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Charcot-Leyden crystal concentration in nasal secretions predicts clinical response to glucocorticoids in patients with chronic rhinosinusitis with nasal polyps

  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Di Wu
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China
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  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Bing Yan
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China

    Beijing Key Laboratory of Nasal Diseases, Beijing Institute of Otolaryngology, Beijing, China
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  • Yang Wang
    Affiliations
    Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China

    Beijing Key Laboratory of Nasal Diseases, Beijing Institute of Otolaryngology, Beijing, China
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  • Luo Zhang
    Affiliations
    Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China

    Beijing Key Laboratory of Nasal Diseases, Beijing Institute of Otolaryngology, Beijing, China
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  • Chengshuo Wang
    Affiliations
    Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China
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  • Author Footnotes
    ∗ These authors contributed equally to this work.
Published:April 16, 2019DOI:https://doi.org/10.1016/j.jaci.2019.03.029
      To the Editor:
      Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous disease characterized by a defective immune barrier and massive inflammatory cell infiltration.
      • Lou H.
      • Meng Y.
      • Piao Y.
      • Wang C.
      • Zhang L.
      • Bachert C.
      Predictive significance of tissue eosinophilia for nasal polyp recurrence in the Chinese population.
      • Wang H.
      • Li Z.Y.
      • Jiang W.X.
      • Liao B.
      • Zhai G.T.
      • Wang N.
      • et al.
      The activation and function of IL-36γ in neutrophilic inflammation in chronic rhinosinusitis.
      • Lu H.
      • Lin X.S.
      • Yao D.M.
      • Zhuang Y.Y.
      • Wen G.F.
      • Shi J.
      • et al.
      Increased serum amyloid A in nasal polyps is associated with systemic corticosteroid insensitivity in patients with chronic rhinosinusitis with nasal polyps: a pilot study.
      Currently, the most effective medical therapy in clinical practice is the use of glucocorticoids.
      • Fokkens W.J.
      • Lund V.J.
      • Mullol J.
      • Bachert C.
      • Alobid I.
      • Baroody F.
      • et al.
      EPOS 2012: European position paper on rhinosinusitis and nasal polyps 2012. A summary for otorhinolaryngologists.
      • Wen W.
      • Liu W.
      • Zhang L.
      • Bai J.
      • Fan Y.
      • Xia W.
      • et al.
      Increased neutrophilia in nasal polyps reduces the response to oral corticosteroid therapy.
      • Wang C.
      • Lou H.
      • Wang X.
      • Wang Y.
      • Fan E.
      • Li Y.
      • et al.
      Effect of budesonide transnasal nebulization in patients with eosinophilic chronic rhinosinusitis with nasal polyps.
      • Milara J.
      • Morell A.
      • Ballester B.
      • Armengot M.
      • Morcillo E.
      • Cortijo J.
      MUC4 impairs the anti-inflammatory effects of corticosteroids in patients with chronic rhinosinusitis with nasal polyps.
      However, many patients have a poor response to the therapy.
      • Wang H.
      • Li Z.Y.
      • Jiang W.X.
      • Liao B.
      • Zhai G.T.
      • Wang N.
      • et al.
      The activation and function of IL-36γ in neutrophilic inflammation in chronic rhinosinusitis.
      • Wang C.
      • Lou H.
      • Wang X.
      • Wang Y.
      • Fan E.
      • Li Y.
      • et al.
      Effect of budesonide transnasal nebulization in patients with eosinophilic chronic rhinosinusitis with nasal polyps.
      • Milara J.
      • Morell A.
      • Ballester B.
      • Armengot M.
      • Morcillo E.
      • Cortijo J.
      MUC4 impairs the anti-inflammatory effects of corticosteroids in patients with chronic rhinosinusitis with nasal polyps.
