Characterization of sputum biomarkers for asthma–COPD overlap syndrome

Figures & data

Table 1 Clinical characteristics and biomarker concentrations of the study subjects across the discovery cohort (n=90)

Discovery cohort	Nonsmokers (n=14)	Healthy smokers (n=14)	Asthma (n=24)	COPD (n=20)	ACOS (n=18)	P-value
Demographics
Female/male	5/9	5/9	15/9	8/12	6/12
Age (years)	46.2 (15.4)	48.4 (10.9)	58.3 (8.3)	59.3 (7.0)	61.8 (6.5)	<0.001
BMI (kg/m^2)	28.2 (4.6)	26.6 (4.8)	26.0 (4.5)	24.6 (4.0)	28.2 (6.5)	0.263
Allergy, n (%)	1 (7)	7 (50)	11 (46)	9 (45)	6 (33)
ICS use, n (%)	–	–	20 (83)	9 (45)	13 (72)
Smoking status
Former/current	–	0/14	9/6	7/12	11/7
Pack-years	–	27.1 (16.1)	13.0 (16.0)	37.2 (17.3)	42.5 (17.7)	<0.001
Prebronchodilator
FVC (L)	4.8 (1.1)	4.7 (1.4)	3.1 (0.7)	3.3 (1.0)	3.0 (1.1)	<0.001
FVC% predicted	103.6 (9.4)	98.6 (15.1)	81.1 (17.7)	81.7 (16.5)	69.3 (18.6)	<0.001
FEV1 (L)	4.0 (1.0)	3.8 (1.1)	2.3 (0.6)	1.9 (0.8)	1.5 (0.6)	<0.001
FEV1% predicted	104.1 (11.0)	96.5 (15.3)	74.0 (15.6)	56.9 (18.7)	44.8 (14.8)	<0.001
FEV1/FVC%	82.1 (3.7)	79.4 (5.2)	74.2 (5.0)	56.4 (13.6)	53.6 (10.7)	<0.001
Post-bronchodilator
FVC (L)	4.8 (1.1)	4.7 (1.4)	3.2 (0.7)	3.4 (0.9)	3.3 (1.0)	<0.001
FVC% predicted	102.6 (8.9)	98.8 (15.9)	83.1 (14.7)	83.4 (16.7)	75.5 (16.9)	<0.001
FEV1 (L)	4.0 (1.0)	3.8 (1.1)	2.5 (0.6)	1.9 (0.8)	1.8 (0.6)	<0.001
FEV1% predicted	105.9 (10.6)	98.5 (15.5)	78.8 (14.0)	58.3 (19.1)	51.6 (13.7)	<0.001
FEV1/FVC%	84.4 (3.7)	80.9 (5.0)	76.8 (4.4)	55.6 (12.4)	55.7 (10.8)	<0.001
DLCO% predicted	99.0 (9.0)	89.8 (10.2)	82.2 (17.8)	53.2 (20.2)	67.9 (18.7)	<0.001
Reversibility test
ΔFEV1a (mL)	72.9 (175.9)	79.3 (203.2)	146.3 (166.8)	53.5 (80.6)	231.1 (187.4)	0.004
ΔFEV1%a	1.9 (4.6)	2.2 (6.0)	6.5 (7.4)	2.8 (4.4)	17.6 (15.6)	<0.001
PEF diurnal variability (%)			25.6 (12.1)	13.5 (5.8)	27.5 (13.6)	0.003
Differential sputum cell counts (%)
Eosinophils	1.9 (3.8)	0.3 (0.6)	5.1 (9.2)	2.4 (4.9)	6.8 (9.8)	0.001
Neutrophils	36.8 (8.0)	42.5 (20.8)	53.6 (23.4)	62.8 (20.3)	64.8 (15.5)	<0.001
Sputum biomarker concentrations
IL-6 (pg/mL)	18.6 (8.1, 49.5)	102.0 (78.0, 137.2)	77.5 (30.8, 121.2)	286.3 (61.6, 412.0)	605.7 (291.8, 808.7)	<0.001
YKL-40 (ng/mL)	22.5 (8.9, 44.0)	15.0 (8.7, 35.0)	47.8 (22.1, 100.5)	50.4 (37.7, 64.4)	142.4 (97.5, 521.2)	<0.001
IL-13 (pg/mL)	53.9 (27.6, 82.4)	40.7 (29.4, 50.1)	159.0 (57.7, 332.4)	141.6 (86.6, 255.7)	256.5 (133.6, 499.1)	<0.001
NGAL (µg/mL)	1.2 (0.9, 3.7)	2.0 (1.1, 3.5)	2.3 (1.4, 3.6)	3.9 (2.4, 5.7)	5.2 (4.1, 14.2)	<0.001
MPO (µg/mL)	2.9 (2.1, 16.8)	4.3 (1.5, 14.0)	6.3 (3.8, 11.7)	12.1 (5.8, 22.7)	21.7 (10.9, 29.6)	0.005

