Biomarker-based clustering of patients with chronic obstructive pulmonary disease

Lowie E.G.W. Vanfleteren; Julie Weidner; Frits M.E. Franssen; Swetlana Gaffron; Niki L. Reynaert; Emiel F.M. Wouters; Martijn A. Spruit

doi:10.1183/23120541.00301-2022

Figures

Tables
Supplementary Materials

Download figure
Open in new tab
Download powerpoint
FIGURE 1
Network interactions of predicted upregulated or downregulated proteins by cluster. Proteins that were similarly and significantly upregulated (clusters 3 and 4) or downregulated (clusters 1 and 2) were entered into String [35], all possible interaction sources were allowed and subjected to a minimum required high-confidence interaction score (0.7). Disconnected nodes are not shown. All clusters had a p-value of ≥3.53×10⁻¹¹. The mode of interaction is depicted as an arrow (positive interaction), inhibition (negative interaction) or ball (unspecified interaction). Gene names, with corresponding biomarker names in parenthesis, used in this figure: ADIPOQ (adiponectin), BDNF (brain-derived neurotrophic factor), B2M (β2-microglobulin), C3 (complement C3), CCL2 (monocyte chemotactic protein-1), CCL5 (RANTES), CRP (C-reactive protein), CXCL10 (interferon (IFN)-γ-induced protein-10), CXCL11 (IFN-γ-inducible T-cell α chemoattractant), EPO (erythropoietin), HP (haptoglobin), ICAM1 (intercellular adhesion molecule-1), IFNG (IFN-γ), IL1RN (interleukin (IL)-1ra), IL2RA (IL-2 receptor-α), IL6 (IL-6), IL12RB1 (IL12p40), BGLAP (osteocalcin), KITLG (stem cell factor), LEP (leptin), MMP3 (matrix metalloproteinase-3), SAA1 (serum amyloid A), SERPINA1 (α1-antitrypsin), SERPINE1 (plasminogen activator inhibitor-1), SPARC (osteonectin), TGFB1 (latency-associated peptide of transforming growth factor-β1), TIMP1 (tissue inhibitor of metalloproteinases-1), TNFRSF11B (osteoprotegerin), TNFRSF1A (tumour necrosis factor receptor (TNFR)I), TNFRSF1B (TNFR2), VEGFA (vascular endothelial growth factor), VCAM1 (vascular cell adhesion molecule-1).

TABLE 1

Systemic levels of biomarkers of inflammation, chemoattraction, cell activation, tissue destruction and tissue repair per cluster

