial eosinophils in biopsies of patients clinically classified as having EoE versus GERD. A, Group 1, more than 15 eos/HPF in the distal esophagus only. B, Group 2, more than 15 eos/HPF in the proximal esophagus only. C, Group 3, more than 15 eos/HPF in the distal and proximal esophagus. D, Total, groups 1 to 3.
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Fig. 4 Correlation of ALOX15 immunostaining score with peak number of intraepithelial eosinophils/HPF. A, Proximal esophagus. B, Distal esophagus.
3.3.2. Group 2 (N15 eos/HPF in the proximal biopsy only) There were 5 patients with more than 15 eos/HPF in the proximal esophagus and less than 15 eos/HPF in the distal esophagus. Following the diagnostic criteria specified above, 4 (80%) were clinically classified as having EoE and 1 (20%) as having GERD. All patients classified as having EoE were ALOX15 positive, and the 1 patient classified as having GERD was ALOX15 negative (Table 2). ALOX15 was detected in the proximal biopsies of all positive cases. In 2 of 4 positive cases, ALOX15 was also detected in the distal biopsy (b15 eos/HPF), where the average peak number of eos/HPF was 11.0 ± 3.0 versus 6.0 ± 3.2 in the ALOX15negative ones (P = .36). 3.3.3. Group 3 (N15 eos/HPF in both the proximal and distal biopsies) There were 33 patients with more than 15 eos/HPF in both proximal and distal esophagus. All patients in this group were classified as having EoE, and 31 (94%) were positive for ALOX15 (Table 2). 3.3.4. Control groups All patients with esophagitis containing 6 to 15 eos/HPF were clinically interpreted as having GERD. ALOX15 was positive in 2 (22%) of 9 patients with biopsies containing 6 to 15 eos/HPF, negative in all patients with GERD with
Table 2
biopsies containing up to 5 eos/HPF, in all biopsies from patients with candida esophagitis, and in all biopsies from patients with normal squamous mucosa.
3.4. Diagnostic implications of ALOX15 expression All patients enrolled in this study received a clinical diagnosis of EoE or GERD by the usual methods without the use of ALOX15 immunohistochemistry. To evaluate a potential clinical use of ALOX15 immunostaining, we compared the prevalence of a clinical diagnosis of EoE with and without ALOX15 immunostain. In the entire study group, 85% of the patients with at least 1 biopsy with more than 15 eos/HPF were clinically diagnosed as having EoE. This prevalence increases to 92% of the patients with biopsies with more than 15 eos/HPF and positive ALOX15 stain. Within group 1, 66% of the patients were diagnosed as having EoE with eosinophil counts alone, and the prevalence increased to 76% in patients with positive ALOX15 stain. Within group 2, 4 of 5 patients were clinically diagnosed as having EoE (80%) with biopsy findings alone, but 100% of patients with positive ALOX15 stain. Within group 3, all patients with both distal and proximal esophageal biopsies exhibiting more than 15 eos/HPF were clinically diagnosed as having EoE, and therefore, prevalence remains unchanged with ALOX15 stain. In summary, the results of this analysis
Correlation between ALOX15 immunohistochemistry and clinical diagnosis in patients with N15 eosinophils/HPF
EoE GERD Sensitivity Specificity Positive predictive value Negative predictive value
Total (n = 62), positive/total
Group 1 (n = 24), positive/total
Group 2 (n = 5), positive/total
Group 3 (n = 33), positive/total
48/53 4/9 90.5% 55% 92% 50%
13/16 4/8 81.2% 50% 76.4% 57.1%
4/4 0/1 100% 100% 100% 100%
31/33 0/0 94% NA 100% 0%
NOTE. Group 1, more than 15 eos/HPF in distal esophagus only; group 2, more than 15 eos/HPF in proximal esophagus only; group 3, more than 15 eos/ HPF in distal and proximal esophagus. Abbreviations: EoE, eosinophilic esophagitis; NA, not applicable.
