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REVIEW
Update on biomarkers in systemic sclerosis: tools
for diagnosis and treatment
Alsya J. Affandi
1,2
& Timothy R. D. J. Radstake
1,2
& Wioleta Marut
1,2
Received: 24 April 2015 /Accepted: 16 June 2015 /Published online: 14 July 2015
#
The Author(s) 2015. This article is published with open access at Springerlink.com
Abstract Systemic sclerosis (SSc) is a complex autoimmune
disease in which immune activation, vasculopathy, and exten-
sive fibrosis of the skin and internal organs are among the
principal features. SSc is a heterogeneous disease with varying
manifestations and clinical outcomes. Currently, patients’ clin-
ical evaluation often relies on subjective meas ures, non-
quantitative methods, or requires invasive procedures as
markers able to predict disease trajectory or response to thera-
py are lacking. Therefore, current research is focusing on the
discovery of useful biomarkers reflecting ongoing inflamma-
tory or fibrotic activity in the skin and internal organs, as well
as being predictive of future disease course. Recently, remark-
able progress has been made towards a better understanding of
numerous mechanisms involved in the pathogenesis of SSc.
This has opened new possibilities for the development of novel
biomarkers and therapy. However, current proposed bio-
markers that could reliably describe various aspects of SSc still
require further investigation. This review will summarize stud-
ies describing the commonly used and validated biomarkers,
the newly emerging and promising SSc biomarkers identified
to date, and consideration of future directions in this field.
Keywords Autoantibodies
.
Biomarker
.
miRNAs
.
Pulmonary fibrosis
.
Skin fibrosis
.
Systemic sclerosis
Systemic sclerosis (SSc) is an autoimmune disease characterized
by fibrosis of the skin and internal organs, preceded by vascular
and immune dysfunction [1]. Depending on the extent of cuta-
neous fibrosis, SSc is classified into two major subtypes: limited
cutaneous (lcSSc) and diffuse cutaneous SSc (dcSSc). In lcSSc,
skin thickening is restricted to the areas distal to the elbows and/
or knees, such as hands and fingers. In dcSSc, the presence of
skin lesions is more extensive and internal organs involvement
is relatively more severe. This classification is supported by the
association with specific autoantibodies that specifically define
the two types of clinical phenotypes. Both SSc phenotypes can
be complicated by severe internal organ dysfunction. Pulmonary
fibrosis and pulmonary arterial hypertensio n (PAH) are the two
most feared complications, representing the major causes of
mortality in SSc patients [2]. Owning to its complex nature
and heterogeneity, SSc remains one of the greatest challenges
to both investigators and physicians. Despite intense investiga-
tion, so far , only a few biomarkers for SSc have been fully
validated and widely accepted. Herein, we present a review of
the literature on promising prognostic biomarkers, biomarkers
of disease activity , skin fibrosis, and lung involvement, with the
aim to provide a comprehensive update on usability of bio-
markers for research and clinical guidance.
Diagnostic and prognostic biomarkers
SSc-specific autoantibodies as predictive markers
The presence of autoantibodies is a central defining aspect of
autoimmune disease s. Autoantibodies are seen at the first
This article is a contribution to the Special Issue on Immunopathology of
systemic sclerosis – Guest Editors: Jacob M. van Laar and John Varga
* Timothy R. D. J. Radstake
T.R.D.J.Radstake@umcutrecht.nl
1
Department of Rheumatology and Clinical Immunology,
University Medical Center Utrecht, Heidelberglaan 100,
3584 CX Utrecht, The Netherlands
2
Laboratory of Translational Immunology, University Medical Center
Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Semin Immunopathol (2015) 37:475–487
DOI 10.1007/s00281-015-0506-4
diagnosis in more than 95 % of SSc patients and have been
associated with distinct disease subtypes and with differences
in disease severity. Antitopoisomerase I (ATAs) and
anticentromere antibodies (ACAs) are the most widely used
diagnostic biomarkers for SSc [3–5].
