Abstract
Background
Central compartment atopic disease (CCAD) and eosinophilic chronic rhinosinusitis (eCRS) are two clinical phenotypes of primary diffuse type 2 chronic rhinosinusitis (CRS) defined in the European Position Paper on Rhinosinusitis 2020 classification. Currently, the distinction between these subtypes relies on phenotypic features alone.
Objective
This study aimed to investigate whether eosinophil activation differed between CCAD and eCRS.
Methods
A cross-sectional study was conducted of adult patients presenting with CCAD and eCRS who had undergone functional endoscopic sinus surgery. Routine pathology results were obtained from clinical records. Eosinophils were counted on haematoxylin and eosin-stained formalin-fixed paraffin-embedded sinonasal tissue. Eotaxin-3, eosinophil peroxidase and immunoglobulin E levels were assessed using immunohistochemistry.
Results
38 participants were included (51.7 ± 15.6 years, 47.4% female), of whom 36.8% were diagnosed with CCAD and 63.2% with eCRS. The eCRS group was characterised by older age (55.8 ± 16.3 vs 44.5 ± 11.8 years, p = 0.029), and on histology exhibited a higher degree of tissue inflammation (τb = 0.409, p = 0.011), greater proportion of patients with >100 eosinophils/high power field (87.5% vs 50%, p = 0.011), and higher absolute tissue eosinophil count (2141 ± 1947 vs 746 ± 519 cells/mm2, p = 0.013). Eotaxin-3 scores were higher in the eCRS group (5.00[5.00–6.00] vs 6.00[6.00–6.75], p = 0.015). Other outcomes were similar.
Conclusions
Eosinophil and eotaxin-3 levels were elevated in eCRS compared with CCAD, suggesting a greater degree of eosinophil stimulation and chemotaxis. Patients with CCAD were younger. Future investigation and biomarkers may better distinguish CRS subpopulations.
Keywords: chronic rhinosinusitis, eosinophils, CCAD, middle turbinate oedema, polyps, allergy, allergic rhinitis, phenotype, endotype, type 2, Th2, central compartment, inhalant allergy
Introduction
The European Position Paper on Rhinosinusitis 2020 (EPOS2020) classification system for chronic rhinosinusitis (CRS), provides some distinction to the pathophysiologic heterogeneity of CRS. The dichotomous historical description of CRS, with or without nasal polyps, offers limited insight in the underlying pathophysiology of CRS. EPOS2020 defines subtypes of primary CRS by functional anatomical distribution and endotype dominance. 1 Within the primary diffuse type 2 dominant CRS category are two phenotypes: central compartment atopic disease (CCAD) and eosinophilic chronic rhinosinusitis (eCRS).
CCAD is a CRS phenotype described in highly allergic patients, with inflammatory changes (obstructive oedema, polypoid changes and soft tissue thickening) affecting the central portion of the sinonasal cavity, largely sparing the peripheral walls, as seen on endoscopic and radiologic assessment.2,3 It is most prevalent in younger patients and strongly associated with inhalant allergen sensitisation,3,4 often requiring allergy-directed immunotherapy as part of disease management.2,5
eCRS is a widely accepted subtype of CRS 6 presenting as nasal polyposis. 1 In contrast to CCAD, it typically arises in middle age with a high concordance with adult-onset asthma, in patients with otherwise no significant history of sinus disease or allergy. Endoscopically, there is diffuse polyp growth and thick eosinophilic mucin characterised by a highly tenacious “chewing gum” appearance.5,7,8 These patients are more likely to require long-term systemic oral corticosteroids or systemic biologic therapies to prevent disease recurrence. 9
As per the EPOS2020 guidelines, both CCAD and eCRS are type 2 dominant and are defined histologically by an elevated tissue eosinophil count > 10 cells/HPF. The distinction between the two eosinophilic subtypes currently relies substantially on clinical, endoscopic and radiological features rather than histology.1,5 This study explores markers of eosinophil stimulation and chemotaxis (eotaxin-3), eosinophil degranulation (eosinophil peroxidase [EPX]) and immunoglobulin E [IgE] to assess if the conditions differ on histology.