ALOX15 in eosinophilic esophagitis
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Table 3 Prevalence of the diagnosis of EoE between eosinophil count alone and with ALOX15 immunohistochemistry
N15 eos/HPF alone ALOX15 positive ALOX15 negative Risk ratio (95% CI) Odds ratio (95% CI) P
Total (n = 62)
Group 1 (n = 24)
Group 2 Group 3 (n = 5) (n = 33)
85%
66%
80%
100%
92%
76%
100%
100%
50%
42%
0%
100%
1.84 (0.99-3.44) 12 (2.40-59.75) .0005
1.78 NA (0.72-4.36) 4.33 NA (0.66-28.11) .1123 .0253
1 (1-1) NA NA
NOTE. Group 1, more than 15 eos/HPF in distal esophagus only; group 2, more than 15 eos/HPF in proximal esophagus only; group 3, more than 15 eos/HPF in distal and proximal esophagus. Abbreviations: CI, confidence interval; NA, not applicable.
show that the addition of ALOX15 immunohistochemistry in biopsies from patients with increased intraepithelial eosinophils could help to better predict a clinical presentation more consistent with EoE or with GERD (Table 3).
4. Discussion The goal of this study was to correlate ALOX15 expression with the clinical classification of EoE or GERD in a group of consecutive pediatric patients biopsied from both the proximal and distal esophagus in which at least 1 of the biopsies exhibited esophageal eosinophilia (N15 eos/ HPF). In many of these patients, the differential diagnosis between EoE and GERD may be quite challenging, particularly in those with increased intraepithelial eosinophils limited to the distal esophagus. This study concludes that the sensitivity of ALOX15 for detecting EoE in patients with more than 15 eos/HPF in the distal esophagus only is relatively high (81.2%); however, the specificity is much lower because a proportion of the ALOX15-positive biopsies were from patients clinically classified as having GERD. Although not statistically significant, these biopsies also had, on average, a higher number of intraepithelial eosinophils when compared with ALOX15-negative GERD cases (38.0 ± 14.3 versus 22.60 ± 3.0; P = .2). It is possible that some of the patients with higher numbers of intraepithelial eosinophils were clinically classified as having reflux esophagitis based on successful response to PPI therapy. Many of these patients may indeed belong to the already recognized category of PPI-responsive esophageal eosinophilia [1]. This is a group of patients that have not yet been fully characterized clinicopathologically. The recent observation that the PPI omeprazole has been
shown to control esophageal eosinophilia through a mechanism that is independent of gastric acid suppression [19,20] may be one explanation for PPI-responsive EoE. In addition, it is possible that some of these patients were indeed responding to a placebo effect of the PPI, as described in a randomized placebo-controlled trial of fluticasone propionate therapy in pediatric patients with EoE who showed histologic remission in up to 18% of the patients treated with placebo [21]. Involvement of the proximal esophagus has long been considered a clinically useful criterion for the diagnosis of EoE because GERD rarely involves the proximal esophagus. This is also confirmed in this study, as most patients with proximal esophageal biopsies with more than 15 eos/HPF were clinically classified as having EoE. Similar to our previous study, ALOX15 immunohistochemistry was negative in all patients with GERD with biopsies containing up to 5 eos/HPF. In addition, in this study, we included a control group of reflux patients with esophageal biopsies containing 6 to 15 eos/HPF. The prevalence of ALOX15-positive immunostaining in this group was still low (22%); however, again, it raises the possibility that some of these patients may have PPIresponsive EoE. The absence of stain in all candida esophagitis samples tested provides further evidence for the role of ALOX15 in mediating the allergic response, as the inflammatory infiltrate in candida esophagitis is primarily composed of polymorphonuclear cells (as opposed to eosinophils) frequently numbering more than 15 per HPF. One limitation of the current study was that esophageal pH testing was not performed, and therefore, reflux was not objectively documented. Because pH monitoring is currently not indicated in the initial evaluation of patients suspected to have reflux disease [22], a prospective study including esophageal pH testing would be necessary to address this issue. This would also help to detect patients with histopathologic features consistent with EoE and with active GERD. Another limitation of this study is the low number of patients who were clinically diagnosed as having GERD. Because the cohort included all consecutive biopsies with more than 15 eos/HPF at any site within the esophagus, most patients were clinically classified as EoE. Similar to other studies that aimed to identify biomarkers of EoE, ALOX15 expression shows a significant overlap between patients with EoE and patients with GERD with ambiguous histopathologic and clinical findings. A recently reported study using a 96-gene expression platform that was highly sensitive and specific for EoE in patients with wellcharacterized disease, when evaluated in a cohort of patients with esophageal biopsies containing between 6 and 15 eos/ HPF and a less definitive clinical presentation, the test result was positive in 47% of patients [18]. In summary, the results of this study strengthened the association of ALOX15 mucosal expression with EoE and identified a small subset of patients with GERD with increased ALOX15 expression. Because immunohistochemistry is an
8 inexpensive test that is easily performed in the pathology laboratory, ALOX15 immunostaining could be clinically useful in cases with discrepant proximal/distal biopsy findings, particularly in those patients with increased intraepithelial eosinophils limited to the distal esophagus.