Anti-Scl-70 antibodies originally identified by Douvas
et al. [6] are directed against DNA topoisomerase I [7]and
therefore should be more accurately termed antitopoisomerase
I antibodies (ATAs). These autoantibodies are seen predomi-
nantly in dcSSc patients; however, their presence is not entire-
ly restricted to this clinical subset since a subgroup of lcSSc
patients was also found to be ATA-positive [8, 9]. ATA has
been associated with poorer prognosis, increased mortality,
pulmonary fibrosis, and cardiac involvement [9–12]. Another
recent study of clinical outcomes in patients with digital ulcers
showed that patients positive for ATAs developed Raynaud’s
phenomenon earlier and had double rate of lung fibrosis as
compared with ACA-positive patients [13]. Some reports in-
dicate that changes in ATA titers over time can be useful in
monitoring disease activity and progression and therefore use-
ful for prognostic purposes [14].
ACAs recognize centromeric protein from CENP-A to
CENP-F, of which CENP-B is reported t o be a major
autoantigen reacting with virtual ly all anti-CENP-positive
SSc sera [15, 16]. ACAs are found in 20 to 30 % of SSc
patients, and in up to 90 % of lcSSc patients [4, 17]. In patients
with Raynaud’s phenomenon, ACAs have been reported to
predict the onset of lcSSc [3, 18]. While severe interstitial
fibrosis and renal crisis occur rarely, pulmonary arterial hyper-
tension occurs in about 20 % of anti-CENP patients [9, 10,
19]. Anti-CENPs are often associated with other antibodies,
such as anti-Sjogren’s-syndrome-related antigen A (anti-Ro)
[20] or antimitochondrial antibodies [21]. Moreover, it has
been reported that ACA positivity correlated with a more fa-
vorable prognosis and lower mortality compared with the pos-
itivity of other SSc-related autoantibodies [22].
Antibodies against RNA polymerase I and III (anti-RNP I
and III) are detected with high specificity in SSc patients (98–
100 %) [23, 24]. Their prevalence varies from 10 to 25 % in
different SSc cohorts. Anti-RNP I and III are associated with
dcSSc involvement and renal crisis [25]. More recently, it has
been shown that the presence of anti-RNP is associated with
rapid onset of the disease and skin thickening. Therefore, they
are still among the best predictive markers available for rapid
skin progression [26]. Autoantibodies to RNP II are uncom-
mon and are not specific for SSc since they can be also be
found in the sera of systemic lupus erythematosus (SLE) and
overlap syndrome [27
].
Ad
ditional SSc-specific autoantibodies with diagnostic or
prognostic utility include the anti-Th/To and anti-U3 RNP
(antifibrillarin) antibodies. Th/To autoantibodies are directed
against subunit of RNase P and RNase MRP [28]. They are
found in 2–5 % of SSc patients and are clinically associated
with lcSSc (8.4 % of lcSSc patients, 0.6 % of dcSSc) [10, 29].
Among lcSSc patients, anti-Th/To are a marker of the worst
survival rate perhaps related to severe pulmonary embolism
(PE) preceding PAH and renal crisis [19]. It has been reported
that the presence of anti-Th/To may assist in identifying sine
SSc subset in patients with pulmonary fibrosis [30]. Anti-U3
RNP antibodies target fibrillarin, a small protein belonging to
the U3 small nuclear ribonucleoprotein (RNP) complex. Al-
though they are considered to be a specific marker for SSc,
they are found in less than 7 % of SSc sera and their confir-
mation using advanced techniques continues to be a challenge
[31]. They are most frequent in males and African Americans
with SSc and are associated with muscle involvement and
increased risk of PAH [32].
Recently, autoantibodies against angiotensin II type 1 re-
ceptor (AT
1
R) and endothelin-1 type A receptor (ET
A
R) have
been shown to be elevated in the sera of most SSc patients,
and associated with vascular and fibrotic complications [33,
34]. They are more frequent in SSc-PAH/connective tissue
disease-PAH compared to other forms of pulmonary hyper-
tension. Therefore, they could serve as new predictive and
prognostic biomarkers of PAH in SSc. These autoantibodies
not only predict development of PAH but also are associated
with higher mortality in SSc patients [33].
