3.1 Study cohort and serum markers associating with SSc and selected disease features
The study cohort included 371 SSc patients with serum samples that could be analyzed for markers of panel A containing immune-related molecules and other proteins linked to pro-fibrotic and inflammatory pathways. Among these, 255 patients (69 %) had serum samples also analyzed for markers of panel B, enriched for molecules previously shown to associate with human cardiac or pulmonary diseases (40, 41). There were no differences between patient characteristics and demographics with or without panel B evaluation with respect to age, sex, subtype or autoantibodies. Most patients were female (n=313, 84 %) and presented with lcSSc (n=269, 73 %). Average age at serum sampling for panel A was 57 ± 14 years and median disease duration was 2.5 years (IQR 0.7-8.1). Demographic and serological features are shown in Table 1. Data on cardiovascular medications are shown in Supplementary Table 1. Seventy-four patients presented with precapillary PH (46 with PAH and 28 with PH-ILD). To assess the first outcome measure, serum levels of the individual panel A and B markers were compared between the SSc patients and healthy controls. Markers with significantly altered serum levels in SSc patients compared to controls are displayed in Figure 1. We identified altered levels of 26 serum markers in SSc with varying levels in dcSSc, and the two major SSc specific autoantibodies, ACA and ATA (Figure 1).
Table 1: Systemic sclerosis specific demographics and cardiac outcome measures of patients evaluated by panel A.
|
Patients with available data,
n (%)
|
Panel A
|
Age at serum sampling, years
|
364/371 (98)
|
57.2 (13.9)
|
Female, n (%)
|
371/371 (100)
|
313 (84)
|
Disease duration, years
|
364/371 (98)
|
2.5 (0.7-8.1)
|
Observation period, years
|
363/371 (98)
|
5.9 (3.1-8.3)
|
Mortality, n (%)
|
371/371 (100)
|
103/371 (28)
|
lcSSc, n (%)
|
362/371 (98)
|
269 (73)
|
ACA, n (%)
|
370/371 (99)
|
196 (53)
|
ATA, n (%)
|
370/371 (99)
|
58 (16)
|
mRSS
|
355/371 (96)
|
5 (2-13)
|
BMI, kg/m2
|
243/371 (65)
|
24 (4)
|
ILD, > 10 % fibrosis
|
356/371 (96)
|
87 (24)
|
Smoking
|
234/371 (63)
|
135 (58)
|
Hypertension
|
246/371 (66)
|
31 (13)
|
Ischemic heart disease
|
246/371 (66)
|
42 (17)
|
GLS > -17.0 %, n (%)
|
111/371 (30)
|
30 (27)
|
Diastolic dysfunction, n (%)
|
135/371 (36)
|
37 (27)
|
TAPSE < 17 mm, n (%)
|
167/371 (45)
|
29 (17)
|
Data are presented as mean (SD), median (IQR) or number (percentage). ACA, anti-centromere antibodies; ATA, anti-topoisomerase I antibodies; BMI, body mass index; GLS, global longitudinal strain; ILD, interstitial lung disease; lcSSc, limited cutaneous systemic sclerosis; mRSS, modified Rodnan skin score; TAPSE, tricuspid annular plane systolic excursion. Parameters of cardiac function are evaluated on echocardiographies performed within three years from blood sampling.
3.2 Patients evaluated for cardiac dysfunction in the study cohort
Echocardiographic data on GLS, LV diastolic dysfunction and/or TAPSE, within three years from serum sampling, was available in 188/371 (51%) study cohort patients (Figure 2). Patients with available echocardiography within three years from serum sampling were older than the patients without available echocardiography (60.0 vs 54.2 years, p<0.001), they had a shorter observation period (4.9 vs 7.5 years, p<0.001) and higher mortality (38 % vs 17 %, p<0.001). There were no differences with respect to sex, disease subtype, antibodies (ATA and ACA) or disease duration at serum sampling.
3.3 Serum markers associated with LV systolic dysfunction
To assess the second outcome, association of circulating biomarkers and cardiac dysfunction including LV systolic dysfunction, LV diastolic dysfunction and RV systolic dysfunction in SSc, we analyzed all patients with available sera and echocardiographic data.