      Thus, developing biomarkers predicting glucocorticoid response in patients with CRSwNP would greatly improve the efficacy of glucocorticoids and lighten the economic burden in clinical practice. Although several biomarkers associated with glucocorticoid resistance have been discovered recently, most of these have been detected in polyp tissue samples.
      • Hong H.
      • Chen F.
      • Sun Y.
      • Yang Q.
      • Gao W.
      • Cao Y.
      • et al.
      Nasal IL-25 predicts the response to oral corticosteroids in chronic rhinosinusitis with nasal polyps.
      • Yao Y.
      • Xie S.
      • Yang C.
      • Zhang J.
      • Wu X.
      • Sun H.
      Biomarkers in the evaluation and management of chronic rhinosinusitis with nasal polyposis.
      Because the procedure for obtaining tissues often leads to unavoidable mucosal trauma and increased risk of bleeding and infection, there is a need for biomarkers that can be detected by using noninvasive procedures.
      Charcot-Leyden crystal (CLC) was first described in the 19th century.
      • Dor P.J.
      • Ackerman S.J.
      • Gleich G.J.
      Charcot-Leyden crystal protein and eosinophil granule major basic protein in sputum of patients with respiratory diseases.
      It is a unique component of eosinophils and basophils and is regarded as a hallmark protein of eosinophilic inflammatory diseases. Previous studies have demonstrated the presence of CLC in sputum of patients given a diagnosis of allergic asthma or pulmonary ascariasis and in the feces of patients with eosinophilic diseases of the digestive system, such as ulcerative colitis and amoebic Trichuris species infection.
      • Dor P.J.
      • Ackerman S.J.
      • Gleich G.J.
      Charcot-Leyden crystal protein and eosinophil granule major basic protein in sputum of patients with respiratory diseases.
      In preliminary experiments we successfully detected CLC in nasal secretions of patients with CRSwNP collected according to the noninvasive method of Watelet et al.
      • Watelet J.B.
      • Gevaert P.
      • Holtappels G.
      • Van Cauwenberge P.
      • Bachert C.
      Collection of nasal secretions for immunological analysis.
      Because rhinorrhea is one of the most common symptoms in these patients and these secreted fluids can readily be collected by using a noninvasive method, we have hypothesized that CLC in nasal secretions might serve as a predictive marker of glucocorticoid response in patients with CRSwNP. Thus this study aimed to investigate the predictive capacity of CLC concentrations in nasal secretions for glucocorticoid response in patients with CRSwNP.
      The study was approved by the Ethics Committee of Beijing TongRen Hospital, and all participants provided written informed consent. Eighty-nine patients given a diagnosis of CRSwNP according to the European Position Paper on Rhinosinusitis and Nasal Polyps 2012
      • Fokkens W.J.
      • Lund V.J.
      • Mullol J.
      • Bachert C.
      • Alobid I.
      • Baroody F.
      • et al.
      EPOS 2012: European position paper on rhinosinusitis and nasal polyps 2012. A summary for otorhinolaryngologists.
      and who had no contraindications to glucocorticoids were enrolled in the study. None of the patients had received any treatment with glucocorticoids or immunomodulatory drugs within 4 weeks before enrollment. Patients with fungal sinusitis, allergic fungal rhinosinusitis, cystic fibrosis, or primary ciliary dyskinesia were excluded from the study, and all eligible patients were evaluated for comorbidity of allergic rhinitis, asthma, and atopy. None of the enrolled patients received a diagnosis of aspirin-exacerbated respiratory disease. Clinical characteristics, such as staging of computed tomography (CT) and objective evaluation of olfactory function, were determined, as described previously (detailed criteria for each clinical characteristics are presented in the Patients and samples section in this article's Online Repository at www.jacionline.org).
      • Fokkens W.J.
      • Lund V.J.
      • Mullol J.
      • Bachert C.
      • Alobid I.
      • Baroody F.
      • et al.
      EPOS 2012: European position paper on rhinosinusitis and nasal polyps 2012. A summary for otorhinolaryngologists.
      • Bousquet J.
      • Khaltaev N.
      • Cruz A.A.
      • Denburg J.
      • FokkenWJ
      • Togias A.
      • et al.
      Allergic rhinitis and its impact on asthma (ARIA) 2008.

