Elevated ALCAM Expression Associated with Endotypes and Postoperative Recurrence in Chronic Rhinosinusitis with Nasal Polyps

Figures & data

Table 1 The Demographic and Clinical Features of Participants

Variables	HC Group (n=40)	neCRSwNP Group (n=70)	eCRSwNP Group (n=50)	P
Age, years	41.0±10.6	41.4±10.7	41.0±11.6	0.974
Gender (male/female)	21/19	39/31	27/23	0.946
Smoking (yes/no)	12/28	18/52	15/35	0.836
BMI, kg/m^2	23.0±1.4	23.0±1.9	23.2±1.7	0.794
Allergic rhinitis (yes/no)	0/40	10/60	16/34	<0.001
Asthma (yes/no)	0/40	5/65	9/41	0.009
Peripheral eosinophil count (10^9/L)	0.1±0.1	0.2±0.1	0.3±0.1	<0.001
Peripheral eosinophil percentage (%)	2.6±1.3	3.1±1.3	3.9±1.1	<0.001
Tissue eosinophil count (n/HPF)	–	16.5±6.5	47.6±20.6	<0.001
Tissue eosinophil percentage (%)	–	6.4±1.9	22.0±8.6	<0.001
Disease duration, years	–	3.0±2.5	3.4±3.5	0.022
Preoperative VAS score	–	5.2±1.6	5.7±1.8	0.130
Preoperative Lund-Mackay score	–	18.4±3.6	18.5±3.4	0.856

Abbreviations: neCRSwNP, non-eosinophilic chronic rhinosinusitis with nasal polyps; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps; HC, healthy control; BMI, body mass index; HPF, high power field; VAS, visual analogue scale.

Table 2 Relationship Between Serum ALCAM and Clinical Variables in CRSwNP Patients

Variables	r	P
Age, years	−0.038	0.675
BMI, kg/m^2	−0.159	0.083
Peripheral eosinophil count (10^9/L)	0.186	0.042
Peripheral eosinophil percentage (%)	0.008	0.388
Disease duration, months	0.083	0.365
Preoperative VAS score	0.112	0.221
Preoperative Lund-Mackay score	−0.149	0.104
Tissue eosinophil count (n/HPF)	0.654	<0.001
Tissue eosinophil percentage (%)	0.680	<0.001

Abbreviations: CRSwNP, chronic rhinosinusitis with nasal polyps; BMI, body mass index; VAS, visual analogue scale; HPF, high power field.

Table 3 Binary Logistic Regression Analysis of Factors Associated with eCRSwNP

Variables	OR	95% CI	P
Allergic rhinitis (yes/no)	1.677	0.544–5.164	0.368
Asthma (yes/no)	1.891	0.475–7.529	0.366
Peripheral eosinophil count (10^9/L)	3.227	0.009–1171.23	0.697
Peripheral eosinophil percentage (%)	1.627	1.079–2.452	0.020
Disease duration, years	1.074	0.928–1.242	0.339
Serum ALCAM level (ng/mL)	1.155	1.074–1.243	<0.001

Abbreviations: CRSwNP, chronic rhinosinusitis with nasal polyps; OR, odds rate; CI, confidence interval.

Figure 1 Comparison of serum ALCAM levels between CRSwNP patients and HCs. (A) ALCAM levels were significantly elevated in the CRSwNP group than the HC group. (B) Serum ALCAM levels were markedly enhanced in the eCRSwNP group than the neCRSwNP and HC groups.

Abbreviations: ALCAM, activated leukocyte cell adhesion molecule; CRSwNP, chronic rhinosinusitis with nasal polyps; HC, healthy control; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps; neCRSwNP, non-eosinophilic chronic rhinosinusitis with nasal polyps.

Figure 2 The ROC curves of potentially predictive biomarkers for discriminating eCRSwNP. (A) serum ALCAM; (B) peripheral eosinophil percentage.

Abbreviations: ROC, receiver operating characteristic; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps; ALCAM, activated leukocyte cell adhesion molecule.

Table 4 Demographic Characteristics Between Primary and Recurrent CRSwNP Patients

Variables	Non-Recurrent Group (n=78)	Recurrent Group (n=32)	P
Age, years	41.2±10.9	42.0±12.0	0.750
Gender (male/female)	44/34	15/17	0.404
Smoking(yes/no), n	24/54	7/25	0.484
BMI, kg/m^2	23.1±1.8	22.6±1.8	0.192
Allergic rhinitis(yes/no), n	12/66	11/21	0.022
Asthma(yes/no), n	8/70	4/28	0.743
Peripheral eosinophil count (10^9/L)	0.3±0.1	0.3±0.1	0.097
Peripheral eosinophil percentage (%)	3.4±1.3	3.6±1.3	0.455
Disease duration, years	3.2±2.9	3.2±3.2	0.989
Preoperative VAS score	5.3±1.7	5.7±1.8	0.315
Preoperative Lund-Mackay score	18.7±3.4	17.9±3.6	0.191
Tissue eosinophil count (n/HPF)	24.3±17.7	43.9±23.1	<0.001
Tissue eosinophil percentage (%)	10.5±7.9	18.8±10.8	<0.001
neCRSwNP/eCRSwNP	52/26	12/20	0.006

Abbreviations: neCRSwNP, non-eosinophilic chronic rhinosinusitis with nasal polyps; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps; BMI, body mass index; HPF, high power field; VAS, visual analogue scale.