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Original contribution
Correlation of ALOX15 expression with eosinophilic or reflux esophagitis in a cohort of pediatric patients with esophageal eosinophilia☆,☆☆ Andres Matoso MD a,1 , Danisha Allen MD a,1 , Michael Herzlinger MD b , Jason Ferreira MD c , Sonja Chen MD a , Shaolei Lu MD, PhD a , Valeria Fabre MD d , Renee Monahan BSc a , Dongfang Yang MSc a , Lelia Noble a , Shamlal Mangray MD a , Murray B. Resnick MD, PhD a,⁎ a
Department of Pathology and Laboratory Medicine, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI 02903 b Department of Pediatrics, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI 02903 c Department of Medicine, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, RI 02903 d Department of Medicine, Johns Hopkins Hospital, Baltimore, MD 21287 Received 18 November 2013; revised 13 January 2014; accepted 17 January 2014
Keywords: Eosinophilic esophagitis; Biomarker; ALOX15
Summary The differential diagnosis between eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) is often challenging. We recently showed that the ALOX15 protein is expressed in 95% of esophageal biopsies from patients with a definitive diagnosis of EoE. Here we correlated ALOX15 expression with the clinical classification of EoE or GERD in a cohort of consecutive pediatric patients (n = 62) with at least 1 esophageal biopsy containing at least 15 eosinophils per high-power field (eos/HPF). The patients were categorized into the following groups: (1) at least 15 eos/HPF in the distal esophagus only (n = 24), (2) at least 15 eos/HPF in the proximal esophagus only (n = 5), and (3) at least 15 eos/HPF in the distal and proximal biopsies (n = 33). Control groups included patients with GERD with biopsies containing 6 to 15 eos/HPF (n = 9), patients with GERD with 5 eos/HPF or less (n = 15), patients with candida esophagitis (n = 15), and patients with normal biopsies (n = 15). ALOX15 was positive in 90.5% of patients with EoE (13/16 in group 1, 4/4 in group 2, 31/33 in group 3) versus 44% of patients with GERD (4/8 in group 1, 0/1 in group 2, and 0/0 in group 3), 2 of 9 (22%) of patients with 6 to 15 eos/HPF, and was negative in all patients with GERD with biopsies containing 5 eos/HPF or less, all patients with candida esophagitis, and all normal controls. In conclusion, ALOX15 is a sensitive marker of EoE; however, subpopulations of patients with GERD with N5 eos/HPF also express ALOX15. Positive ALOX15 expression is more prevalent in EoE than in GERD and may prove to be a useful diagnostic marker in patients with discrepant biopsy findings between the proximal and distal esophagus. © 2014 Elsevier Inc. All rights reserved.