      Global Initiative For Asthma. Global strategy for asthma management and prevention, Revised 2014 Vancouver, GINA, 2014. Available at: www.ginasthma.org

      • Lou H.
      • Meng Y.
      • Piao Y.
      • Wang C.
      • Zhang L.
      • Bachert C.
      Predictive significance of tissue eosinophilia for nasal polyp recurrence in the Chinese population.
      • Lund V.
      • Kennedy D.
      Staging for rhinosinusitis.
      • Meng Y.
      • Lou H.
      • Wang C.
      • Zhang L.
      Predictive significance of computed tomography in eosinophilic chronic rhinosinusitis with nasal polyps.
      Bilateral nasal cavity secretions and polyp tissues were collected for comparison from each patient on admission to the hospital, as described previously (detailed information on collection of nasal secretions is presented in the Patients and samples section in this article's Online Repository).
      • Watelet J.B.
      • Gevaert P.
      • Holtappels G.
      • Van Cauwenberge P.
      • Bachert C.
      Collection of nasal secretions for immunological analysis.
      After enrollment, all patients started a 2-week course of oral glucocorticoid therapy (24 mg of methylprednisolone by mouth every morning) and at the end of this treatment period were divided into 2 groups according to the Nasal Polyp Size Score (NPSS) system (see Table E1 in this article's Online Repository at www.jacionline.org): glucocorticoid responders (n = 48, patients whose change in NPSS was more than 1 point) and glucocorticoid nonresponders (n = 41, patients whose change in NPSS was no more than 1 point). Data for the clinical characteristics for each patient were expressed as medians and interquartile ranges (IQRs), except for binary variables and age (expressed as the mean and SD, see Table E2 in this article's Online Repository at www.jacionline.org).
      CLC concentrations in nasal secretions were analyzed by using an ELISA. Statistical analysis was performed with GraphPad Prism 6 software (GraphPad Software, La Jolla, Calif), and receiver operating characteristic (ROC) curves were drawn with SPSS 24.0 (IBM, Armonk, NY; details are presented in the Methods section in this article's Online Repository). Statistical significance was considered at a P value of less than .05.
      The 2 groups were not significantly different in terms of sex, age, smoking history, nasal obstruction, rhinorrhea, facial pain, or subjective olfactory disorder. However, patients in the glucocorticoid responder group had higher rates of comorbid allergic rhinitis (P = .025; odds ratio [OR], 3.600; 95% CI, 1.184-10.94) and atopy (P = .020; OR, 2.824; 95% CI, 1.190-6.699) than patients in the glucocorticoid nonresponder group. Similarly, patients in the glucocorticoid responder group had significantly greater severity of olfactory disorders (P = .035), greater total ethmoid sinus score/total maxillary sinus score (E/M) ratios of Lund-Mackay scores (P < .001),
      • Meng Y.
      • Lou H.
      • Wang C.
      • Zhang L.
      Predictive significance of computed tomography in eosinophilic chronic rhinosinusitis with nasal polyps.
      and greater percentages of peripheral blood eosinophils (P = .016) than patients in the glucocorticoid nonresponder group. There was no significant difference in neutrophil percentages between the 2 groups (P = .172). Histologic evaluation of polyp tissue revealed that patients in the glucocorticoid responder group had significantly greater numbers of infiltrating eosinophils (P < .001) and lower numbers of infiltrating neutrophils (P < .001) compared with patients in the glucocorticoid nonresponder group.
      Analysis of the average concentration of CLC in nasal secretions showed that this was significantly greater (105.3 ng/mL; IQR, 51.08-153.5 ng/mL) in patients in the glucocorticoid responder group than in patients in the glucocorticoid nonresponder group (0.6460 ng/mL; IQR, 0-29.34 ng/mL; P < .001; see Table E2). Four patients in the glucocorticoid responder group had much higher CLC concentrations (outliers are marked red in Fig 1, B; with 3 being 1000 ng/mL and 1 being 651.8 ng/mL). Despite excluding these data, differences between median values (90.02 ng/mL; IQR, 50.18-136.3 ng/mL) for the glucocorticoid responder group were still found to be significantly greater than for those of the glucocorticoid nonresponder group (P < .001).
      Figure thumbnail gr1
      Fig 1CLC concentrations in nasal secretions and predictive values. A, The area under curve (AUC) for CLC concentrations in nasal secretions was 0.897 (OR, 1.043; 95% CI, 1.020-1.066), whereas the AUC for eosinophil percentages in polyp tissues was 0.855 (OR, 1.056; 95% CI, 1.025-1.087). B, Distribution of CLC concentrations in nasal secretions in the glucocorticoid (GC) responder group (n = 48, 4 outliers are marked in red) and the glucocorticoid nonresponder group (n = 41, P < .001). The optimal cutoff value determined by using the Youden index was 30.065 ng/mL. C, Distributions of eosinophil percentages in polyp tissues (P < .001) in the glucocorticoid responder group (n = 48) and the glucocorticoid nonresponder group (n = 41). The optimal cutoff value determined by using the Youden index was 41.50%. D and E, Change in nasal CLC concentration before and after glucocorticoid therapy in the glucocorticoid responder group (n = 12; Fig 1, D) and the glucocorticoid nonresponder group (n = 17; Fig 1, E).