More recently , antiestrogen receptor antibodies (anti-ERα)
were detected in sera of 42 % SSc patients; whereas, no anti-
ERα antibodies were found in healthy controls. Anti-ERα anti-
bodies were significantly associated with disease activity and
were mainly found among patients with the diffuse form of the
disease, the ANAs positivity, and the late capillaroscopy pattern.
However , it is important to note that anti-ERα antibodies are not
specific for SSc since they were also detected in patients with
SLE but not in other patients with other autoimmune diseases
such as rheumatoid arthritis (RA) or Behcet’s disease [35].
Other autoantibodies of relevance to SSc but less common-
ly present in SSc patients include the anti-U11/U12 RNP an-
tibodies. They are highly associated with severe lung fibrosis,
and anti-PM-Scl antibodies found in patients with SSc overlap
and with myositis [36]. Remarkably, many of the autoanti-
bodies in SSc show correlation with other type of biomarkers
such as those for skin fibrosis and pulmonary complications,
as we will discuss below (Table 1).
Circulating miRNA
miRNAs are a class of endogenous and evolutionary con-
served short, noncoding RNAs that bind to the 3′ untranslated
region of target genes. Once bound, miRNAs repress target
gene translation or promote mRNA destabilization and degra-
dation. They are expressed in a tissue-specific and cell type-
specific manner but can also circulate in the bloodstream, and
such circulating miRNAs are remarkably stable [37]. This has
raised the possibility that miRNAs may be probed in the
476 Semin Immunopathol (2015) 37:475–487
circulation and can serve as novel diagnostic markers. It has
been shown that the elevated expression of pro-fibrotic
miRNAs and reduced expression of antifibrotic miRNAs are
important factors in the developments of fibrosis in SSc. Fur-
thermore, several studies have already demonstrated that the
levels of selected miRNAs were altered in the serum of SSc
patients [38, 39].
The levels of miR-150 were found to be downregulated in
the serum of SSc patients versus healthy controls and were
correlated with more severe clinical manifestations. For in-
stance, h igher incid ence of antit opoisomerase I antibodies
and a higher prevalence of pitting scars were seen in patients
with lower miR-150 levels than in those without. Although
the difference did not reach statistical significance, patients
with lower miR-150 levels had a higher ratio of dcSSc to
lcSSc and a higher modified Rodnan skin score (mRSS) when
compared with patients with normal levels of miR-150 [40].
Similar correlation was observed for another miRNA—
miR196a, where patients with lower serum miR-196a levels
had significantly higher ratio of dcSSc to lcSSc and higher
mRSS; in addition, they showed higher prevalence of pitting
scars than those without [41]. Other studies evidenced that the
serum level of miR-30b [42] and let-7a [43]werehighlyde-
creased in SSc patients versus healthy controls. Both miRNAs
were again downregulated more strongly in diffuse subset of
SSc than in limited SSc. Interestingly, miR-30b and let-7a
were inversely correlated with mRSS [42, 43].
On the contrary, serum levels of miR-92a [44] and miR-142-
3p [45] were markedly higher in SSc when compared to healthy
controls or SLE, dermatomyositis, and scleroderma spectrum
disorder (SSD) patients. Therefore, these miRNAs may provide
as useful diagno stic markers for the differentiation of SSc from
other scleroderma spectrum disorders (Table 2).
Biomarkers for disease activity
One of the main challenges of SSc studies is to develop a
sufficient tool for a global measurement for disease activity
that represents an ongoing disease activity and/or response to
treatment. Unlike other autoimmune diseases such as SLE or
RA, for many SSc patients, ongoing inflammation is difficult
to assess and vascular and tissue fibroses are not easy to quan-
titate especially in the early stage of the disease. Currently, the
Valentini disease activity index, developed by The European
Scleroderma Study Group (EScSG), is the most widely used
activity score in SSc studies [46]. This activity index includes
mRSS, DLCO, and E SR, w ithout a specific biochemical
marker. The Medsger disease severity scale is also frequently
used as a measure of disease activity [47]. However, this as-
sessment may more reflect damage or severity rather than the
ongoing disease activity.