	Cluster 1:	Cluster 2:	Cluster 3:	Cluster 4:
	lower-level cluster I	lower-level cluster II	higher-level cluster I	higher-level cluster II
Patients, n	64	64	46	39
Acute phase proteins and complement system
Complement C3, mg·mL⁻¹	1.2±0.2	1.1±0.2	1.3±0.2	1.5±1.0
CRP, ng·mL⁻¹	3011±2795	1773±1859	8214±8153	9725±8480
Fibrinogen, μg·mL⁻¹	6417±2826	6870±4533	6465±2374	8879±7911
Serum amyloid A, ng·mL⁻¹	3059±2705	1889±1403	6239±7311	10 745±7810
Cytokines and chemokines and their receptors
Eotaxin-2, pg·mL⁻¹	1022±632	1398±940	1371±954	1337±965
IL-1β, pg·mL⁻¹	4.8±1.2	4.3±1.3	4.2±1.1	5.0±1.6
IL-12 subunit p40, ng·mL⁻¹	0.6±0.2	0.5±0.1	0.5±0.1	0.6±0.1
IL-23, ng·mL⁻¹	1.4±0.4	1.2±0.3	1.2±0.3	1.4±0.3
IL-6, pg·mL⁻¹	2.9±1.6	1.9±1.7	3.4±1.7	6.3±8.2
IL-8, pg·mL⁻¹	12±5	13±5	14±6	14±5
IFN-γ-inducible T-cell α-chemoattractant, pg·mL⁻¹	43±22	44±24	55±25	64±47
IFN-γ-inducible protein-10, pg·mL⁻¹	93±41	73±39	90±30	142±98
IFN-γ, pg·mL⁻¹	0.5±0.3	0.4±0.4	0.5±0.2	1.3±2.6
Macrophage migration inhibitory factor, ng·mL⁻¹	0.1±0.1	0.1±0.1	0.1±0.1	0.1±0.1
MCP-1, pg·mL⁻¹	511±190	469±130	606±159	513±200
MCP-4, pg·mL⁻¹	688±235	678±207	762±210	778±455
T-cell-specific protein RANTES, ng·mL⁻¹	19±8	23±10	31±10	25±14
Thymus and activation-regulated chemokine, pg·mL⁻¹	524±414	636±384	784±620	770±828
IL-2 receptor-α, pg·mL⁻¹	2427±694	2020±535	2695±927	3429±1240
TNF-R2, ng·mL⁻¹	6.0±1.6	4.9±1.2	6.0±1.8	9.8±5.0
TNF-R1, pg·mL⁻¹	1850±476	1531±436	1905±588	2603±919
Osteoprotegerin, pM	7.0±1.9	6.7±1.1	7.2±1.3	8.9±2.0
Inhibitors of cytokine signalling
IL-1 receptor antagonist, pg·mL⁻¹	423±132	336±74	372±99	388±118
Adhesion molecules
sICAM-1, ng·mL⁻¹	282±76	292±76	321±102	389±113
sVCAM-1, ng·mL⁻¹	520±116	493±106	517±142	719±396
Immunoglobulins
IgA, mg·mL⁻¹	2.9±1.6	2.4±1.2	3.1±1.4	3.6±2.9
IgM, mg·mL⁻¹	2.1±1.4	1.8±1.9	1.7±1.1	2.5±4.2
Growth factors
Angiopoietin-2, ng·mL⁻¹	3.7±1.8	3.8±1.3	4.4±1.4	5.1±1.3
BDNF, ng·mL⁻¹	18.1±4.9	22±5	23±7	18±7
Stem cell factor, pg·mL⁻¹	397±93	330±83	354±88	473±163
Vascular endothelial growth factor, pg·mL⁻¹	242±111	258±122	354±159	290±170
Tissue remodelling
α1-Antitrypsim, mg·mL⁻¹	1.9±0.3	2.0±0.4	1.9±0.4	2.3±0.6
LAP TGF-β1, ng·mL⁻¹	9.4±2.8	12±3	13±3	10±3
MMP-3, ng·mL⁻¹	19±14	13±7	14±7	23±16
MMP-9, ng·mL⁻¹	147±72	130±62	153±69	159±83
Tissue inhibitor of metalloproteinases-1, ng·mL⁻¹	150±23	163±27	195±30	199±65
Hormones and adipokines
Adiponectin, μg·mL⁻¹	4.4±2.1	7.0±3.3	5.1±2.7	7.4±4.7
Leptin, ng·mL⁻¹	14.8±12.3	8.2±8.8	16.8±14.6	13.7±14.8
Erythropoietin, IU·mL⁻¹	11.3±5.2	8.4±2.6	11±7	13±4
Osteocalcin (OCN/BGLAP), ng·mL⁻¹	117±20	127±18	116±17	135±25
Coagulation
α2-Macroglobulin (A2Macro), mg·mL⁻¹	1.7±0.4	1.7±0.4	1.7±0.6	1.9±0.7
Factor VII, ng·mL⁻¹	447±140	406±99	403±127	422±119
PAI-1, ng·mL⁻¹	195±45	228±53	290±56	220±112
Plasma carrier proteins
Fetuin A, μg·mL⁻¹	574±292	706±364	603±303	652±420
Haptoglobin, mg·mL⁻¹	1.7±0.9	1.6±1.1	2.9±1.4	3.5±3.1
IGFBP-1, ng·mL⁻¹	2978±2432	4368±2771	3204±2178	4504±2870
IGFBP-2, ng·mL⁻¹	79±34	110±53	80±35	132±45
Vitamin D-binding protein, μg·mL⁻¹	288±108	300±91	291±122	396±727
Other
Ferritin, ng·mL⁻¹	180±142	151±128	190±160	183±203
β2-Microglobulin, μg·mL⁻¹	2.1±0.5	1.8±0.3	2.1±0.4	2.9±1.0
Myoglobin, ng·mL⁻¹	69±37	74±111	54±28	96±62
Osteonectin, ng·mL⁻¹	1145±344	1394±230	1720±322	1318±394
Osteopontin, ng·mL⁻¹	22±7	22±8	22±7	36±14
Bilirubin, mmol·L⁻¹	11.6±4.9	14±6	10±5	12±3
Leukocytes, ×10⁹ cells·L⁻¹	6.7±1.4	6.9±1.9	8.1±1.4	8.5±2.4
LDL, mmol·L⁻¹	2.7±0.9	3.1±0.9	3.2±1.2	2.9±1.0
HDL, mmol·L⁻¹	1.7±0.4	1.7±0.5	1.7±0.5	1.5±0.5