Materials and Methods
A cross-sectional study was conducted of adult patients (≥18 years) who were diagnosed with primary diffuse type 2 dominant CRS and had undergone functional endoscopic sinus surgery performed in a tertiary rhinology clinic. Mucosa and blood were collected at the time of surgery. Patients had refrained from systemic corticosteroids for at least 4 weeks prior to surgery.
All patients had to have a tissue eosinophil count >10 cells/HPF to be included and thus defined as type 2 dominant. Overall inflammation was graded as mild, moderate or severe as per Snidvongs et al. 10 Demographic characteristics, including age, sex, smoking status (within 12 months) and current use of biologics or sublingual immunotherapy, were recorded. Detailed histopathological assessment and scoring for immunohistochemistry were performed in a blinded manner.
The study received ethics approval from the St Vincent's Hospital Sydney Human Research Ethics Committee (REGIS identifier 2019/PID14562) and tissue was collected for analysis under the Rhinology Tissue Bank Program (REGIS identifier 2019/PID13319).
Population Definitions
The patients recruited were not consecutively enrolled but selected as having either a classic CCAD or eCRS phenotype as per agreement by two senior authors (RJH and LK) within the eosinophilic CRS patient population. It is acknowledged that there are many diffuse type 2 CRS patients whose clinical characteristics may not clearly fulfil the currently two accepted phenotypes.
Those CRS patients that had history of allergic symptoms at other sites than the nose (conjunctiva, childhood asthma, eczema) plus a positive epicutaneous allergen test, endoscopic features of middle turbinate head oedema and/or polypoid change, and centrally limited mucosal changes on computed tomography (CT) were defined as having CCAD.2,4
Patients were defined as having eCRS when presenting with a clinical history of early smell reduction and adult-onset asthma, endoscopic findings of bilateral polyp growth arising from the middle meatus, presence of eosinophilic mucin (defined by a viscous “chewing gum”-like appearance on endoscopy) and diffuse inflammatory mucosal changes on CT.1,11
Assessment of Eosinophils
A preoperative blood sample was taken and total blood eosinophil count (×109 cells/L) was measured. Eosinophils were also assessed on mucosal tissue from surgery.
Blocks of formalin-fixed paraffin-embedded (FFPE) sinonasal tissue were assessed on haematoxylin and eosin (H&E) and immunohistochemistry. One slide per sinonasal biopsy underwent regressive Harris-type H&E stain with H&E, 0.5% alcohol (Australian Biostain Pty Ltd, Traralgon, Australia) using the Tissue-Tek Prisma Plus Automated Slide Stainer (Sakura, San Diego, USA). Slides were then dehydrated in ethanol and xylene, mounted and cover-slipped using Tissue-Tek Glas Coverslipper (Sakura). Slides were digitalised using the Ventana DP 200 slide scanner (Roche agnostics, Risch-Rotkreuz, Switzerland) at 40× magnification. All histopathology variables were individually assessed on digital whole slide image on QuPath v0.2.2 software 12 and scored in a blinded manner by a single observer (AS) under the instruction of an anatomical pathologist (PE).
Eosinophil Levels
The degree of tissue eosinophilia was assessed via two techniques. Firstly, tissue was examined under light microscopy, and eosinophil count was ordinally classified as 10–100 eosinophils/high power field (HPF) (400× magnification) or >100 eosinophils/HPF, by assessment of at least three dense collections of eosinophils. Secondly, formal quantification of tissue eosinophils was performed by counting in three 0.1 mm2 fields in subepithelial layers of each digitalised H&E-stained tissue section, selected at low power (40×). Fields were chosen to best represent the overall level of active inflammation on the slide and were subsequently reviewed at higher power (100×) to ensure that areas of crush or atypical clusters of inflammation were not selected. Eosinophils were independently counted in the subepithelial stroma as nucleated cells with ruby red granules, or as well-circumscribed, anucleated cells with ruby red granules (Figure 1). Eosinophils in the epithelium and intravascular space were excluded. The eosinophil cell count was calculated as the average number of eosinophils across the three 0.1 mm2 areas then expressed as eosinophils per 1 mm2.