The enhanced liver fibrosis (ELF) test was developed as a
clinical grade serum test for chronic liver diseases, including
procollagen-III aminoterminal-propeptide (PIIINP), tissue in-
hibitor of matrix metalloproteinase-1 (TIMP-1), and
hyaluronic acid (HA) in its algorithm. Each of these three
serum markers is increased in SSc patients as compared to
healthy controls and associated with more severe complica-
tions or increased mortality [48–50]. Recently, ELF test was
tested in SSc patients and showed significant correlations with
both disease activity and severity [51]. ELF score also corre-
lated with mRSS, Health Assessment Questionnaire-Disability
Index (HAQ-DI), and inversely correlated with DLCO, but it
did not correlate with vasculopathy features such as PAH [51].
Other candidate biochemical markers for disease activity
and severity in SSc have been derived from their association
with an organ-specific involvement. The markers for pulmo-
nary involvement in SSc, serum vWF and KL6, were signif-
icantly associated with disease severity and activity, respec-
tively [52, 53]. The serum level of cartilage oligomeric matrix
protein (COMP), a molecule that has been associated with
skin and lun g fibrosis as we describe below, also showed
correlation with disease severity [54]. The angiopoietin/Tie2
axis has gained some interests due to their roles in angiogen-
esis. Serum levels of angiopoietin-2 (Ang-2), but not
angiopoietin-1, have been shown to correlate with disease
activity [55]. Another study found a similar but not significant
Table 1 Diagnostic and prognostic biomarkers—autoantibodies
Biomarker Source Association Reference
Antitopoisomerase I (ATAs) Serum dcSSc, poor prognosis, increased mortality, lung fibrosis,
cardiac involvement
[8–14]
Anticentromere (ACAs) Serum lcSSc, PAH, more favorable prognosis, lower mortality [4, 9, 10, 15–17, 19]
Anti-RNA polymerase I and III (anti-RNAP I, III) Serum dcSSc, skin progression, renal crisis [23–26]
Anti-Th/To Serum lcSSc, a marker of worst survival rate, muscle involvement, PAH [10, 19, 29, 30]
Anti-U3 RNP Serum Activity [31, 32]
Anti-AT
1
R, anti- ET
A
R Serum Activity, PAH, vascular and fibrotic complications, higher mortality [33]
Anti-ERα Serum Activity, dcSSc [35]
Anti-U11/U12 RNP Serum Severe lung fibrosis [36]
Semin Immunopathol (2015) 37:475–487 477
trend in plasma, although the authors found a stronger corre-
lation using the ratio of Ang-2 and its soluble receptor Tie2
with disease activity [56].
The classic inflammatory cytokine IL-6 is increased in the
sera of SSc patients and has been associated with multiple
organ involvement including skin [57], the occurrence of pul-
monary fibrosis [58], FVC decline, and increased mortality
[59]. Plasma IL-6 level was found to be higher in ATA-
positive and anti-RNAP III-positive patients but not in
ACA-positive SSc patients [60]. In one study, serum IL-6
was shown to correlate with disease activity [61], although
this was not found by others [62]. In a genetic association
study, IL-6 polymorphism in SSc patients was shown to be
associated with disease activity and HAQ-DI, unfortunately
circulating IL-6 was not measured [63].
Growth differentiation factor 15 (GDF-15) is a distant
member of the TGFβ superfamily and found to be elevated
in the serum of SSc patients compared to healthy controls [64,
65]. In SSc patients, serum GDF-15 levels showed strong
correlation with mRSS, disease activity, and disease severity
[65], in particular those with pulmonary involvement, as we
will discuss below.
It is important to note that many of these studies assessing
disease activity are cross-sectional and limited to small co-
horts at single centers. Future multicenter validation and lon-
gitudinal study are necessary to assess their sensitivity for
changes over time in a larger population. Multibiomarker ap-
proach such as the ELF score should also be considered
(Table 3).
Biomarkers correlating with skin fibrosis
Skin fibrosis, the hallmark of SSc, is defined as an excess
deposition and accumulation of extra cellular matrix in the
dermis. Despite our growing understanding of this process
and many available targets, our therapeutic success in amelio-
rating skin fibrosis in SSc is still minimal. Even today, the
gold standard for measuring SSc skin fibr osis is mRSS, a
relatively simple determination of skin thickness, which has
significant inter-observer variability and is rather subjective.