      A binary logistic regression model method of forward Wald mode was used for further comparison between groups to determine specific factors associated with polyp recurrence. All the clinical characteristics, including CLC concentrations in nasal secretions; percentages of eosinophils, neutrophils, lymphocytes, and plasma cells in polyp tissues; percentages of eosinophils in peripheral blood; Lund-Mackay score E/M ratios; objective olfactory function; and comorbid rates of allergic rhinitis and atopy, which had been found to be significantly different by preliminary analyses, were introduced as variables in the binary logistic regression model method. The multivariate analysis revealed that only CLC concentrations in nasal secretions (P < .001; OR, 1.043; 95% CI, 1.020-1.066) and eosinophil percentages in polyp tissues (P < .001; OR, 1.056; 95% CI, 1.025-1.087) showed the potential for predicting glucocorticoid response in patients with CRSwNP (see Table E3 in this article's Online Repository at www.jacionline.org). ROC curves for these 2 parameters (shown in Fig 1, A) demonstrated corresponding areas under the curve to be 0.897 and 0.855 for CLC concentrations in nasal secretions and eosinophil percentages in polyp tissues, respectively. According to the Youden index (see Table E4 in this article's Online Repository at www.jacionline.org), the optimal cutoff value for CLC concentrations in nasal secretions to predict glucocorticoid response was 30.065 ng/mL (Youden index = 67.6%, with a sensitivity of 89.6% and a specificity of 78.0%; Fig 1, B), and the optimal cutoff value for percentage of eosinophils in polyp tissues to predict glucocorticoid response was 41.50% (Youden index = 66.6%, with a sensitivity of 81.3% and a specificity of 85.4%; Fig 1, C).
      In regard to the effect on glucocorticoids, NPSSs of glucocorticoid responders and glucocorticoid nonresponders demonstrated no difference before glucocorticoid treatment (P = .968), but there were significant differences in NPSSs of postglucocorticoid treatment (P < .001) and change in NPSSs before and after glucocorticoid therapy (P < .001). Analysis of CLC concentrations in 29 patients (12 of whom were glucocorticoid responders and 17 of whom were glucocorticoid nonresponders) with available samples indicated that CLC was detectable in both subgroups before and after glucocorticoid treatment and that glucocorticoid treatment significantly decreased CLC concentrations in the glucocorticoid responder group (P = .002) but not in the glucocorticoid nonresponder group (P = .211; Fig 1, D and E).
      Additional analysis of results for CLC concentrations indicated that patients with CLC concentrations of less than 30.065 ng/mL (n = 37) showed significantly greater differences in response to glucocorticoid therapy compared with those in patients with CLC concentrations of greater than the cutoff value (n = 52; P < .001; Fig 2, A). Although the NPSS was not significantly different between the 2 groups before glucocorticoid therapy (P > .999), this was significantly decreased after glucocorticoid therapy in patients with higher CLC concentrations compared with patients with lower CLC concentrations (P < .001; Fig 2, B and C). Moreover, patients with greater CLC concentrations in nasal secretions displayed more severe infiltration of eosinophils in both polyp tissues (P < .001) and peripheral blood than patients with lower CLC concentrations (P = .014; Fig 2, D and E), whereas infiltration of neutrophils was not significantly different in either polyp tissues (P = .302) or peripheral blood (P = .071) of patients between the 2 groups (Fig 2, F and G). In contrast, CLC concentrations in nasal secretions were not significantly associated with distribution of sex, age, smoking history, comorbid asthma, rhinitis, or atopy (see Fig E1 in this article's Online Repository at www.jacionline.org). Similarly, any of the 4 subjective symptoms (see Fig E2 in this article's Online Repository at www.jacionline.org), objective olfactory test results, Lund-Mackay scores of the paranasal sinuses, or E/M ratios (see Fig E3 in this article's Online Repository at www.jacionline.org) were also not significantly associated with CLC concentrations in patients with CRSwNP.
      Figure thumbnail gr2
      Fig 2Differences in clinical characteristics between patients with nasal CLC concentrations less than (n = 37) or greater than (n = 52) the cutoff value. A, Number of glucocorticoid responders (black columns)/nonresponders (gray columns). B, NPSSs after glucocorticoid therapy. C, Changes in NPSSs before and after glucocorticoid therapy. D, Eosinophil percentages in polyp tissues. E, Eosinophil percentages in peripheral blood. F, Neutrophil percentages in polyp tissues. G, Neutrophil percentages in peripheral blood.
      In this study we investigated the predictive significance of CLC concentrations in nasal secretions for glucocorticoid response in patients with CRSwNP and found that this noninvasive sampling method had a similar predictive capacity as measuring eosinophil percentages in polyp tissues. CLC concentrations in nasal secretions were associated with glucocorticoid responses, and glucocorticoid therapy significantly decreased CLC concentrations. Nevertheless, all the other parameters, including sex, age, smoking history, comorbid asthma, comorbid allergic rhinitis, comorbid atopy, symptom severity, or CT scan staging, had little effect on CLC concentrations in nasal secretions.