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Disclosures: This study was supported by the Molecular Pathology Core of the COBRE Center for Cancer Research Development, funded by the National Institute of General Medical Sciences of the National Institute of Health (P20GM103421). ☆☆ Conflict of interest: The authors declare no competing interest. ⁎ Corresponding author. Department of Pathology, 593 Eddy St, Providence, RI 02903. E-mail address: [email protected] (M. B. Resnick). 1 These authors contributed equally to this work. 0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2014.01.021
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1. Introduction The clinical diagnosis of eosinophilic esophagitis (EoE) is based on the correlation of clinical, endoscopic, and histopathologic findings. According to the most recent consensus recommendations in EoE [1], 1 or more biopsy specimens must show eosinophil-predominant inflammation, with a minimum threshold of 15 eosinophils per high-power field (eos/HPF). The histopathologic findings that support the diagnosis of EoE, including superficial clustering of intraepithelial eosinophils, basal cell hyperplasia, and elongation of papillae, are not entirely specific of EoE, and there is a considerable overlap with features of gastroesophageal reflux disease (GERD) [2-7]. For instance, some patients with strong clinical evidence of EoE may have biopsies with many of the features mentioned, but with a peak eosinophil count of less than 15/HPF [1]. In addition, a subgroup of patients with typical symptoms and biopsy findings of EoE who have had GERD excluded may show favorable clinicopathological response to treatment with proton pump inhibitors (PPI-responsive esophageal eosinophilia) [8-10]. The distinction between EoE and GERD is clinically important because medical treatment differs between these 2 entities; while reflux disease is treated with antacid medication, EoE responds to topical steroids and dietary allergen elimination. Several studies have been performed to better characterize the molecular signature of EoE [11-16]. We previously conducted a validation study to confirm the presence of several novel biomarkers in EoE detectable by immunohistochemistry [17]. In this study, we found that 95% of the biopsies of patients with clinically proven EoE were positive for the protein arachidonate-15 lipoxigenase (ALOX15). More recently, Wen et al [18] introduced an EoE diagnostic panel based on a 96-gene expression profile that included overexpression of ALOX15. In Wen’s study, ALOX15 was expressed more than 1000-fold higher in biopsies of patients with EoE compared with normal controls. Although the overexpression of ALOX15 was highly sensitive and specific for EoE, all of the biopsies included in our previous study were from patients with straightforward diagnoses who presented with all of the histopathologic and clinical features of EoE, including involvement of the proximal esophagus. In the present study, we aimed to evaluate ALOX15 expression in a cohort of consecutive pediatric patients with esophageal eosinophilia and correlated the findings with their clinical classification as EoE versus GERD.
2. Materials and methods
A. Matoso et al. tal. The cohort included consecutive pediatric patients with biopsies from the distal and proximal esophagus obtained from 2009 through 2012 (ages 0-18 years) with at least 1 biopsy with 15 or more intraepithelial eos/HPF. Control groups included patients with GERD with biopsies containing 6 to 15 eos/HPF, patients with GERD with biopsies containing up to 5 eos/HPF, patients with candida esophagitis, and patients with normal squamous mucosa. Clinical diagnosis of EoE was made as defined by the 2011 consensus guidelines [1]. Specifically, patients were required to have symptoms of esophageal dysfunction, a trial with PPIs without remission of symptoms, clinical history of other allergies and/or food impaction, endoscopic findings more consistent with EoE (furrows, rings, and ridging), 1 or more esophageal biopsies with a minimum of 15 eos/HPF, and other causes of esophageal eosinophilia excluded. Clinical diagnosis of GERD included clinical presentation and histopathology consistent with reflux in patients with good symptomatic response to acid suppression. Two pathologists (A. M. and M. R.) reviewed the hematoxylin and eosin slides to confirm the histologic diagnosis. A pediatric gastroenterologist (M. H.) reviewed the patients' medical records to ensure that these patients fit the diagnostic selection criteria for either EoE or GERD.
2.2. Immunohistochemistry Immunohistochemistry was performed using the antibody against ALOX15 (11-K; Santa Cruz Biotechnology, Santa Cruz, CA; 1:100 dilution) as described previously [17]. Known cases of EoE were used as a positive control. Negative controls were achieved by replacing the primary antibody with normal serum. Results were scored using a semiquantitative system, which takes into consideration the extent and intensity of staining. For extent, points were assigned as follows: 0, negative; 1, up to 10% positive cells; 2, 10% to 50% positive cells; and 3, more than 50% positive cells. For intensity, points were assigned as follows: 0, negative (no stain); 1, weakly positive (pale brown cytoplasmic stain); 2, moderately positive (dark-brown cytoplasmic stain in part or all the cytoplasm); and 3, intensely positive (very dark-brown stain covering the entire cytoplasm of all positive cells). A total score of 3 or higher was considered positive and 2 or lower was considered negative. Only staining of squamous cells was considered positive staining. Owing to the discontinuous nature of EoE, “patchy” lesions were graded on areas of histologic changes consistent with EoE. A. M. and M. R. independently scored each section without knowledge of the clinical follow-up. Concordance was high, and discrepant cases were reviewed to reach consensus.