LV systolic dysfunction assessed by GLS was available in 111/188 (59 %), of whom 30/111 (27 %) had systolic dysfunction defined as GLS>-17.0 % (Figure 2). Compared to patients with normal GLS, the patients with low GLS were more often male (43 % vs 11 %, p<0.001), but did not differ regarding age, subtype, ACA, ATA, precapillary PH, disease duration or observation period. By adjusting for age and sex in logistic regression, systolic dysfunction by GLS showed significant association to two of the disease-linked serum markers displayed in Figure 1; angiopoietin 2 (ANGPT2) and OPN (Table 2).
Table 2: Association between echocardiographic ventricular dysfunction and serum markers adjusted for age and sex.
Serum marker
|
OR
|
95 % CI
|
p-value
|
Left ventricular systolic dysfunction, GLS >-17%
|
ANGPT2 (n=100)
|
3.42
|
1.52-7.71
|
0.003
|
OPN (n=93)
|
1.95
|
1.08-3.52
|
0.026
|
Left ventricular diastolic dysfunction
|
ANGPT2 (n=122)*
|
1.38
|
0.98-1.94
|
0.062*
|
Endostatin (n=113)*
|
1.60
|
0.95-2.67
|
0.075*
|
TRAIL (n=131)
|
0.41
|
0.21-0.78
|
0.007
|
Right ventricular systolic dysfunction, TAPSE <17 mm
|
ANGPT2 (n=153)
|
1.67
|
1.11-2.50
|
0.014
|
Endostatin (n=143)
|
1.86
|
1.22-2.84
|
0.004
|
OPN (n=143)
|
1.86
|
1.25-2.77
|
0.002
|
TRAIL (n=163)
|
0.32
|
0.15-0.66
|
0.002
|
ANGPT2, angiopoietin 2; CI, confidence interval; GLS, global longitudinal strain; OPN, osteopontin; OR, odds ratio; TAPSE, tricuspid annular plane systolic excursion; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. *indicates numerical associations. HR of ANGPT2, Endostatin, OPN and TRAIL represent an increase of one standard deviation.
3.4 Serum markers associated with left ventricular diastolic dysfunction
Echocardiographic evaluation of LV diastolic dysfunction by e’, E/e’, left atrial volume index and tricuspid regurgitant velocity according to recommendations, was performed within three years of serum sampling in 135 of the study cohort patients with available echo (72 %). Among these, 37/135 (27 %) presented with diastolic dysfunction. There were no differences between patients segregated by diastolic function with respect to disease duration, sex, SSc subtype, ACA or ATA. Patients with diastolic dysfunction had higher age (64.6 vs 55.8 years, p<0.001), more frequent coexisting precapillary PH (67 % vs 21 %, p<0.001) and shorter observation period (2.6 vs 6.1 years, p<0.001) than patients with normal diastolic function. By adjusting for age and sex in logistic regression, diastolic dysfunction was associated with TRAIL, while showing numerical associations with ANGPT2 and endostatin (Table 2).
3.5 Serum markers associated with RV systolic dysfunction
RV systolic dysfunction was assessed by TAPSE in 167/188 patients (89 %), with TAPSE <17 mm, indicating RV systolic dysfunction, present in 29/167 patients (17 %). Patients with low TAPSE presented numerical higher age (63.1 vs 58.3 years, p=0.074), more frequent precapillary PH during the observation period (64 % vs 30 %, p=0.001) and shorter observation period (2.8 vs 5.3 years, p<0.001) than patients with normal TAPSE. There were no differences between patients segregated by low/normal TAPSE with respect to disease duration, sex, SSc subtype, ACA or ATA. In multivariable logistic regression adjusting for age and sex, RV systolic dysfunction was independently associated with ANGPT2, endostatin, OPN and TRAIL.
Sera levels of ANGPT2, OPN and TRAIL of SSc patients compared to healthy controls are shown in Supplementary Figure 1.
3.6 Serum markers associated with mortality
Over the observation period of median 5.9 years (IQR 3.1-8.3), 103/371 patients (28 %) deceased. Patients who died were older (65.5 vs 53.9 years, p<0.001), had longer disease duration at serum sampling (3.9 vs 2.0 years, p=0.004), shorter observation period (2.6 vs 7.5 years, p<0.001), were more often male (22.3 % vs 13.1 %, p=0.028), and presented more often dcSSc (34.3 % vs 22.3 %, p=0.019), compared to survivors.