      Also, CLC concentrations in nasal secretions were correlated with eosinophil percentages in tissue polyps (Spearman correlation model: P < .001, R = 0.5). The possibility that nasal secretions are as valuable as polyp tissues for predicting glucocorticoid response in patients with CRSwNP is particularly important because collection of nasal secretions is much easier, safer, and more tolerable than collection of polyp tissues, and the risks of trauma and hemorrhage are lower. Additionally, use of nasal secretions is an efficient and accurate method of both detecting and counting various inflammatory cells.
      In the case of the 4 glucocorticoid responders with much greater nasal CLC concentrations (outliers) than the rest of the group (marked red in Fig 2, B), further analysis demonstrated that in these patients the clinical indices of eosinophil percentages in polyp tissues, comorbidity of asthma and allergic rhinitis, or inflammatory cell percentages in tissue polyps were not significantly different compared with those of the other patients in the group.
      Considering the efficacy and accuracy of glucocorticoid therapy, it is important to predict the glucocorticoid response of patients before administration of glucocorticoid therapy for long periods. Furthermore, predicting the response to glucocorticoid therapy in patients with CRSwNP is particularly important because glucocorticoid insensitivity is often observed in a large number of this group of patients. In this regard our finding that 46.1% (41/89) of patients with CRSwNP were insensitive to glucocorticoid therapy is in accordance with the findings of a more recent study by Hong et al,
      • Hong H.
      • Chen F.
      • Sun Y.
      • Yang Q.
      • Gao W.
      • Cao Y.
      • et al.
      Nasal IL-25 predicts the response to oral corticosteroids in chronic rhinosinusitis with nasal polyps.
      which reported 44.2% (23/52) of patients with CRSwNP to be nonresponsive to glucocorticoid therapy and demonstrates that CLC concentration in nasal secretions is likely to be a suitable biomarker for predicting glucocorticoid response for patients with CRSwNP.
      Recently, several studies have indicated different biomarkers that might be associated with decreased response to glucocorticoids in patients with CRSwNP.
      • Yao Y.
      • Xie S.
      • Yang C.
      • Zhang J.
      • Wu X.
      • Sun H.
      Biomarkers in the evaluation and management of chronic rhinosinusitis with nasal polyposis.
      For example, increased neutrophilia in nasal polyps
      • Wen W.
      • Liu W.
      • Zhang L.
      • Bai J.
      • Fan Y.
      • Xia W.
      • et al.
      Increased neutrophilia in nasal polyps reduces the response to oral corticosteroid therapy.
      ; increased levels of serum amyloid A, an apolipoprotein associated with high-density lipoprotein
      • Lu H.
      • Lin X.S.
      • Yao D.M.
      • Zhuang Y.Y.
      • Wen G.F.
      • Shi J.
      • et al.
      Increased serum amyloid A in nasal polyps is associated with systemic corticosteroid insensitivity in patients with chronic rhinosinusitis with nasal polyps: a pilot study.
      ; upregulation of mucin 4; and downregulation of mucin 1
      • Haruna S.
      • Ohtori N.
      • Moriyama H.
      • Nakanishi M.
      Olfactory dysfunction in sinusitis with infiltration of numerous activated eosinophils.
      • Milara J.
      • Morell A.
      • Ballester B.
      • Armengot M.
      • Morcillo E.
      • Cortijo J.
      MUC4 impairs the anti-inflammatory effects of corticosteroids in patients with chronic rhinosinusitis with nasal polyps.
      and IL-25
      • Hong H.
      • Chen F.
      • Sun Y.
      • Yang Q.
      • Gao W.
      • Cao Y.
      • et al.
      Nasal IL-25 predicts the response to oral corticosteroids in chronic rhinosinusitis with nasal polyps.
      have been associated with glucocorticoid insensitivity. However, all these biomarkers have been investigated in polyp tissues, thus making use of CLC concentrations in nasal secretions a suitable easy and faster alternative for predicting glucocorticoid response in patients with CRSwNP.
      A major limitation of the present study is that it was conducted in Asian patients, whose response to steroids is poor because of a greater prevalence of noneosinophilic nasal polyps. Therefore the finding for a predictive capability of nasal CLC secretion for nasal polyp response to glucocorticoids is not generalizable to Western patients and needs to be investigated further in this population. Nevertheless, the determination of specific secreted proteins in nasal secretions might serve as a reliable noninvasive biomarker in the future to predict the response of patients with CRSwNP to glucocorticoids.
      In summary, our study has demonstrated that the CLC concentration in nasal secretions is a suitable noninvasive biomarker that could predict glucocorticoid response for patients with CRSwNP. Routine use of this biomarker in clinical practice might lead to better strategies for glucocorticoid therapy in the future.