Moreover, the mRSS may not be sensitive enough to find
smaller but important and early changes in skin thickening
[66]. Therefore, there is a need for other specific and more
precise markers for assessing skin fibrosis.
Peripheral blood biomarkers
There is a large number of potential circulating biomarkers for
skin fibrosis which include COMP, MMP-9, MMP-12, LOX,
IL-6, IL-10, and CXCL4 (Table 4). Here, we will discuss those
biomarkers that are most robustly shown to be of potential
relevance.
Cartilage oligomeric protein 1 (COMP) is a non-
collagenous glycoprotein, mostly synthesized by
chondrocytes, oste oblasts, tenocytes, synovial fibroblas ts,
and dermal fibroblasts. This protein, highly regulated by
TGF-β, is not detectable in the healthy skin but is highly
overexpressed in skin biopsies and fibroblasts of SSc patients
[67, 68]. Moreover, COMP was found to be increased in SSc
sera and correlated with the extent of skin involvement, as
assessed by mRSS and ultrasound [69]. More recent study
confirmed high levels of COMP in the serum of SSc patients,
and its level was higher in dcSSc subset than in lcSSc [54].
Matrix metalloproteinases (MMPs), responsible for the
degradation of collagens and other extra cellular matrix
(ECM) proteins, are also involved in the release and activation
of many cytokines and growth factors [70]. Several inhibitors
are known to control their activity. Both, MMPs and their
inhibitors, were extensively studied in the pathogenesis of
SSc. MMP-9 and MMP-12 w ere found to be a potential
markers for skin fibrosis.
Table 2 Prognostic biomarkers—circulating miRNA
Biomarker Expression in SSc Source Association Reference
miR-150 Downregulated Serum dcSSc, skin fibrosis [40]
miR196a Downregulated Serum dcSSc, skin fibrosis, higher prevalence of pitting scars [41]
miR-30b Downregulated Serum dcSSc, inversely correlated with skin fibrosis [42]
let-7a Downregulated Serum dcSSc, inversely correlated with skin fibrosis [43]
miR-92a Upregulated Serum SSc [44]
miR-142-3p Upregulated Serum SSc [45]
Table 3 Biomarker in SSc disease activity
Biomarker Source Association Reference
ELF test Serum Activity, severity [51]
vWF Serum Severity [52]
KL-6 Serum Activity [53]
Ang-2 Serum Activity [55]
COMP Serum Activity [54]
IL-6 Serum Activity [61, 62]
GDF-15 Serum Activity, severity [65]
478 Semin Immunopathol (2015) 37:475–487
MMP-9, whose substrates include type IV collagen in
basement membrane, has been associated with chronic in-
flammatory autoimmune diseases, including rheumatoid ar-
thritis [71]andSLE[72]. Moreover, its overexpression has
been reported in various pathologic conditions characterized
by excessive fibrosis, including idiopathic pulmonary fibrosis
[73] and chronic pancreatitis [74]. In SSc, fibroblasts isolated
from SSc patients expressed more MMP-9 than healthy con-
trols. Furthermore, serum level of MMP-9 was elevated in
SSc, with higher concentration in dcSSc compared to lcSSc,
and correlated well with mRSS [75].
MMP-12, also known as macrophage metalloelastase
(MME), has a broad substrate specificity for matrix mac-
romolecules, recognizing elastin, type IV collagen, fibro-
nectin, or v itronectin. MMP-12 has been implicated in
different pathological conditions including atherosclerosis,
cancers, and skin diseases [76, 77]. In SSc patients, der-
mal fibro blasts expresse d an d re leased MMP-12 [78].
More recent studies reported that serum levels of MMP-
12 were significantly increased in SSc patients, also cor-
relating well with skin fibrosis, with dcSSc having higher
levels of MMP-12 [79].
Lysyl oxidase (LOX) is an extracellular copper enzyme
that cross-links collagen and elastin, thus stabilizing collagen
fibrils. Consistent with its expression in the skin and fibro-
blasts in the context of SSc, the levels of LOX were elevated
in the serum of SSc patients versus healthy controls. Further
analysis revealed a correlation of LOX concentration with the
mRSS in patients without lung fibrosis, indicating its specific
correlation with skin fibrosis. Moreover, LOX levels were
higher in SSc patients with dcSSc than in those with lcSSc,
which may reflect a more advance fibrosis in diffuse subset of
SSc [80].