In our previous publication, we have presented two multivariable prediction models for SSc mortality (9). When we added serological markers separately to Model A (including diastolic dysfunction, sex, age at echocardiography, RV systolic function, diffusion capacity of the lungs, NT-proBNP and extent of skin affliction), ANGPT-2 and OPN remained independent predictors of mortality in their respective models (Table 3), yet excluding NT-proBNP from Model A due to insignificant p-value. When we added cardiac markers separately to Model B (including precapillary PH, sex, age at echocardiography, NT-proBNP and extent of skin affliction), ANGPT-2 and TRAIL remained independent predictors of mortality, retaining significance for all parameters of the original model (Table 4).
Table 3: Prediction of mortality by angiopoietin 2 and OPN in multivariable cox regression.
Multivariable cox regression on mortality
|
|
HR
|
95 % CI
|
p-value
|
|
HR
|
95 % CI
|
p-value
|
ANGPT2
|
1.52
|
1.20-1.93
|
0.001
|
OPN
|
1.39
|
1.01-1.92
|
0.044
|
Age
|
1.04
|
1.01-1.08
|
0.024
|
Age
|
1.05
|
1.01-1.09
|
0.023
|
Diastolic dysfunction
|
3.71
|
1.62-8.48
|
0.002
|
Diastolic dysfunction
|
3.73
|
1.76-7.90
|
0.001
|
DLCO, %
|
0.95
|
0.93-0.97
|
<0.001
|
DLCO, %
|
0.95
|
0.93-0.97
|
<0.001
|
Male sex
|
0.94
|
0.37-2.36
|
0.888
|
Male sex
|
0.79
|
0.30-2.11
|
0.638
|
mRSS
|
1.07
|
1.03-1.11
|
<0.001
|
mRSS
|
1.05
|
1.01-1.08
|
0.006
|
TAPSE
|
0.32
|
0.13-0.83
|
0.018
|
TAPSE
|
0.26
|
0.12-0.57
|
0.001
|
C-index
|
0.87
|
C-index
|
0.89
|
ANGPT2, angiopoietin 2; CI, confidence interval; DLCO, diffusion capacity of the lungs for carbon monoxide; HR, hazard ratio; mRSS, modified Rodnan skin score; OPN, osteopontin; TAPSE, tricuspid annular plane systolic excursion. HR of ANGPT2 and OPN represent an increase of one standard deviation.
Table 4: Prediction of mortality by ANGPT2 and TRAIL in multivariable cox regression.
Multivariable cox regression on mortality
|
|
HR
|
95 % CI
|
p-value
|
|
HR
|
95 % CI
|
p-value
|
ANGPT2
|
1.33
|
1.07-1.67
|
0.012
|
TRAIL
|
0.66
|
0.44-0.99
|
0.045
|
Age
|
1.07
|
1.04-1.10
|
<0.001
|
Age
|
1.07
|
1.04-1.09
|
<0.001
|
Male sex
|
1.18
|
0.63-2.22
|
0.598
|
Male sex
|
1.23
|
0.67-2.25
|
0.510
|
mRSS
|
1.05
|
1.02-1.08
|
<0.001
|
mRSS
|
1.04
|
1.01-1.06
|
0.005
|
NT-proBNP
|
1.01
|
1.01-1.01
|
<0.001
|
NT-proBNP
|
1.01
|
1.01-1.01
|
<0.001
|
Precapillary PH
|
2.54
|
1.44-4.48
|
0.001
|
Precapillary PH
|
2.03
|
1.19-3.46
|
0.009
|
C-index
|
0.83
|
C-index
|
0.82
|
ANGPT2, angiopoietin 2; CI, confidence interval; HR, hazard ratio; mRSS, modified Rodnan skin score; NT-proBNP, N-terminal prohormone of brain natriuretic peptide; PH, pulmonary hypertension; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. The HR of ANGPT2 and TRAIL represent an increase of one standard deviation.
3.7 Serum markers and relation to pulmonary disease
By logistic regression, neither OPN nor TRAIL showed association to ILD. OPN and TRAIL showed no correlation with mPAP, PVR, cardiac output (CO) nor cardiac index (CI). Angiopoietin showed a numerical association with ILD (p=0.16) and a weak correlation with mPAP, PVR and CI, but not with CO.