      Methods

       Patients and samples

      Eighty-nine patients were given a diagnosis of CRSwNP according to the European Position Paper on Rhinosinusitis and Nasal Polyps 2012,
      • Fokkens W.J.
      • Lund V.J.
      • Mullol J.
      • Bachert C.
      • Alobid I.
      • Baroody F.
      • et al.
      EPOS 2012: European position paper on rhinosinusitis and nasal polyps 2012. A summary for otorhinolaryngologists.
      scheduled to undergo functional endoscopic sinus surgery in Beijing TongRen Hospital, and enrolled in this study. Patients with contraindications for glucocorticoids or who had used glucocorticoids or immunomodulatory drugs within 4 weeks before the study were excluded. Patients with aspirin-exacerbated respiratory disease, fungal sinusitis, allergic fungal rhinosinusitis, cystic fibrosis, or primary ciliary dyskinesia were also excluded. Allergic rhinitis was diagnosed according to the Allergic Rhinitis and its Impact on Asthma 2008
      • Bousquet J.
      • Khaltaev N.
      • Cruz A.A.
      • Denburg J.
      • FokkenWJ
      • Togias A.
      • et al.
      Allergic rhinitis and its impact on asthma (ARIA) 2008.
      criteria. Asthma was diagnosed as defined by the Global Initiative For Asthma 2014 guidelines.

      Global Initiative For Asthma. Global strategy for asthma management and prevention, Revised 2014 Vancouver, GINA, 2014. Available at: www.ginasthma.org

      Atopy was evaluated by measurement of serum specific IgE levels (cutoff value, 0.35 kUA/L; Phadia, Uppsala, Sweden). This study was approved by the Ethics Committee of Beijing TongRen Hospital, and all participants provided written informed consent before entry into the study.
      All eligible patients were asked to self-evaluate their symptoms according to a 0- to 10-point visual analogue scale.
      • Lou H.
      • Meng Y.
      • Piao Y.
      • Wang C.
      • Zhang L.
      • Bachert C.
      Predictive significance of tissue eosinophilia for nasal polyp recurrence in the Chinese population.
      In addition, all the participants underwent CT scans of the paranasal sinuses, and the outcomes of CT scans were evaluated by using the Lund-Mackay staging system.
      • Lund V.
      • Kennedy D.
      Staging for rhinosinusitis.
      • Meng Y.
      • Lou H.
      • Wang C.
      • Zhang L.
      Predictive significance of computed tomography in eosinophilic chronic rhinosinusitis with nasal polyps.
      The objective olfactory test was performed as described by Haruna et al.
      • Haruna S.
      • Ohtori N.
      • Moriyama H.
      • Nakanishi M.
      Olfactory dysfunction in sinusitis with infiltration of numerous activated eosinophils.
      Endoscopic examinations were conducted to investigate the polyp size of each side of the nasal cavity.
      • Milara J.
      • Peiró T.
      • Armengot M.
      • Frias S.
      • Morell A.
      • Serrano A.
      • et al.
      Mucin 1 downregulation associates with corticosteroid resistance in chronic rhinosinusitis with nasal polyps.
      Endoscopic images were evaluated and scored independently by 2 otolaryngologists who were blinded to the trial design. As a result of the examination, all the patients scored at least 4 in total before the glucocorticoid therapy was undertaken. Peripheral blood data were obtained from a routine blood test.
      On admission to the hospital, bilateral nasal cavity secretions from each patient were collected by the method described by Watelet et al.
      • Watelet J.B.
      • Gevaert P.
      • Holtappels G.
      • Van Cauwenberge P.
      • Bachert C.
      Collection of nasal secretions for immunological analysis.
       Each sample was diluted with 0.5 mL of 0.9% natural saline and kept at room temperature for 1 hour, after which samples were centrifuged into a Falcon tube and stored at 4°C until further measurements. Polyp tissues (3 × 3 mm for each participant) were also obtained immediately after nasal secretions were collected. The patients received a 2-week therapy of oral glucocorticoid (24 mg of methylprednisolone by mouth every morning) and at the end of treatment were divided into glucocorticoid responder (n = 48) and glucocorticoid nonresponder (n = 41) groups based on the change in polyp size scores. Patients who did not have a reduction of more than 1 polyp score based on the NPSS system after glucocorticoid therapy were defined as glucocorticoid nonresponders.
      • Fokkens W.J.
      • Lund V.J.
      • Mullol J.
      • Bachert C.
      • Alobid I.
      • Baroody F.
      • et al.
      EPOS 2012: European position paper on rhinosinusitis and nasal polyps 2012. A summary for otorhinolaryngologists.
      • Milara J.
      • Peiró T.
      • Armengot M.
      • Frias S.
      • Morell A.
      • Serrano A.
      • et al.
      Mucin 1 downregulation associates with corticosteroid resistance in chronic rhinosinusitis with nasal polyps.

       Histologic evaluation

      Histologic criteria were determined by using hematoxylin and eosin (H&E) staining.
      • Fischer A.H.
      • Jacobson K.A.
      • Rose J.
      • Zeller R.
      Hematoxylin and eosin staining of tissue and cell sections.
      Tissue samples were dehydrated, embedded in paraffin, and then cut at 5-μm thicknesses by using a Leica RM2235 cryostat (Leica Microsystems, Bannockburn, Ill). All sections were examined by using optical microscopy at a magnification of ×400, and absolute numbers and percentages of eosinophils, neutrophils, plasma cells, and lymphocytes were calculated. For each section, absolute numbers and percentages of each cell type were recorded as mean data of 3 nonoverlapping regions, and the final evaluation for each patient's tissue sample was recorded as mean data of 5 sections. Inflammatory cell counts were performed by 2 pathologists who were blind to the study design.