Figure 1.
Visual guide for identification of eosinophils. Representative microscopic images of nucleated and anucleated eosinophils on haematoxylin and eosin (H&E) stained sinonasal tissue, visualised on digital whole slide imaging at high power (40×).
Eosinophil Activation
Three immunohistochemistry stains were optimised to assess eosinophil chemotaxis (eotaxin-3), degranulation (EPX) and immunoglobulin E (IgE).
Immunohistochemical staining was performed on serial 3 µm-thick FFPE sinonasal tissue using the Roche Ventana Benchmark Ultra automated staining system (Ventana Medical Systems, Inc., Tucson, USA). Sections were deparaffinised with the Roche-Ventana EZ Prep and rehydrated. No antigen retrieval was required for EPX, while treatment was required for IgE (in protease-1 [Ventana], 4 min at room temperature) and eotaxin-3 (in protease-1, 4 min at room temperature). The following primary antibodies were applied: rabbit polyclonal anti-eotaxin-3 antibody (1:300, BS-15512R, Bioss Antibodies, Woburn, MA, USA), rabbit polyclonal anti-human EPX (1:400, ab238506, Abcam, Cambridge, UK) and anti-IgE antibody MH25-1 (1:20, sc-52335, Santa Cruz Biotechnologies, Santa Cruz, CA, USA); and incubated for 32 min at 36 °C. Immunoreactivity was enhanced with OptiView detection (eotaxin-3, EPX) or UltraView detection (IgE) (Ventana). Slides were counter-stained with haematoxylin and dehydrated at the end of the procedure. Positive cells and cell products stained brown. Paraffin-embedded specimens of tonsil tissue were used as controls.
A semi-quantitative scoring system for eotaxin-3 staining was adapted from methods previously described for eosinophilic esophagitis. 13 Scores were assessed in the interstitium of the subepithelial stroma. Two parameters were described: (i) predominant intensity of staining (0 = no staining, 1 = minimal, 2 = moderate, 3 = strong) (Figure 2) and (ii) the percentage area of eotaxin-3 staining in the tissue (0 = no positive staining, 1 = 0%–10%, 2 = 11%–30%, 3 = 31%–60%, 4 = >60%). Then, these two scores were totalled for an overall score from 0 to 8 for each of the three regions; the greater the score, the greater the degree of eosinophil stimulation and chemotaxis.
Figure 2.
Grading of intensity of eotaxin-3 staining. Representative microscopic images of sinonasal tissue stained for eotaxin-3 on immunohistochemistry, visualised on digital whole slide imaging at high power (20×). Intensity of staining was scored according to a 4-point scale: (A) 0 = no staining, (B) 1 = minimal (light brown), (C) 2 = moderate (medium brown) and (D) 3 = excessive (dark brown). The black line represents a scale of 50 µm.
EPX staining was scored using a semi-quantitative scoring system adapted from previous methods in the literature for eosinophilic esophagitis and eosinophilic asthma.14,15 Scores were assessed in the interstitium of the subepithelial stroma. Three parameters were described: (i) overall intensity of staining in the sub-epithelial tissue (0 = minimal, 0.5 = moderate, 1 = excessive), (ii) percent area of EPX staining in the sub-epithelial tissue (0 = <1%, 1 = 1%–24%, 2 = 25%–49%, 3 = 50–74%, 4 = 75%–100%) and (iii) level of deposition in the subepithelial tissue (0 = no eosinophils/no EPX release, 1 = eosinophils present/no detectable EPX release, 2 = EPX release limited to areas surrounding eosinophils, 3 = nominal deposition, 4 = considerable deposition, 5 = dense deposition). Then, these three scores were totalled for an overall score from 0 to 10; the greater the score, the greater the degree of eosinophil degranulation.
Assessment of IgE
IgE was assessed in blood as total serum IgE (IU/L) and in mucosa. Tissue IgE-positive mast cell counts were performed in three 0.4 mm2 hotspot fields, that is, selected regions on each slide containing more IgE-positive mast cells. As previously described, 16 IgE-positive mast cells were classified into two types: mast cells that appeared IgE-positive only in the membrane were termed “membrane IgE-positive mast cells,” while mast cells with pan-cytoplasmic IgE positivity were termed “cytoplasmic IgE-positive mast cells” (Figure 3). The number of each cell type was counted separately, and the average across the three fields was determined for each slide and expressed in cells per mm2.