Data are presented as mean±sd. Eight biomarkers were measured, but excluded from the statistical analysis, because of >30% missing values (B-lymphocyte chemoattractant, eotaxin-1, eotaxin-3, interleukin (IL)-1α, IL-12 subunit p70, IL-15, IL-17 and major histocompatibility complex (MHC) class I chain-related protein-A). ▪ A significantly higher level compared to the remaining three clusters (p-value <0.01); ▪ a tendency for a significantly higher level compared to the remaining three clusters (p-value between 0.05 and 0.01); ▪ a significantly lower level compared to the remaining three clusters (p<0.01); ▪ a tendency for a significantly lower level compared to the remaining three clusters (p-value between 0.05 and 0.01). CRP: C-reactive protein; IFN: interferon; MCP: monocyte chemotactic protein; TNF-R: tumour necrosis factor receptor; BDNF: brain-derived neurotrophic factor; sICAM: soluble intercellular adhesion molecule; sVCAM: soluble vascular adhesion molecule; LAP TGF-β1: latency-associated peptide of transforming growth factor-β1; MMP: matrix metalloproteinase; PAI: plasminogen activator inhibitor; IGFBP: insulin-like growth factor-binding protein; LDL: low-density lipoprotein; HDL: high-density lipoprotein.

TABLE 2

General characteristics of study subjects

Subjects, n	213
Age, years	63.6±7.0
Male	59
BMI, kg·m⁻²	26.2±5.1
FFMI, kg·m⁻²	17.0±2.4
mMRC dyspnoea grade	2.1±1.1
Current smoker	28
Smoking pack-years	46±26
Long-term oxygen therapy	17
FEV₁, L	1.40±0.54
FEV₁, % predicted	51.2±16.9
FEV₁/FVC	0.40±0.11
ITGV, % predicted	148±33
D_LCO, % predicted	56±17
6MWD, m	470±106
SGRQ, total score	51.3±17.5
Updated BODE score	2.9±2.5
Framingham 10-year risk, %	9.4±6.7

Data are presented as mean±sd or %, unless otherwise stated. BMI: body mass index; FFMI: fat-free mass index; mMRC: modified Medical Research Council; FEV₁: forced expiratory volume in 1 s; FVC: forced vital capacity; ITGV: intrathoracic gas volume; D_LCO: diffusion capacity of the lung for carbon monoxide; 6MWD: 6-min walk distance; SGRQ: St George's Respiratory Questionnaire; BODE: body mass index, obstruction, dyspnoea, exercise capacity index.

TABLE 3

The clinical characteristics of each cluster

	Cluster 1: lower-level cluster I	Cluster 2: lower-level cluster II	Cluster 3: higher-level cluster I	Cluster 4: higher-level cluster II
Female	34	47	48	33
Age, years	65±7	61±6	62±6	69±6
Lung function
FEV₁, L	1.5±0.5	1.4±0.5	1.4±0.5	1.3±0.6
FEV₁, % predicted	54±16	51±19	50±15	48±16
FEV₁/FVC, %	41±12	40±11	40±11	38±9
D_LCO, % predicted	60±17	54±16	58±17	48±14
ITGV, % predicted	139±32	156±35	147±34	152±29
RV, % predicted	153±45	175±54	162±44	165±39
TLC, % predicted	116±17	123±16	119±17	116±16
P_aCO₂, kPa	5.3±0.6	5.2±0.5	5.5±0.7	5.4±0.6
P_aO₂, kPa	9.6±1.1	9.6±1.2	9.3±0.9	9.2±0.9
S_aO₂, %	95.1±1.9	95.2±1.7	94.7±2.0	94.4±1.8
P_Imax, % predicted	85±24	77±25	78±22	75±18
P_Emax, % predicted	64±20	62±22	60±17	56±17
COPD-specific characteristics
mMRC dyspnoea grade	2.0±1.1	2.0±1.1	2.1±0.9	2.4±1.3
Long-term oxygen therapy	11	14	17	31
Smoking pack-years	44±21	47±28	48±30	47±23
Current smoker	14	31	37	36
Hospital admissions for COPD in past 12 months, n	0.51±1.17	0.42±1.17	0.49±0.84	0.69±1.28
Steroid/antibiotic courses for COPD in past 12 months, n	1.53±1.95	1.42±1.61	1.01±1.32	1.92±1.92
Exacerbations in past 12 months, n	2.05±2.60	1.85±2.41	1.54±1.81	2.55±2.18
GOLD group A/B/C/D	19/30/20/30	18/26/15/40	11/40/7/40	15/18/15/51
Physical fitness
6MWD, m	493±91	497±101	461±91	402±126
Peak work rate, W	86±30	76±23	75±27	58±26
Peak work rate, % predicted	63±25	62±25	61±29	47±22
Constant work-rate test, s	409±264	408±329	296±191	234±187
Health status
SGRQ symptoms domain	54±19	53±21	57±18	57±25
SGRQ activity domain	65±22	68±22	73±17	69±27
SGRQ impact domain	35±19	38±18	47±16	43±26
SGRQ total score	48±19	50±17	57±12	53±23
Prognostic indices
Updated BODE index	2.2±1.8	2.7±2.3	2.6±2.0	4.6±3.7
Framingham risk score	9.7±6.1	7.5±5.9	9.3±7.2	12.3±7.2