Table 5 Binary Logistic Regression Analysis of Factors Associated with the Recurrence of CRSwNP

Variables	OR	95% CI	P
Allergic rhinitis (yes/no)	2.549	0.816–7.961	0.107
Tissue eosinophil count (n/HPF)	1.053	0.990–1.120	0.103
Tissue eosinophil percentage (%)	0.887	0.751–1.048	0.160
neCRSwNP/eCRSwNP	1.642	0.282–9.542	0.581
Serum ALCAM level (ng/mL)	1.233	1.099–1.383	<0.001

Abbreviations: CRSwNP, chronic rhinosinusitis with nasal polyps; OR, odds rate; CI, confidence interval.

Figure 3 The serum ALCAM levels in the non-recurrent CRSwNP patients and the recurrent CRSwNP patients. (A) Serum ALCAM concentrations were elevated in the recurrent CRSwNP group than the non-recurrent CRSwNP group. (B) Serum ALCAM levels were significantly enhanced in the recurrent eCRSwNP group than the non-recurrent eCRSwNP and non-recurrent neCRSwNP groups.

Abbreviations: ALCAM, activated leukocyte cell adhesion molecule; CRSwNP, chronic rhinosinusitis with nasal polyps; HC, healthy control; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps; neCRSwNP, non-eosinophilic chronic rhinosinusitis with nasal polyps.

Figure 4 The ROC curve (A) and Kaplan-Meier survival analysis (B) for serum ALCAM level in the prediction of CRSwNP recurrence.

Abbreviations: ROC, receiver operating characteristic; ALCAM, activated leucocyte cell adhesion molecule; CRSwNP, chronic rhinosinusitis with nasal polyps.

Figure 5 Comparison of ALCAM mRNA levels in the tissue of CRSwNP patients and HCs. (A) ALCAM mRNA expressions were elevated in the CRSwNP group than the HC group. (B) ALCAM mRNA levels were significantly increased in the eCRSwNP group than the neCRSwNP group. (C) no significant difference was observed in mRNA levels between the non-recurrent CRSwNP group and the recurrent CRSwNP group.

Abbreviations: ALCAM, activated leukocyte cell adhesion molecule; CRSwNP, chronic rhinosinusitis with nasal polyps; HC, healthy control; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps; neCRSwNP, non-eosinophilic chronic rhinosinusitis with nasal polyps.

Figure 6 The ALCAM protein expression in the tissue based on IHC. Representative images of ALCAM staining from HCs (A and B), eCRSwNP patients (C and D) and neCRSwNP patients (E and F).

Abbreviations: ALCAM, activated leukocyte cell adhesion molecule; HC, healthy control; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps; neCRSwNP, non-eosinophilic chronic rhinosinusitis with nasal polyps; IHC, immunohistochemistry.

Figure 7 The ALCAM protein expression in the nasal polyps tissue based on IHC. Representative images of ALCAM staining from non-recurrent CRSwNP patients (A and B) and recurrent CRSwNP patients (C and D).

Abbreviations: ALCAM, activated leukocyte cell adhesion molecule; CRSwNP, chronic rhinosinusitis with nasal polyps; IHC, immunohistochemistry.

Figure 8 Quantitative analysis of ALCAM immunoreactivity in nasal poly and HC samples. (A) ALCAM positive cell number was significantly greater in the eCRSwNP group than the neCRSwNP and HC groups. (B) ALCAM positive cell number was significantly greater in the recurrent CRSwNP group than the non-recurrent CRSwNP group and.

Abbreviations: ALCAM, activated leukocyte cell adhesion molecule; CRSwNP, chronic rhinosinusitis with nasal polyps; HC, healthy control; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps; neCRSwNP, non-eosinophilic chronic rhinosinusitis with nasal polyps.

Figure 9 The ALCAM protein expression in the tissue based on WB. (A) WB images of ALCAM among eCRSwNP, neCRSwNP and HC groups. (B) WB images of ALCAM between the recurrent CRSwNP and non-recurrent CRSwNP groups. (C) comparison of ALCAM relative protein level among eCRSwNP, neCRSwNP and HC groups. (D) comparison of ALCAM relative protein level between the recurrent CRSwNP and non-recurrent CRSwNP groups.

Abbreviations: ALCAM, activated leukocyte cell adhesion molecule; WB, Western blotting; CRSwNP, chronic rhinosinusitis with nasal polyps; HC, healthy control; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps; neCRSwNP, non-eosinophilic chronic rhinosinusitis with nasal polyps.

Figure 10 Hypothetical model showing association between ALCAM expression and eosinophil infiltration and recurrence in CRSwNP. On allergens derived from irritants and bacteria challenge, DCs were activated and the production of ALCAM was increased, releasing its soluble form (sALCAM). This promoted the ALCAM–CD6 interactions and enhanced Th2 immune responses, then induced the eosinophil tissue infiltration and aggravation of eosinophilic inflammation, resulting in tissue eosinophilia and recurrence in CRSwNP.

Abbreviations: ALCAM, activated leukocyte cell adhesion molecule; CRSwNP, chronic rhinosinusitis with nasal polyps; DC, dendritic cell; sALCAM, soluble activated leukocyte cell adhesion molecule.