Figure 3.
Visual guide for identification of immunoglobulin E (IgE)-positive mast cells. Representative microscopic images of sinonasal tissue stained for IgE on immunohistochemistry, showing (A) cytoplasmic IgE-positive mast cells and (B) membrane IgE-positive mast cells, visualised on digital whole slide imaging at high power (20×). The black line represents a scale of 50 µm.
Statistical Analysis
Statistical analyses were performed using SPSS version 26 (IBM, USA). Independent variables were compared between CCAD and eCRS patients. Univariate analyses were performed with nominal data compared using Pearson's chi-square and continuous data using Student's t-test. Ordinal subdomains of histopathology scoring were tested with Kendall's tau-b test. Mean and standard deviation (SD) were reported for parametrically distributed continuous variables. Median and interquartile range were summarised for ordinal variables. A two-tailed p-value of less than 0.05 was considered statistically significant.
Results
Population Characteristics
A total of 38 participants were included in this study (51.7 ± 15.6 years, 47.4% female), of whom 36.8% had a diagnosis of CCAD and 63.2% had a diagnosis of eCRS (Table 1). The CCAD group was younger (44.5 ± 11.8 years vs 55.8 ± 16.3 years, p = 0.029), however, no differences were observed in sex or smoking status. At the time of surgery, biologics were used in 25% of eCRS patients but none of the CCAD patients (p = 0.041), whereas sublingual immunotherapy was used in 50% of CCAD patients and 8.3% of eCRS patients (p < 0.01). On histology, the overall degree of inflammation was significantly higher in patients with eCRS compared with patients with CCAD (τb = 0.409, p = 0.011) (Table 2).
Table 1.
Comparison of Baseline Characteristics Between Study Populations.
| CCAD (n = 14) | eCRS (n = 24) | p-value | |
|---|---|---|---|
| Age (years) (mean ± SD) | 44.5 ± 11.8 | 55.83 ± 16.26 | 0.029 |
| Gender (% female) | 35.7 | 54.2 | 0.272 |
| Smoking (%) | 14.3 | 20.8 | 0.615 |
| Biologics at time of surgery (%) | 0 | 25 | 0.041 |
| Sublingual immunotherapy at time of surgery (%) | 50 | 8.3 | 0.003 |
| Serum IgE (kU/L) (mean ± SD) | 230.1 ± 272.4 | 260.2 ± 270.8 | 0.742 |
CCAD: central compartment atopic disease; eCRS: eosinophilic chronic rhinosinusitis; SD: standard deviation; IgE: Immunoglobulin E. Bold text denotes a statistically significant result (p < 0.05).
Table 2.
Comparison of Tissue Inflammatory Marker Levels Between Study Populations.
| CCAD (n = 14) | eCRS (n = 24) | p-value | |
|---|---|---|---|
| Overall degree of inflammation (%) | 0.011 | ||
| Mild | 7.2 | 4.2 | |
| Moderate | 71.4 | 29.1 | |
| Severe | 21.4 | 66.7 | |
| Blood eosinophilia (×109 cells/L) (mean ± SD) | 0.33 ± 0.24 | 0.28 ± 0.17 | 0.436 |
| Degree of tissue eosinophilia (%) | 0.011 | ||
| 10–100 eos/HPF | 50 | 12.5 | |
| >100 eos/HPF | 50 | 87.5 | |
| Tissue eosinophil count on WSI (cells/mm2) (mean ± SD) | 746 ± 519 | 2141 ± 1947 | 0.013 |
CCAD: central compartment atopic disease; eCRS: eosinophilic chronic rhinosinusitis; eos/HPF: eosinophils per high power field; HPF: high power field; WSI: whole slide image. Bold text denotes a statistically significant result (p < 0.05).
