The EPX-mAb based immunohistochemical assay described in this report represents a novel tool and systematic method to assess esophageal tissues for evidence of eosinophilic inflammation in both children and adults. That is, the previously limited availability of eosinophil-specific antibody-staining options for immunohistochemical assays that are sensitive, reproducible, and useful in the most commonly available format (i.e., archived formalin-fixed paraffin-embedded tissues) had prevented the development and use of this strategy to evaluate patients. However, development of an eosinophil granule protein-specific monoclonal antibody, together with a novel histological scoring system, allowed for not only the specific detection of eosinophils and eosinophil degranulation but also provided a sensitive and cost effective method that specifically identified patients with EoE vs. GERD on the basis of a single histological evaluation. In addition, it allowed for the differentiation of “Indeterminate” patients in whom the diagnosis of EoE was not certain.
This study assessed children and adults with previously established diagnoses and not consecutive patients. In turn, this may have led to a selection bias ultimately leading to either higher EPX-mAb
based scores for patients with EoE or lower EPX-mAb
based scores for GERD and/or control subjects. However, in this rapidly evolving field, the utility of our retrospective analysis of very well defined patients has proven to be quite valuable, especially in light of the fact that a number of challenges have led to difficulty in obtaining a true diagnosis of EoE 4, 8
. As a consequence, we propose the current scoring system as an initial effort to evaluate tissues that can now be used to prospectively study consecutive patients with esophageal diseases.
These findings address several critical and timely unmet needs in the care of patients which display eosinophilic esophageal inflammation: (i)
The use of this EPX-mAb
based scoring system allowed for the rapid identification of patients with EoE. (ii)
Esophageal eosinophilia as a pure numerical value of intact eosinophils was improved by the additional sensitivity of EPX-mAb
based immunohistochemical detection of tissue infiltrating eosinophils. This issue was highlighted in our assessments of Indeterminate pediatric patients where we found that although traditional H&E histopathological assessments failed to reveal a focus of ≥15 eosinophils/40× hpf,
75% of these cases displayed this guideline prerequisite as determined by EPX-mAb
based immunohistochemistry. The sensitivity of this strategy was further enhanced by expanding the term “eosinophilia” to include assessment and quantification of eosinophil degranulation. (iii)
Because an increasing body of evidence suggests that tissues from patients with GERD may have similar number of esophageal eosinophils as with EoE, this system allowed for differentiation between these two diseases 1, 3
. The inclusion of multiple EPX-mAb
based histological scoring parameters provided not only a statistically-significant quantitative means by which to identify EoE patients but also qualitative measures that appear to reliably differentiate these subjects from patients with GERD. In particular, a subset of EPX-mAb
based parameters may be sufficient to achieve a quick, but accurate, differential diagnosis between these esophageal diseases. For example, 24 of 26 EoE patients displayed extensive degranulation in multiple biopsies compared to only 2 of 7 biopsies from GERD patients showing a similar level of degranulation. In addition, low power assessments of the fraction of patient biopsies displaying eosinophil degranulation (Reproducibility) and the fractional area of the maximally affected tissue fragment that displayed evidence of degranulation (Patchiness) together were alone sufficient to accurately identify EoE patients relative to subjects with GERD. (iv)
Indeterminate diagnoses were clarified with EPX-mAb
based immunohistochemistry. That is, this strategy allowed for a diagnosis of EoE and GERD in patients who achieved many, but not all, of the clinical, endoscopic, and histopathologic features of these diseases. Indeed, our study provides examples of indeterminate EoE patients who nonetheless failed to achieve the prerequisite ≥15 eosinophils/40× hpf
It is interesting that some EoE and GERD patients had biopsies with many intact eosinophils but minimal levels of degranulation (e.g., patient #6 and #10 and patient #27 vs. #29 (S-Table 3
), respectively), whereas other patients displayed widespread degranulation in the presence of very few intact eosinophils (e.g., patient #44, #46, and #47). These observations suggest that the degranulation detected in the tissue sections of this study was specific/unique to individual patient biopsies and unlikely to be a consequence of artefactual events associated with tissue processing and/or handling 9
. Thus, while current guidelines propose that identification of ≥15 eosinophils/40× hpf
(in the proper clinical/endoscopic context) is required for the diagnosis of EoE, the EPX-mAb
based scoring system’s utilization of multiple parameters such as eosinophil degranulation, provides an alternative diagnostic strategy for pathologists/clinicians.
based immunohistochemical evaluations address long held technical issues related to assessment of eosinophil degranulation. Previous studies of a limited number of subjects identified that the mucosa affected by EoE displayed enhanced eosinophil degranulation (i.e., deposition of eosinophil derived neurotoxin (EDN 10
) or major basic protein (MBP 9
) relative to GERD patients. However, issues arise with the use of antibodies reactive to each of these granule protein constituents. For example, although EDN is a prominent eosinophil secondary granule protein, several studies have demonstrated that unlike eosinophil peroxidase, EDN is not eosinophil specific and is expressed in both other leukocytes (e.g., neutrophils 11
) and tissue/organs (e.g., liver 12
). In addition, the cationic character of MBP, together with its propensity to “stick” to virtually any substratum as well as its near insolubility in environments at neutral pH, have been problematic issues that may have biased earlier studies by artefactually limiting the extent of observable areas of degranulation. Moreover, these intensely staining local aggregates may also have been responsible for the perception that tissue handling/processing in earlier studies led to eosinophil degranulation 9
. In contrast, the nominal cationic character of eosinophil peroxidase (pI ~8.9 13
) together with its greater solubility at neutral pH would prevent aggregation and allow this granule protein to disperse to a greater extent.
The practical utility of this method lies in its simplicity, durability and cost-saving features. Immunohistochemical staining with EPX-mAb is straightforward, can be performed on archived formalin-fixed paraffin-embedded tissues and requires no unique technology. The assessment and scoring evaluations of the EPX-mAb based staining patterns in children and adults were reproducible as evidenced by negligible inter-observer variability. The added time and cost for this assessment will be minimal when taken in the context of achieving the correct diagnosis in a rapid time frame. For example, current diagnostic guidelines support the strategy that a diagnosis of GERD must be ruled out as a cause for esophageal eosinophilia before assigning a diagnosis of EoE. In this light, a patient would need to undergo either two (2) months of proton pump inhibition or a pH/impedance monitoring of the distal esophagus both of which can be time consuming, costly and potentially uncomfortable. However, in the context of a preponderance of clinical symptoms, endoscopic results, and evaluations of histopathology, the EPX-mAb based scoring algorithm could provide a quantitatively definitive evaluation of initial biopsies permitting an immediate diagnosis of EoE, including potentially the differentiation between difficult to diagnose EoE vs. GERD patients (i.e., Indeterminate cases). That is, despite guideline driven diagnoses that had been unclear, EPX-mAb based immunohistochemistry appears capable of identifying subsets of patients as either EoE or GERD. These observations suggest either an overlap between these two types of esophageal patients based on clinicopathologic findings or that a unique subset of EoE patients are instead simply difficult to diagnose GERD patients. In summary, we anticipate that validation and future use of this system will improve care of children and adults and allow for greater understanding of esophageal inflammatory diseases.