Data are presented as mean±sd or %, unless otherwise stated. ▪ A significantly higher level compared to the remaining three clusters (p<0.01); ▪ a tendency for a significantly higher level compared to the remaining three clusters (p-value between 0.05 and 0.01); ▪ a significantly lower level compared to the remaining three clusters (p<0.01); ▪ a tendency for a significantly lower level compared to the remaining three clusters (p-value between 0.05 and 0.01). FEV₁: forced expiratory volume in 1 s; FVC: forced vital capacity; D_LCO: diffusion capacity of the lung for carbon monoxide; ITGV: intrathoracic gas volume; RV: residual volume; TLC: total lung capacity; P_aCO₂: arterial carbon dioxide partial pressure; P_aO₂: arterial oxygen partial pressure; S_aO₂: arterial haemoglobin oxygen saturation; P_Imax: maximal inspiratory mouth pressure; P_Emax: maximal expiratory mouth pressure; mMRC: modified Medical Research Council; GOLD: Global Initiative for Chronic Obstructive Lung Disease; 6MWD: 6-min walk distance; SGRQ: St George's Respiratory Questionnaire; BODE: body mass index, obstruction, dyspnoea, exercise capacity index.

TABLE 4

Comorbidities per biomarker-based cluster

	Cluster 1: lower-level cluster I	Cluster 2: lower-level cluster II	Cluster 3: higher-level cluster I	Cluster 4: higher-level cluster II
Comorbidities, n	3.5±1.5	3.4±1.5	3.7±1.7	4.3±1.8
Charlson Comorbidity Index	1.6±0.7	1.5±0.9	1.6±0.8	1.8±1.1
eGFR, mL·min⁻¹	78±21	83±23	87±25	66±20
Creatinine, μmol·L⁻¹	93±20	78±15	84±14	99±32
Renal impairment	23	14	15	41
Haemoglobin, mmol·L⁻¹	9.0±0.6	9.1±0.7	9.1±0.9	8.7±0.7
Haematocrit	44±4	44±4	44±5	42±4
Anaemia	2	2	11	10
Systolic blood pressure, mmHg	140±17	136±25	138±15	145±28
Diastolic blood pressure, mmHg	83±8	82±11	83±9	83±12
Hypertension	45	44	48	62
BMI, kg·m⁻²	27.4±4.8	24.2±4.9	28.1±5.4	25.2±4.2
Obese	28	16	33	18
Underweight	9	25	4	15
FFMI, kg·m⁻²	17.6±2.0	16.1±2.5	17.5±2.8	16.9±2.0
Muscle wasting	14	50	20	26
Glucose, mmol·L⁻¹	5.8±1.0	5.7±0.8	5.9±1.0	5.6±0.9
Hyperglycaemia	56	55	61	44
Triglycerides, mmol·L⁻¹	1.6±0.7	1.3±0.5	2.0±1.3	1.4±0.7
Cholesterol, mmol·L⁻¹	5.0±1.0	5.4±1.0	5.8±1.5	5.1±1.1
Cholesterol/HDL ratio	3.2±1.0	3.3±0.9	3.8±1.3	3.6±1.1
Dyslipidaemia	41	22	52	33
Thrombocytes, ×10⁹ cells·L⁻¹	235±57	253±65	334±138	260±72
HADS Anxiety	5.5±3.	6.7±4.3	7.0±3.8	6.0±3.7
Anxiety	16	23	25	22
HADS Depression	5.1±3.5	5.5±3.9	6.7±3.3	5.7±3.2
Depression	16	15	20	14
c-IMT, mm	1.0±0.2	0.9±0.2	1.0±0.2	1.0±0.2
Atherosclerosis	61	35	57	62
Cardiac Infarction Injury Score	10.4±7.2	11.2±5.4	11.3±5.7	13.3±7.0
T-score total body	−0.6±1.1	−1.4±1.3	−0.8±1.2	−1.4±1.5
Osteoporosis	22	41	22	41

Supplementary Materials

Figures
Tables

Supplementary Material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplement on data analysis and statistical methods 00301-2022.supplementary_methods
Tables S1-S9 00301-2022.supplementary_tables