Eosinophils
Blood eosinophil counts were similar between patients with CCAD and patients with eCRS (0.33 ± 0.24 × 109 cells/L vs 0.28 ± 0.17 × 109 cells/L, p = 0.436). The degree of tissue eosinophilia on conventional microscopy was significantly elevated in patients with eCRS, with a higher proportion of eCRS patients with >100 eosinophils/HPF (87.5% vs 50%, p = 0.011) and on eosinophil count (2141 ± 1947 cells/mm2 vs 746 ± 519 cells/mm2, p = 0.013) (Table 2).
Total eotaxin-3 score in the subepithelial stroma was found to be elevated in patients with eCRS compared with patients with CCAD (5.00[5.00–6.00] vs 6.00[6.00–6.75], p = 0.015) (Table 3).
Table 3.
Comparison of Eotaxin-3 Scores in the Subepithelial Stroma Between Patients with CCAD and Patients with eCRS.
| CCAD (n = 14) | eCRS (n = 24) | p-value | |
|---|---|---|---|
| Staining intensity (%) | 0.085 | ||
| Minimal | 21.4 | 8.3 | |
| Moderate | 35.7 | 20.8 | |
| Excessive | 42.9 | 70.9 | |
| Percent area of staining (%) | 0.274 | ||
| 10–30 | 21.4 | 8.3 | |
| 30–60 | 42.9 | 41.7 | |
| >60 | 35.7 | 50 | |
| Total eotaxin-3 score (%) | 0.015 | ||
| 0–3 | 0 | 0 | |
| 4 | 14.3 | 0 | |
| 5 | 42.9 | 20.8 | |
| 6 | 35.7 | 54.2 | |
| 7 | 7.1 | 25.0 |
CCAD: central compartment atopic disease; eCRS: eosinophilic chronic rhinosinusitis. Bold text denotes a statistically significant result (p < 0.05).
Total EPX scores were similar in patients with CCAD and patients with eCRS (6.25[6.00–7.00] vs 7.00[5.75–7.50], p = 0.183) (Table 4). There were no significant differences between the patient groups in any of the three sub-categories that comprised the EPX score (staining intensity, percent area of staining and deposition).
Table 4.
Comparison of EPX Scores in the Subepithelial Stroma Between Patients with CCAD and Patients with eCRS.
| CCAD (n = 14) | eCRS (n = 24) | p-value | |
|---|---|---|---|
| Staining intensity (%) | 0.593 | ||
| Minimal | 7.1 | 4.2 | |
| Moderate | 42.9 | 37.5 | |
| Excessive | 50 | 58.3 | |
| Percent area of staining (%) | 0.223 | ||
| <1 | 14.3 | 0 | |
| 1–24 | 50 | 54.2 | |
| 25–49 | 21.4 | 13.3 | |
| 50–74 | 14.3 | 12.5 | |
| 75–100 | 0 | 12.5 | |
| Deposition (%) | |||
| Limited EPX release | 7.1 | 4.2 | |
| Nominal deposition | 7.1 | 8.3 | 0.336 |
| Considerable deposition | 50 | 33.3 | |
| Dense deposition | 35.8 | 54.2 | |
| Total EPX score (%) | 0.183 | ||
| 3.5 | 7.1 | 0 | |
| 4 | 0 | 0 | |
| 4.5 | 0 | 4.2 | |
| 5.0 | 7.1 | 4.2 | |
| 5.5 | 7.1 | 16.6 | |
| 6.0 | 28.7 | 4.2 | |
| 6.5 | 21.5 | 16.6 | |
| 7.0 | 14.3 | 29.2 | |
| 7.5 | 7.1 | 0 | |
| 8 | 7.1 | 8.3 | |
| 9 | 0 | 4.2 | |
| 10 | 0 | 12.5 |
None of the results were significant at p < 0.05. CCAD: central compartment atopic disease; eCRS: eosinophilic chronic rhinosinusitis; EPX: eosinophil peroxidase.