       ELISA

      Detection and estimation of CLC concentrations in nasal secretions were performed by using commercial ELISA kits (Cloud-Clone, Wuhan, China). The detection range of this kit was 0.312 to 20 ng/mL CLC. Before the assay, all samples of nasal secretions were diluted 50-fold with 0.9% natural saline, and the assay was conducted strictly according to the manufacturer's instructions.

       Statistical analysis

      All data were expressed as medians and IQRs, except for binary variables and age, which were expressed as means and SDs. Statistical analysis was performed with GraphPad Prism 6 software. All parametric variants were analyzed by using Student t tests, and nonparametric variants were analyzed by using Mann-Whitney U tests. Binary variables were analyzed by using Fisher and χ2 tests, and binary logistic regression was used to find the potential predictive markers for glucocorticoid response. ROC curves were generated to determine the optimal cutoff point by using SPSS version 24.0. The predictive ability of CLC concentrations in nasal secretions for glucocorticoid sensitivity was determined by the area under the curve, and the optimal cutoff point was determined by using the Youden index. P values of less than .05 were considered statistically significant. All data were analyzed by using 2-tailed tests.
      Figure thumbnail fx1
      Fig E1Effect of sex (black columns for male and gray columns for female subjects; A), age (B), comorbid asthma (C), comorbid allergic rhinitis (D), comorbid atopy (E), and smoking history (F) on nasal CLC concentrations less than or greater than the cutoff value (black columns for yes and gray columns for no in Fig E1, C-F).
      Figure thumbnail fx2
      Fig E2Severity and incidence of chronic rhinosinusitis symptoms in patients with nasal CLC concentrations less than or greater than the cutoff value: A, nasal obstruction; B, rhinorrhea; C, facial pain; and D, subjective olfactory disorder.
      Figure thumbnail fx3
      Fig E3Difference of patients with nasal CLC concentrations less than or greater than the cutoff value in objective olfactory test results (A), Lund-Mackay scores for paranasal sinuses on CT scans (B), and ratios of Lund-Mackay scores of ethmoid sinus/Lund Mackay scores of the maxillary sinus (C).
      Table E1NPSS system
      Polyp scorePolyp size
      0No polyps
      1Small polyps in the middle meatus not reaching below the inferior border of the middle concha
      2Polyps reaching below the lower border of the middle turbinate
      3Large polyps reaching the lower border of the attachment of inferior turbinate or polyps medial to the middle concha
      4Large polyps causing almost complete congestion/obstruction of the inferior meatus
      Table E2Demographic and clinical characteristics of glucocorticoid responders and glucocorticoid nonresponders
      Glucocorticoid responders (n = 48)Glucocorticoid nonresponders (n = 41)P value
      Sex (male/female)29/1928/13.510
      Age (y), mean ± SD44.69 ± 11.5244.63 ± 11.66.983
      Asthma (yes/no)18/3016/251
      Allergic rhinitis (yes/no)16/325/36.025
      Atopy (yes/no)32/1617/24.020
      Smoking history (yes/no)9/397/341
      Nasal obstruction, median (IQR)7.0 (6.0-8.0)7.0 (6.0-8.0).895
      Rhinorrhea, median (IQR)6.0 (4.3-6.8)5.0 (3.5-6.5).101
      Facial pain, median (IQR)0 (0-2.8)0 (0-2.0).514
      Subjective olfactory disorder, median (IQR)6.0 (4.0-10.0)4.0 (0-8.5).080
      Objective olfactory function, median (IQR)5.0 (3.0-5.0)3.0 (1.0-5.0).035
      Lund-Mackay score, median (IQR)19.5 (14.3-22.0)17.0 (14.0-20.0).154
      Lund-Mackay score E/M ratio, median (IQR)2.6 (2.0-3.5)2.0 (1.9-2.7)<.001
      NPSS (preglucocorticoid therapy), median (IQR)5.0 (4.0-6.0)5.0 (4.0-6.0).767
      NPSS (postglucocorticoid therapy), median (IQR)2.0 (2.0-3.0)5.0 (4.0-5.0)<.001
      NPSS (postglucocorticoid minus preglucocorticoid), median (IQR)−2.0 (−3.0 to −2.0)0 (−1.0 to 0)<.001
      Tissue eosinophils (%), median (IQR)60.75 (47.25-74.38)10.52 (0-37.17)<.001
      Tissue neutrophils (%), median (IQR)0 (0-1.255)1.400 (0-4.600).003
      Tissue lymphocytes (%), median (IQR)21.50 (13.54-33.30)48.70 (31.96-65.85)<.001
      Tissue plasma cells (%), median (IQR)14.95 (6.75-21.20)21.30 (9.65-40.15)<.001
      Peripheral blood eosinophils (%), median (IQR)5.950 (4.625-8.775)4.500 (1.650-7.900).016
      Peripheral blood neutrophils (%), median (IQR)53.45 (47.30-58.10)56.00 (49.45-63.05).172
      Nasal secretion eosinophils, median (IQR)1.0 (0-2.0)0 (0-1.0).032
      Nasal secretion CLC concentration, median (IQR)105.3 (51.08-153.5)0.6460 (0-29.34)<.001
      Table E3Binary logistic regression for potential markers associated with glucocorticoid response
      MarkersOR95% CIP value
      CLC concentration (nasal secretions)1.0431.020-1.066<.001
      Eosinophil percentage (tissue)1.0561.025-1.087<.001
      Table E4Determination of the optimal cutoff value for each marker
      Cutoff valueSensitivitySpecificityYouden index
      CLC concentration (nasal secretions)28.425