2.1. Patients and tissue samples Archival esophageal biopsies were obtained from the Pathology Department of the Rhode Island Hospital (Providence, RI), following a protocol approved by the institutional review board of Lifespan/Rhode Island Hospi-
2.3. Statistical analysis Statistical analysis was performed using the STATA (version 13) software (Stata, College Station, TX). P values
ALOX15 in eosinophilic esophagitis
3 etc) were excluded from analysis. Control groups included patients with GERD with biopsies containing 6 to 15 eos/HPF (n = 9), patients with GERD with biopsies containing up to 5 eos/HPF (n = 15), patients with candida esophagitis (n = 15), and patients with normal esophageal biopsies (n = 15). Because involvement of the proximal esophagus is characteristic of EoE and involvement of the distal esophagus is more characteristic of GERD, patients were grouped as follows: group 1, more than 15 eos/HPF in the distal esophagus only (n = 24); group 2, more than 15 eos/ HPF in the proximal esophagus only (n = 5); and group 3, more than 15 eos/HPF in both distal and proximal esophagus (n = 33). The clinicopathological characteristics of all patients are summarized in Table 1.
were calculated using the Fisher exact test, t test, Woolf test, and Spearman rank test. Differences were expressed as mean ± SEM and considered significant at a P value of equal or less than .05.
3. Results 3.1. Study population A total of 77 patients were identified in the pathology database. All subjects underwent upper endoscopy for evaluation of gastrointestinal symptoms suggestive of esophageal dysfunction. As per protocol in our institution, biopsies were taken from the proximal and distal esophagus; all of the study patients had at least 1 esophageal biopsy with 15 or more intraepithelial eos/HPF. Only first-time biopsies from patients without history of any EoE-specific therapy were included in the study. Medical records were reviewed for documentation of symptoms, endoscopic findings, and medical management, and patients were classified as having EoE or GERD based on the criteria outlined in Materials and Methods. Instances in which there were insufficient data to determine a clinical diagnosis of either EoE or GERD (n = 8) or in which there was a confounding gastrointestinal disorder (n = 7, celiac, Crohn disease, Helicobacter pylori infection,
Table 1
3.2. ALOX15 immunohistochemistry Expression of ALOX15 was detected in the cytoplasm of squamous epithelial cells and eosinophils as previously described [17]. Although scoring was restricted to epithelial expression in squamous cells, positive staining of eosinophils was used as an internal control in cases of negative squamous staining, as they were consistently positive. Glandular cells at the gastroesophageal junction and subepithelial stromal cells were consistently negative (Fig. 1A and B). There were occasional biopsies with less
Clinicopathological characteristics of patients
Age (y), mean ± SEM Sex (male/female) Symptoms Abdominal pain (%) Vomiting (%) Dysphagia (%) Food impaction (%) Heartburn (%) Failure to thrive (%) Endoscopy Normal (%) Erythema (%) Rings (%) Ridging (%) Furrows (%) White plaques (%) Allergies Food allergy (%) Asthma (%) Rhinitis or dermatitis (%) Eosinophils/HPF, mean ± SEM Distal Proximal
Group 1 (≥15 eos/HPF in distal only; n = 24)
Group 2 (≥15 eos/HPF in proximal only; n = 5)
Group 3 (≥15 eos/HPF in proximal and distal; n = 33)
GERD with 6-15 eos/HPF (n = 9)
10.9 ± 1.13 16:8
9.8 ± 1.8 2:3
12.1 ± 0.8 21:12
10.4 ± 1.6 5:4
7 (29) 10 (41) 15 (63) 6 (25) 6 (25) 10 (41)
0 0 3 4 2 3
(0) (0) (60) (80) (40) (60)
8 (24) 16 (48) 20 (60) 10 (30) 7 (21) 19 (58)
2 4 3 3 2 1
(22) (44) (33) (33) (22) (11)
7 (29) 5 (21) 2 (8) 9 (37) 12 (50) 3 (13)
1 1 1 4 2 3
(20) (20) (20) (80) (40) (60)
12 (36) 3 (9) 12 (36) 21 (64) 15 (45) 2 (6)
2 0 0 1 1 0
(22) (0) (0) (11) (11) (0)
11 (46) 8 (33) 8 (33)
2 (40) 1 (20) 1 (20)
12 (36) 3 (9) 17 (51)
1 (11) 3 (33) 2 (22)
38.2 ± 4.5 4.5 ± 0.9
8.2 ± 2.5 38.6 ± 8.4
43.9 ± 3.9 49.4 ± 5.0
10.6 ± 0.9 1.6 ± 0.6
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A. Matoso et al.