IgE
Serum IgE levels were similar (230.1 ± 272.4 kU/L vs 260.2 ± 270.8 kU/L, p = 0.742) between groups. No significant differences were observed between the CCAD group and the eCRS group in the number of cytoplasmic IgE-positive mast cells (89.7 ± 25.3 cells/mm2 vs 107.4 ± 40.6 cells/mm2, p = 0.67), membrane IgE-positive mast cells (13.9 ± 15.6 cells/mm2 vs 9.9 ± 9.4 cells/mm2, p = 0.32) or total IgE-positive mast cells (103.6 ± 34.2 cells/mm2 vs 117.5 ± 45.0 cells/mm2, p = 0.32).
Discussion
CCAD and eCRS are two distinct clinical variants of primary diffuse type 2 CRS however they are both type-2 dominant or eosinophil dominant. Although recent studies have successfully described their differences in clinical, radiologic and endoscopic presentation,2–5,17 clear distinctions are not always possible. This study investigated eosinophil and IgE features in CCAD and eCRS patients to see if a difference could be observed. Within this cohort, CCAD patients tended to be younger than the eCRS group, in keeping with current knowledge of their allergic basis; this is consistent with the incidence of inhalant allergy or rhinitis seen in younger patients.18,19 In the eCRS group, the key histopathological differences were higher tissue eosinophil count and increased eotaxin-3 expression suggesting greater eosinophil stimulation and chemotaxis, with a higher degree of overall inflammation. However, despite this correlation, prior studies have described that eosinophil count alone does not usefully distinguish CCAD and eCRS. 5 The specific features of these eosinophil subpopulations may need further characterisation.
All patients in this study underwent FESS at the time of tissue collection. Although this study is only cross-sectional in nature, a higher tissue eosinophil count, as seen in the eCRS cohort, has been shown to predict the likelihood of disease recurrence post-FESS, with one meta-analysis identifying a cut-off of > 55 cells/HPF. 11 On the other hand, CCAD has more recently been associated with more favourable post-surgical outcomes compared with other subtypes of CRSwNP, including significantly lower rates of polyp recurrence and revision FESS. 20 Since CCAD is thought to be driven by underlying inhalant allergy, immunotherapy is often provided to achieve optimal postoperative outcomes and symptom control, whereas post-op eCRS management involves topical corticosteroid irrigations with the addition of biologics if appropriate. 5 Managing underlying allergy is important to all patients with sinonasal disease. Additionally, concomitant allergy can add to the symptom burden in CRS patients regardless of the CRS phenotype. Although sublingual immunotherapy may play a core part in CCAD management, it is still utilised in eCRS patients (8.3% in this study) to manage concomitant allergic symptoms.
Eotaxin-3, a marker of eosinophil chemotaxis, was significantly elevated in the subepithelial stroma of eCRS tissue compared to CCAD. This is consistent with prior studies where eotaxin-3 upregulation in CRS has been associated with nasal polyposis and poorer CT and endoscopic scores,21–23 as is more likely to be seen in eCRS, where pan-sinus opacification is a common radiologic feature.3,17 These studies measured eotaxin-3 expression using quantitative polymerase chain reaction and immunoassay methods, confirming the immunohistochemistry findings of the present study. As eCRS is pathophysiologically driven by Type 2 eosinophilic infiltration, 5 elevated levels of eotaxin-3 in eCRS may be explained by its role as a potent cytokine which promotes the recruitment and persistence of activated eosinophils in disease tissue. Analogous to other type 2-mediated diseases such as eosinophilic esophagitis, 13 there is potential utility of eotaxin-3 in defining eosinophilic subpopulations of CRS.
No significant difference was observed in EPX score between patients with CCAD and patients with eCRS. EPX was selected for investigation as it is one of four highly cytotoxic granule proteins secreted by degranulating eosinophils and has a pathologic role in CRS by contributing to tissue damage, polypoid oedema, necrosis of the mucosal epithelium and the release of histamine from mast cells.24,25 It has previously been correlated with disease activity in type 2-mediated diseases including allergic rhinitis and asthma.26–29 There is, however, some controversy surrounding its accuracy as a marker of eosinophilic activation in disease states. A study by Sanz et al. documented a significant correlation between EPX and asthma disease severity, however, found that eosinophil count was a better disease marker than EPX. 28 Prior research in CRS has more commonly used other eosinophil granule proteins as markers of eosinophil activity, such as eosinophil cationic protein,30–32 which may in fact represent another potential marker to distinguish CCAD and eCRS phenotypes.