      29.340

      30.065

      32.188

      35.292
      0.896

      0.896

      0.896

      0.875

      0.854
      0.732

      0.756

      0.780

      0.780

      0.780
      0.628

      0.652

      0.676

      0.655

      0.635
      Eosinophil percentage (tissue)38.870

      40.500

      41.500

      43.000

      44.500
      0.833

      0.813

      0.813

      0.792

      0.771
      0.805

      0.829

      0.854

      0.854

      0.854
      0.638

      0.642

      0.666

      0.645

      0.624
      Determination of the optimal cutoff value for each marker is shown in boldface.

      References

        • Wang H.
        • Li Z.Y.
        • Jiang W.X.
        • Liao B.
        • Zhai G.T.
        • Wang N.
        • et al.
        The activation and function of IL-36γ in neutrophilic inflammation in chronic rhinosinusitis.
        J Allergy Clin Immunol. 2018; 141: 1646-1658
        • Lu H.
        • Lin X.S.
        • Yao D.M.
        • Zhuang Y.Y.
        • Wen G.F.
        • Shi J.
        • et al.
        Increased serum amyloid A in nasal polyps is associated with systemic corticosteroid insensitivity in patients with chronic rhinosinusitis with nasal polyps: a pilot study.
        Eur Arch Otorhinolaryngol. 2018; 275: 401-408
        • Wen W.
        • Liu W.
        • Zhang L.
        • Bai J.
        • Fan Y.
        • Xia W.
        • et al.
        Increased neutrophilia in nasal polyps reduces the response to oral corticosteroid therapy.
        J Allergy Clin Immunol. 2012; 129: 1522-1528
        • Wang C.
        • Lou H.
        • Wang X.
        • Wang Y.
        • Fan E.
        • Li Y.
        • et al.
        Effect of budesonide transnasal nebulization in patients with eosinophilic chronic rhinosinusitis with nasal polyps.
        J Allergy Clin Immunol. 2015; 135: 922-929
        • Milara J.
        • Morell A.
        • Ballester B.
        • Armengot M.
        • Morcillo E.
        • Cortijo J.
        MUC4 impairs the anti-inflammatory effects of corticosteroids in patients with chronic rhinosinusitis with nasal polyps.
        J Allergy Clin Immunol. 2017; 139: 855-862
        • Hong H.
        • Chen F.
        • Sun Y.
        • Yang Q.
        • Gao W.
        • Cao Y.
        • et al.
        Nasal IL-25 predicts the response to oral corticosteroids in chronic rhinosinusitis with nasal polyps.
        J Allergy Clin Immunol. 2018; 141: 1890-1892
        • Yao Y.
        • Xie S.
        • Yang C.
        • Zhang J.
        • Wu X.
        • Sun H.
        Biomarkers in the evaluation and management of chronic rhinosinusitis with nasal polyposis.
        Eur Arch Otorhinolaryngol. 2017; 274: 3559-3566
        • Dor P.J.
        • Ackerman S.J.
        • Gleich G.J.
        Charcot-Leyden crystal protein and eosinophil granule major basic protein in sputum of patients with respiratory diseases.
        Am Rev Respir Dis. 1984; 130: 1072-1077
        • Watelet J.B.
        • Gevaert P.
        • Holtappels G.
        • Van Cauwenberge P.
        • Bachert C.
        Collection of nasal secretions for immunological analysis.
        Eur Arch Otorhinolaryngol. 2004; 261: 242-246