Fig. 1 ALOX15 immunohistochemistry in esophageal mucosa. A, Low-power view of a biopsy from the gastroesophageal junction shows diffuse and strong ALOX15 immunostaining in the squamous epithelium (arrow) and negative staining in the glandular (cardia-type) epithelium (arrowhead). B, Strong cytoplasmic staining of the squamous cells, whereas subepithelial stroma is negative (arrowhead). C, Positive staining both the cytoplasm of squamous cells (arrow) and in eosinophils (arrowheads). D, An esophageal biopsy showing negative staining in the squamous cells but positive staining in eosinophils (arrowheads).
Fig. 2 Relationship between ALOX15 immunostaining and eosinophil count. Peak number of intraepithelial eosinophils in biopsies of patients of ALOX15-positive versus ALOX15-negative cases. A, Group 1, more than 15 eos/HPF in the distal esophagus only. B, Group 2, more than 15 eos/HPF in the proximal esophagus only. C, Group 3, more than 15 eos/HPF in the distal and proximal esophagus. D, Total, groups 1 to 3.
ALOX15 in eosinophilic esophagitis than 15 eos/HPF and positive ALOX15 immunostaining in squamous cells (Fig. 1C), and a subset of biopsies with more than 15 eos/HPF that were negative for ALOX15 immunostaining in squamous cells but exhibiting positive staining of eosinophils (Fig. 1D).
3.3. Relationship between ALOX15 immunohistochemistry results and clinical diagnosis A total of 53 (85%) patients were classified clinically as having EoE, and 9 (15%) patients were classified as having GERD. ALOX15 immunostaining was positive in biopsies of 48 (90.5%) patients classified as having EoE versus 4 (44.4%) biopsies of patients classified as having GERD (P = .003). ALOX15-positive biopsies had a higher peak number of eos/HPF when compared with ALOX15-negative samples (50.5 ± 3.8 versus 28.6 ± 4.8; P b .01; Fig. 2). Similarly, the average peak number of eos/HPF was higher in patients clinically classified as having EoE than in patients classified as having GERD (49.9 ± 3.7 versus 29.4 ± 6.6; P = .03; Fig. 3). Within the patient group classified as having GERD, ALOX15-positive biopsies showed a tendency to having a higher average number of peak eosinophils/HPF (38.0 ± 14.3 versus 22.60 ± 3.0; P = .2). However, within the group of patients classified as having EoE, ALOX15-positive and
5 ALOX15-negative biopsies had similar average numbers of peak eosinophils/HPF (42.8 ± 4.9 versus 38.8 ± 10.1; P = .7). Overall, there was a correlation between the peak number of eos/HPF and ALOX15 immunostaining regardless of the clinical diagnosis in both the proximal (r = 0.23, P b .01) and distal esophagus (r = 0.55, P b .01; Fig. 4). The minimum number of eos/HPF in which there was ALOX15-positive immunostaining was 7 eos/HPF. 3.3.1. Group 1 (N15 eos/HPF in the distal biopsy only) There were 24 patients with more than 15 eos/HPF in the distal esophagus and less than 15 eos/HPF in the proximal esophagus. Following the diagnostic criteria specified above, 16 (67%) were classified as having EoE and 8 (33%) as having GERD. Of the 16 patients classified as having EoE, 13 were positive for ALOX15 (81%) versus 4 of 8 (50%) patients classified as having GERD (Table 2). ALOX15 expression was detected in the distal biopsy (N15 eos/HPF) in all positive cases and in the distal and proximal in 2 cases. Both cases with ALOX15-positive proximal esophageal biopsies (b15 eos/HPF) were classified clinically as EoE. Although this group of patients was characterized by more than 15 eos/HPF in the distal esophagus only, the proximal esophagus frequently showed intraepithelial eosinophils, with an average peak number of eos/HPF of 8.5 ± 1.5 in the ALOX15-positive cases and 4.1 ± 1.0 in the ALOX15negative ones (P = .20).
Fig. 3 Relationship between clinical diagnosis and eosinophil count. Peak number of intraepithelial eosinophils in biopsies of patients clinically classified as having EoE versus GERD. A, Group 1, more than 15 eos/HPF in the distal esophagus only. B, Group 2, more than 15 eos/HPF in the proximal esophagus only. C, Group 3, more than 15 eos/HPF in the distal and proximal esophagus. D, Total, groups 1 to 3.
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A. Matoso et al.
Fig. 4 Correlation of ALOX15 immunostaining score with peak number of intraepithelial eosinophils/HPF. A, Proximal esophagus. B, Distal esophagus.