In SSc patients, there is a strong relationship between in-
flammation and fibrosis supported by the upregulation of
both, pro-inflammatory and pro-fibrotic markers in the serum
as well as in skin. The role of different cytokines and
chemokines has been analyzed in skin fibrosis of SSc in sev-
eral studies. For instance, IL-6 and IL-10 serum levels were
found to be elevated in SSc patients and significantly corre-
lated with skin fibrosis assessed by mRSS [57]. However,
recently, Codullo et al. confirmed that SSc patients expressed
high level of IL-6 but did not find clear associations with
mRSS or other clinical parameters [62].
CXCL4, largely viewed as a pro-inflammatory chemo-
kine, in addition to its chemoattractant activity, regulates
an array of immune cells, including T cells, monocytes,
dendritic cells, as well as non-immune cells like endothe-
lial cells. Recently, van Bon et al. used a proteomic ap-
proach and identified CXCL4 as a potential biomarker
associated with multiple organ involvement in SSc. Circu-
lating CXCL4 levels strongly correlated with the extent of
skin fibrosis more with dcSSc subsets than lcSSc. In a
prospective cohort study, elevated CXCL4 in the serum
of SSc predicted a faster progression of skin fibrosis [81].
Gene expression profiling
Gene expression profiling from skin biopsies is another
interesting approach to identify biomarkers for skin fi-
brosis. Skin biopsies, although more difficult to obtain,
allow for a more direct insight into the ongoing fibrotic
reaction. Moreover, they can lead to the discovery of
genes specific f or different subsets of SSc and to predict
if patients will develop more severe subset of the dis-
ease. For in stance, in 2008, Milano et al. reported a
177-gene signature that was associated with severity of
skin disease in diffuse subsets of SSc [82]. This identi-
fication not only allows for a better understanding of
the disease pathogenesis but also provides important in-
formation for novel therapeutic targets.
TGF-β is one of the most potent pro-fibrotic cytokines
in SSc and also one of the strongest stimulators for the
differentiation of fibroblasts into activated myofibroblasts.
Therefore, a group of researchers examined the expression
of genes highly upregulated by TGF-β and found that
some of these genes were highly correlated with the skin
score. When they expande d their studies to interferon-
regulated genes, they found that several of these genes
also strongly correlated with the mRSS. Therefore, the
combination of both TGF-β and IFN-regulated genes,
namely COMP a nd thrombospondin-1 (TSP-1) (TGF-β
Table 4 Emerging biomarker in
SSc skin fibrosis
Biomarker Source Association Reference
COMP Skin, serum dcSSc, skin fibrosis [54, 67–69]
MMP-9, MMP-12 Skin, serum dcSSc, skin fibrosis [75, 78, 79]
LOX Skin, serum dcSSc, skin fibrosis [80]
IL-6, IL-10 Serum Skin fibrosis [57, 62]
CXCL4 Serum dcSSc, skin fibrosis [81]
TSP-1, IFI44, Siglec-1 Skin Skin fibrosis [83]
LH2 Skin Skin fibrosis activity [84]
Semin Immunopathol (2015) 37:475–487 479
regulated genes), and IFN-inducible 44 (IFI44) and
sialoadhesin (Siglec-1) resulted in a particularly strong cor-
relation with skin score [83].
Lysyl hydroxylase-2 (LH2), an enzyme involved in colla-
gen biosynthesis, was found to be elevated in the skin biopsies
and isolated fibroblasts of SSc patients and could represent a
marker for the skin fibrotic activity in these patients. LH-2
overexpression was found to be accompanied by an associated
increase in the Pyr cross-links present in the accumulated col-
lagen in the SSc patients. These Pyr cross-links are critical for
the mechanical stability and tensile strength of collagen [84]
(Table 4).