Notes: The demographics are presented as mean (SD) unless otherwise stated. The biomarker concentrations are presented as medians (IQR). P-value for the differences was analyzed by Kruskal–Wallis H test. Δ, change in.

a Improvement in FEV₁ after 400 µg of salbutamol.

Abbreviations: ACOS, asthma–COPD overlap syndrome; BMI, body mass index; DLCO, diffusing capacity of the lung for carbon monoxide; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; ICS, inhaled corticosteroids; IL, interleukin; IQR, interquartile range; MPO, myeloperoxidase; NGAL, neutrophil gelatinase-associated lipocalin; PEF, peak expiratory flow; SD, standard deviation; YKL-40, chitinase-3-like protein 1.

Table 2 Clinical characteristics and biomarker concentrations of the study subjects across the replication cohort (n=135)

Replication cohort	Nonsmokers (n=22)	Healthy smokers (n=40)	Asthma (n=21)	COPD (n=35)	ACOS (n=17)	P-value
Demographics
Females/males	13/9	5/35	17/4	1/34	3/14
Age (years)	58.2 (10.8)	63.4 (11.2)	42.0 (15.4)	72.2 (8.1)	65.9 (8.9)	<0.001
BMI (kg/m^2)	23.8 (3.5)	24.0 (3.2)	24.6 (5.4)	23.5 (3.0)	24.2 (3.2)	0.952
ICS use, n (%)	–	–	16 (76)	–	15 (88)
Pack-years	–	37.1 (23.7)	4.3 (12.5)	47.6 (21.9)	40.7 (16.6)	<0.001
Prebronchodilator
FVC (L)	2.9 (0.7)	3.6 (1.0)	3.1 (1.0)	3.1 (0.8)	3.2 (0.6)	0.079
FVC% predicted	95.6 (17.3)	100.0 (16.6)	100.4 (17.5)	93.2 (17.4)	94.9 (13.2)	0.302
FEV1 (L)	2.4 (0.6)	2.8 (0.8)	2.5 (0.7)	1.9 (0.5)	1.8 (0.5)	<0.001
FEV1% predicted	96.0 (16.7)	94.1 (17.7)	94.6 (16.2)	69.6 (16.5)	64.3 (15.3)	<0.001
FEV1/FVC%	83.2 (7.1)	77.1 (5.7)	80.7 (6.1)	59.8 (8.6)	55.2 (11.5)	<0.001
Differential sputum cell counts (%)
Eosinophils	2.6 (4.1)	2.0 (3.2)	12.0 (17.4)	3.2 (8.7)	9.2 (17.1)	0.051
Neutrophils	64.2 (27.7)	70.0 (20.9)	46.2 (26.0)	72.4 (17.4)	66.1 (24.8)	0.008
Sputum biomarker concentrations
IL-6 (pg/mL)	4.4 (1.5, 11.4)	8.3 (4.0, 18.2)	11.2 (4.0, 20.1)	11.6 (3.4, 28.1)	21.8 (7.4, 54.2)	0.017
YKL-40 (ng/mL)	10.1 (4.5, 27.7)	18.3 (5.0, 29.4)	17.4 (8.2, 21.7)	21.3 (11.4, 31.1)	20.6 (13.4, 30.6)	0.138
IL-13 (pg/mL)	28.7 (18.6, 43.5)	39.9 (25.7, 70.4)	14.7 (7.4, 47.4)	39.6 (33.1, 52.0)	39.1 (22.4, 59.2)	0.032
NGAL (ng/mL)	53.9 (23.7, 91.8)	82.4 (45.8, 167.0)	50.1 (33.4, 163.5)	106.2 (60.8, 178.6)	209.3 (160.0, 304.9)	<0.001
MPO (ng/mL)	165.2 (101.4, 300.0)	170.3 (111.3, 329.0)	139.4 (98.3, 254.6)	251.4 (166.0, 365.5)	152.1 (118.7, 307.4)	0.330

Notes: The demographics are presented as mean (SD) unless otherwise stated. The biomarker concentrations are presented as medians (IQR). P-value for the differences was analyzed by Kruskal–Wallis H test. Comparisons between groups were evaluated by the Kruskal–Wallis test followed by the Mann–Whitney U test.