Despite the allergic clinical predisposition of CCAD, no significant difference in total IgE was observed between the two patient groups in cytoplasmic, membrane or total IgE-positive mast cells. However, local infiltration of IgE-positive mast cells in the nasal mucosa positively correlated with clinical severity and histopathological factors in eCRS, according to the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC) criteria. 16 Non-specific IgE infiltrates have been reported in eCRS. 6 We did not measure allergen-specific IgE and it is recognised that patients with CRSwNP often demonstrate polyclonal IgE responses with poor allergen specificity or clonal selection. 33 On the other hand, a more recent study investigating the cytokine profile of CCAD and eCRS via immunoassay also found no significant difference in IgE between the groups but found that interleukin-5 and interleukin-13 levels were increased in CCAD. 34 The degree to which the phenotype is defined might underlie the lack of difference in these studies.
While prior studies have successfully correlated biomarkers of eosinophilic activation and allergic drive with various measures of clinical severity, our main findings were that eCRS patients have significantly elevated tissue eosinophil and eotaxin-3 levels compared to CCAD, however, these differences are not sufficient to confidently distinguish between the disease subtypes, potentially due to the limitations of our sample population. As more research into the clinical features of CCAD and eCRS emerges, increased knowledge of their underlying histopathological characteristics is warranted. Furthermore, although the pathologists were blinded to the patient groups in our study, there may also be future opportunities to expand the application of digital analysis technology for biomarker analysis, beyond bias reduction, such as automated cell counting and specific quantification of staining intensity.
In this study, a clear objective histologic difference between the phenotypes was not defined. It is acknowledged that the phenotypes, CCAD and eCRS, will still require a collection of features to distinguish them, including age of onset (young adult vs middle age), endoscopic location of polypoid change (turbinate vs sinus), radiologic change (central vs diffuse), degree of tissue eosinophilia and other allergic comorbidities. The timing of lower airway involvement (childhood vs adult onset), smell loss (late vs early) and other biomarkers are all areas for future research. It is not anticipated that all eosinophilic CRS patients be characterised by only these two phenotypes. There also needs to be scope for ambiguity and even other phenotypes to be defined in the future. However, the use of simple eosinophil density that is estimated on H&E stain from three most dense collections of eosinophils in the stroma, with <10/HPF (may have one field only >10), 10–100 per HPF (10–100 eosinophils per HPF, in two or more areas) and >100 per HPF (>100 eosinophils per HPF, in two or more areas), may assist in defining the more eosinophil dense patients. 10
Conclusion
Eosinophil and eotaxin-3 levels were significantly elevated in eCRS compared with CCAD, suggesting a greater degree of eosinophil stimulation and chemotaxis. Patients with CCAD were younger. Although histology alone cannot separate these phenotypes, future application of tissue biomarkers may better distinguish CRS subpopulations.
Acknowledgments
The authors would like to thank Mr Gary Gracie, Ms Tanya Wyatt and the laboratory staff of the SydPath Department of Anatomical Pathology at St Vincent's Hospital, Sydney, for their valuable assistance with tissue retrieval, preparation and staining.
Footnotes
Ethics: Ethical approval from the St Vincent's Hospital Sydney Human Research Ethics Committee (REGIS identifier 2019/PID14562).
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Richard J Harvey is a consultant/advisory board with Medtronic, Novartis, Glaxo-Smith-Kline and Meda Pharmaceuticals. He has been on the speakers' bureau for Glaxo-Smith-Kline, AstraZeneca, Meda Pharmaceuticals and Seqiris. Janet Rimmer has honoraria with Sanofi Aventis, Novartis, Mundipharma, BioCSL and Stallergenes. Larry Kalish is on the speakers' bureau for Care Pharmaceuticals and Mylan Pharmaceuticals. All other authors have no financial disclosures or conflicts of interest.
Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.
ORCID iDs: Andrea Sit https://orcid.org/0000-0003-3551-4004
William Sewell https://orcid.org/0000-0002-3586-8761
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