Biomarkers involved in SSc lung involvement
Pulmonary complications are common in SSc patients. They
are most often manifested by the fibrotic interstitial lung dis-
ease (ILD), or pulmonary vascular disease leading to pulmo-
nary arterial hypertension (PAH), or co-occurrence of both.
Together, SSc-associated ILD and PAH are the major cause
of disease-related mortality in SSc [85, 86].
Interstitial lung disease
The majority of SSc patients have evidence of pulmonary
fibrosis, based on autopsy and radiographic findings [87]. In
the European League Against Rheumatism Scleroderma Tri-
als and Research (EUSTAR) registry, pulmonary fibrosis ap-
peared more common in diffuse than in limited SSc (53.4 vs
34.7 %) [9]. To detect ILD in SSc, a chest imaging using high-
resolution computed tomography (HRCT) and pulmonary
function tests (PFT), including the measurement of forced
vital capacity (FVC) and diffusing capacity of lung for carbon
monoxide (DLCO), are being used. A new quantitative HRCT
has improved visual radiographic assessment of ILD but not
yet widely available [88]. Moreover, repeated exposure to
radiation can be detrimental. Although FVC and DLCO show
correlation with HRCTand adequately measure lung function,
they are not specific for ILD or the ongoing fibrotic process.
Therefore, additional biomarkers that are more accessible, re-
peatable, and complement both HRCT and PFT are needed
[89].
Several different studies have examined the use of the lung-
epith elial-derived protein surfactant protein-D (SP-D) and
glycoprotein Krebs von den Lungen-6 (KL-6). As reviewed
by others previously, most studies showed increased serum
SP-D and KL-6 and their correlation with decline in FVC
and DLCO in SSc patients, however with varying degrees of
correlation [90, 91]. A recent study showed that serum KL-6
correlated strongly with HRCT-fibrosis score, serum ATA ti-
ters, and correlated inversely with FVC and DLCO [53]. They
found a moderate correlation of SP-D with HRCT-fibrosis
score, but no significant association with other clinical param-
eters tested. Other studies also found correlation of serum KL-
6, as well as COMP, with lung fibrosis [92]. Additionally, SSc
patients with elevated SP-D and KL-6 had far more frequent
ATA positivity and less frequent ACA compared to those with
normal level [93]. Serum KL-6 also showed strong correlation
with mRSS and disease activity index, indicating it to be a
multipurpose biomarker candidate in SSc [53].
CCL18 is a chemokine produced by antigen presenting
cells, particularly by alveolar macrophages in different inter-
stitial lung diseases [94]. In SSc, the level of CCL18 was
elevated in the bronchoalveolar lavage (BAL) fluid, lung, se-
rum, and associated with lung involvement [95, 96]. One
study showed moderate but significant negative correlation
of serum CCL18 with DLCO and FVC in SSc [97]. In their
retrospective cohort analysis, serum CCL-18 level was de-
creased in SSc patients having an improvement of pulmonary
fibrosis (as measured by HRCT, PFT, and BAL analysis) and
comparable to the decrease of KL-6 and SP-D [97]. Another
study found a similar observation where serum CCL18 in SSc
correlated with DLCO decline and total lung capacity (TLC)
decline, and changes of TLC over a period of at least 6 months
[95]. A longitudinal study of a 4-year period showed that a
cut-off serum CCL18 value at 187 ng/ml is able to predict
worsening ILD [98]. This was later reproduced in an indepen-
dent cohort with a similar cut-off value and hazard ratios [99],
but another study challenged this finding suggesting that cor-
relation between CCL18 and changes in FVC could only be
seen at a short term (1 year) but not at a longer period [100].
Interestingly, a recent microarray analysis of SSc-ILD lung
showed that lung CCL18 RNA expression correlated with
changes of HRCT-score FibMax and negatively correlated,
although not strongly, with % predicted FVC [96].
van Bon et al. showed that chemokine CXCL4 was asso-
ciated with lung disease manifestations in SSc [
81].
Patients
who had high circulating CXCL4 (>10 ng/ml) developed lung
fibrosis earlier compared to those who had low CXCL4, as
measured by >30 % decrease of FVC and HRCT. In the pro-
spective cohort, patients with a high CXCL4 baseline showed
a significantly faster decline in DLCO and a higher prevalence
of HRCT-confirmed lung fibrosis. Earlier study has showed a
significant increase of CXCL4 in BAL fluid from SSc patients
exclusively those with ILD [101].