3.3.2. Group 2 (N15 eos/HPF in the proximal biopsy only) There were 5 patients with more than 15 eos/HPF in the proximal esophagus and less than 15 eos/HPF in the distal esophagus. Following the diagnostic criteria specified above, 4 (80%) were clinically classified as having EoE and 1 (20%) as having GERD. All patients classified as having EoE were ALOX15 positive, and the 1 patient classified as having GERD was ALOX15 negative (Table 2). ALOX15 was detected in the proximal biopsies of all positive cases. In 2 of 4 positive cases, ALOX15 was also detected in the distal biopsy (b15 eos/HPF), where the average peak number of eos/HPF was 11.0 ± 3.0 versus 6.0 ± 3.2 in the ALOX15negative ones (P = .36). 3.3.3. Group 3 (N15 eos/HPF in both the proximal and distal biopsies) There were 33 patients with more than 15 eos/HPF in both proximal and distal esophagus. All patients in this group were classified as having EoE, and 31 (94%) were positive for ALOX15 (Table 2). 3.3.4. Control groups All patients with esophagitis containing 6 to 15 eos/HPF were clinically interpreted as having GERD. ALOX15 was positive in 2 (22%) of 9 patients with biopsies containing 6 to 15 eos/HPF, negative in all patients with GERD with
Table 2
biopsies containing up to 5 eos/HPF, in all biopsies from patients with candida esophagitis, and in all biopsies from patients with normal squamous mucosa.
3.4. Diagnostic implications of ALOX15 expression All patients enrolled in this study received a clinical diagnosis of EoE or GERD by the usual methods without the use of ALOX15 immunohistochemistry. To evaluate a potential clinical use of ALOX15 immunostaining, we compared the prevalence of a clinical diagnosis of EoE with and without ALOX15 immunostain. In the entire study group, 85% of the patients with at least 1 biopsy with more than 15 eos/HPF were clinically diagnosed as having EoE. This prevalence increases to 92% of the patients with biopsies with more than 15 eos/HPF and positive ALOX15 stain. Within group 1, 66% of the patients were diagnosed as having EoE with eosinophil counts alone, and the prevalence increased to 76% in patients with positive ALOX15 stain. Within group 2, 4 of 5 patients were clinically diagnosed as having EoE (80%) with biopsy findings alone, but 100% of patients with positive ALOX15 stain. Within group 3, all patients with both distal and proximal esophageal biopsies exhibiting more than 15 eos/HPF were clinically diagnosed as having EoE, and therefore, prevalence remains unchanged with ALOX15 stain. In summary, the results of this analysis
Correlation between ALOX15 immunohistochemistry and clinical diagnosis in patients with N15 eosinophils/HPF
EoE GERD Sensitivity Specificity Positive predictive value Negative predictive value
Total (n = 62), positive/total
Group 1 (n = 24), positive/total
Group 2 (n = 5), positive/total
Group 3 (n = 33), positive/total
48/53 4/9 90.5% 55% 92% 50%
13/16 4/8 81.2% 50% 76.4% 57.1%
4/4 0/1 100% 100% 100% 100%
31/33 0/0 94% NA 100% 0%
NOTE. Group 1, more than 15 eos/HPF in distal esophagus only; group 2, more than 15 eos/HPF in proximal esophagus only; group 3, more than 15 eos/ HPF in distal and proximal esophagus. Abbreviations: EoE, eosinophilic esophagitis; NA, not applicable.
ALOX15 in eosinophilic esophagitis
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Table 3 Prevalence of the diagnosis of EoE between eosinophil count alone and with ALOX15 immunohistochemistry
N15 eos/HPF alone ALOX15 positive ALOX15 negative Risk ratio (95% CI) Odds ratio (95% CI) P
Total (n = 62)
Group 1 (n = 24)
Group 2 Group 3 (n = 5) (n = 33)
85%
66%
80%
100%
92%
76%
100%
100%
50%
42%
0%
100%
1.84 (0.99-3.44) 12 (2.40-59.75) .0005
1.78 NA (0.72-4.36) 4.33 NA (0.66-28.11) .1123 .0253
1 (1-1) NA NA
NOTE. Group 1, more than 15 eos/HPF in distal esophagus only; group 2, more than 15 eos/HPF in proximal esophagus only; group 3, more than 15 eos/HPF in distal and proximal esophagus. Abbreviations: CI, confidence interval; NA, not applicable.
show that the addition of ALOX15 immunohistochemistry in biopsies from patients with increased intraepithelial eosinophils could help to better predict a clinical presentation more consistent with EoE or with GERD (Table 3).