Abbreviations: ACOS, asthma–COPD overlap syndrome; BMI, body mass index; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; ICS, inhaled corticosteroids; IL, interleukin; IQR, interquartile range; MPO, myeloperoxidase; NGAL, neutrophil gelatinase-associated lipocalin; SD, standard deviation; YKL-40, chitinase-3-like protein 1.

Table 3 ROC analysis for sputum biomarkers in the discovery cohort

Discovery cohort	IL-6 (pg/mL)	YKL-40 (ng/mL)	IL-13 (pg/mL)	NGAL (µg/mL)	MPO (µg/mL)
ACOS versus non-ACOS
AUC (95% CI)	0.887 (0.813–0.962)	0.854 (0.749–0.959)	0.753 (0.636–0.870)	0.814 (0.697–0.932)	0.737 (0.590–0.883)
Optimal threshold	>200.5	>90.4	>99.7	>4.0	>14.5
Sensitivity/specificity	0.938/0.757	0.833/0.845	0.882/0.515	0.833/0.750	0.706/0.789
P-value	<0.001	<0.001	0.001	<0.001	0.003
ACOS versus COPD
AUC (95% CI)	0.772 (0.617–0.926)	0.794 (0.638–0.951)	0.665 (0.486–0.844)	0.711 (0.541–0.881)	0.647 (0.460–0.834)
Optimal threshold	>479.8	>78.9	>175.2	>4.0	>14.3
Sensitivity/specificity	0.625/0.850	0.833/0.800	0.647/0.700	0.833/0.600	0.706/0.650
P-value	0.006	0.002	0.088	0.026	0.128
ACOS versus asthma
AUC (95% CI)	0.930 (0.853–1.000)	0.814 (0.681–0.947)	0.638 (0.463–0.812)	0.833 (0.704–0.963)	0.767 (0.598–0.937)
Optimal threshold	>199.5	>90.4	>377.8	>3.8	>14.2
Sensitivity/specificity	0.938/0.875	0.833/0.750	0.412/0.864	0.833/0.792	0.706/0.917
P-value	<0.001	0.001	0.145	<0.001	0.004

Abbreviations: ACOS, asthma–COPD overlap syndrome; AUC, area under the curve; CI, confidence interval; IL, interleukin; MPO, myeloperoxidase; NGAL, neutrophil gelatinase-associated lipocalin; ROC, receiver operating characteristic; YKL-40, chitinase-3-like protein 1.

Table 4 Multivariate stepwise analysis of all subjects with sputum biomarker as the dependent variable in patients with asthma, COPD, and ACOSa

Discovery cohort	β	t	P-value
Sputum IL-6
Pre-FEV1% predicted	−0.309	−2.381	0.021
Pack-years	0.262	2.018	0.049
Sputum NGAL
Pre-FEV1% predicted	−0.420	3.498	0.001
Sputum YKL-40
Age (years)	0.283	2.257	0.028
Pre-FEV1% predicted	−0.273	2.183	0.033
Sputum MPO
Pack-years	0.390	3.304	0.002
Age (years)	0.282	2.388	0.020

Notes:

a Multivariate analysis was adjusted for age, body mass index, pack-years, and all the significant variables in the univariate analysis.

Abbreviations: ACOS, asthma–COPD overlap syndrome; FEV₁, forced expiratory volume in 1 second; IL, interleukin; MPO, myeloperoxidase; NGAL, neutrophil gelatinase-associated lipocalin; YKL-40, chitinase-3-like protein 1.

Figure 1 Correlation of sputum NGAL with pre-FEV₁% predicted in patients with asthma, COPD, and ACOS in the discovery (A) and in the replication cohorts (B).

Abbreviations: ACOS, asthma–COPD overlap syndrome; FEV₁, forced expiratory volume in 1 second; NGAL, neutrophil gelatinase-associated lipocalin.

Figure 2 Expression levels of sputum biomarkers in patients with asthma, COPD, and ACOS in the discovery (A) and replication cohorts (B).

Notes: The results represent standardized data (Z-scores), and the solid lines show the median value.
Abbreviations: ACOS, asthma–COPD overlap syndrome; IL, interleukin; MPO, myeloperoxidase; NGAL, neutrophil gelatinase-associated lipocalin; YKL-40, chitinase-3-like protein 1.