4. Discussion The goal of this study was to correlate ALOX15 expression with the clinical classification of EoE or GERD in a group of consecutive pediatric patients biopsied from both the proximal and distal esophagus in which at least 1 of the biopsies exhibited esophageal eosinophilia (N15 eos/ HPF). In many of these patients, the differential diagnosis between EoE and GERD may be quite challenging, particularly in those with increased intraepithelial eosinophils limited to the distal esophagus. This study concludes that the sensitivity of ALOX15 for detecting EoE in patients with more than 15 eos/HPF in the distal esophagus only is relatively high (81.2%); however, the specificity is much lower because a proportion of the ALOX15-positive biopsies were from patients clinically classified as having GERD. Although not statistically significant, these biopsies also had, on average, a higher number of intraepithelial eosinophils when compared with ALOX15-negative GERD cases (38.0 ± 14.3 versus 22.60 ± 3.0; P = .2). It is possible that some of the patients with higher numbers of intraepithelial eosinophils were clinically classified as having reflux esophagitis based on successful response to PPI therapy. Many of these patients may indeed belong to the already recognized category of PPI-responsive esophageal eosinophilia [1]. This is a group of patients that have not yet been fully characterized clinicopathologically. The recent observation that the PPI omeprazole has been
shown to control esophageal eosinophilia through a mechanism that is independent of gastric acid suppression [19,20] may be one explanation for PPI-responsive EoE. In addition, it is possible that some of these patients were indeed responding to a placebo effect of the PPI, as described in a randomized placebo-controlled trial of fluticasone propionate therapy in pediatric patients with EoE who showed histologic remission in up to 18% of the patients treated with placebo [21]. Involvement of the proximal esophagus has long been considered a clinically useful criterion for the diagnosis of EoE because GERD rarely involves the proximal esophagus. This is also confirmed in this study, as most patients with proximal esophageal biopsies with more than 15 eos/HPF were clinically classified as having EoE. Similar to our previous study, ALOX15 immunohistochemistry was negative in all patients with GERD with biopsies containing up to 5 eos/HPF. In addition, in this study, we included a control group of reflux patients with esophageal biopsies containing 6 to 15 eos/HPF. The prevalence of ALOX15-positive immunostaining in this group was still low (22%); however, again, it raises the possibility that some of these patients may have PPIresponsive EoE. The absence of stain in all candida esophagitis samples tested provides further evidence for the role of ALOX15 in mediating the allergic response, as the inflammatory infiltrate in candida esophagitis is primarily composed of polymorphonuclear cells (as opposed to eosinophils) frequently numbering more than 15 per HPF. One limitation of the current study was that esophageal pH testing was not performed, and therefore, reflux was not objectively documented. Because pH monitoring is currently not indicated in the initial evaluation of patients suspected to have reflux disease [22], a prospective study including esophageal pH testing would be necessary to address this issue. This would also help to detect patients with histopathologic features consistent with EoE and with active GERD. Another limitation of this study is the low number of patients who were clinically diagnosed as having GERD. Because the cohort included all consecutive biopsies with more than 15 eos/HPF at any site within the esophagus, most patients were clinically classified as EoE. Similar to other studies that aimed to identify biomarkers of EoE, ALOX15 expression shows a significant overlap between patients with EoE and patients with GERD with ambiguous histopathologic and clinical findings. A recently reported study using a 96-gene expression platform that was highly sensitive and specific for EoE in patients with wellcharacterized disease, when evaluated in a cohort of patients with esophageal biopsies containing between 6 and 15 eos/ HPF and a less definitive clinical presentation, the test result was positive in 47% of patients [18]. In summary, the results of this study strengthened the association of ALOX15 mucosal expression with EoE and identified a small subset of patients with GERD with increased ALOX15 expression. Because immunohistochemistry is an
8 inexpensive test that is easily performed in the pathology laboratory, ALOX15 immunostaining could be clinically useful in cases with discrepant proximal/distal biopsy findings, particularly in those patients with increased intraepithelial eosinophils limited to the distal esophagus.
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