CXCL8 (IL-8) functions as the main chemotactic factor for
neutrophils and other granulocytes. CXCL8 gene polymor-
phisms were associated with an increased susceptibility to
SSc [102, 103]. Circulating CXCL8 level has been reported
to be elevated in SSc patients [62, 104], but this was found not
in all studies [60]. Two studies showed an increase of CXCL8
level in the BAL fluid from SSc patients that correlated with a
more extensive lung fibrosis based on HRCT [92] and in-
versely correlated with DLCO, FVC, and TLC [105], but nei-
ther study measured circulating CXCL8. Other investigations
480 Semin Immunopathol (2015) 37:475–487
using CXCL8 level in the serum of SSc patients showed
no significant association with PFT or any future pulmo-
nary involvement [62, 106]; whereas, one report showed
serum CXCL8 association w ith DLCO decrease [104].
These findings suggest that CXCL8 level may more
strongly reflect disease progression locally rather than
systemically.
S100A8 (MRP-8, calgranulin A) and S100A9 (MRP-14,
calgranulin B) are members of the S100 calcium-binding pro-
teins. Together they form a complex, S100A8/9 (calprotectin,
calgranulinA/B), that is able to modulate inflammatory pro-
cesses mainly by binding to toll-like receptor 4 (TLR4) [107].
Earlier studies have shown increased of S100A8/9 or their
homodimeric form ats in the sera [108, 109], f eces [110,
111], saliva [112], BAL fluids [92], and skin [109, 113]of
SSc patients compared to healthy individuals. High level of
S100A8/9 in the BAL fluid [92] and serum [109]inpatients
has been associated with an extensive lung fibrosis and ATA
positivity, although no direct correlation to PFT was found. A
recent proteomic analysis of serum samples from SSc patients
also showed S100A8/9 to be increased particularly in lcSSc
having lung fibrosis and ATA-positive patients [114]. As re-
cent evidences point to the role of TLR4 signaling in fibrosis
[115–117], S100A8/9 is a potentially interesting molecule to
investigate further.
The serum level of GDF-15 has been shown to be strongly
associated with multiple organ involvement in SSc especially
in the lung. Serum GDF-15 level was higher in patients with
ILD compared to those without, and its level in the serum
negatively correlated with DLCO and FVC [64, 65]. Patients
with high level of serum GDF-15 had a more frequent ATA
and a less frequent ACA positivity [64]. Importantly, SSc
patients with higher baseline GDF-15 leve l showed lower
DLCO and worsened l ung diseases severity score over a
follow-up period of up to 30 months, suggesting its value as
a predictive prognostic marker of lung function and fibrosis in
SSc [65](Table5).
Pulmonary arterial hypertension
Although PAH appears to be more frequent in the lcSSc than
dcSSc (9.2 vs 5.9 % according to EULAR registry [9]), it can
occur in all forms of SSc. Patients with SSc-PAH has a poor
prognosis; therefore, an early detection of SSc-PAH and initi-
ation of therapy are essential in disease management. Trans-
thoracic echocardiography (TTE) of pulmonary artery systolic
pressure is the most widely used screening tool for PAH; how-
ever, the method has considerable measurement variability
and may not be sufficiently sensitive for the detection of early
disease [120]. The more invasive right heart catheterization
(RHC) is still the golden standard to confirm PAH in SSc
patients.
N-terminal pro-brain natriuretic peptide (NT-proBNP), a
biomarker of myocardial stress, has been intensively studied
as a diagnostic marker for SSc- PAH . Serum NT-pro-BNP
levels have been showed to strongly correlate with mean pul-
monary arterial pressure (PAP) and pulmonary vascular resis-
tance (PVR) [121]. They also showed association of NT-
proBNP with severity of PAH and risk of mortality [121].
Later studies also showed similar correlation of NT-proBNP
with increased PAP based on TTE and RHC, as well as neg-
ative correlation with DLCO and the presence of ATA
[122
–12
6]. More recently, NT-proBNP has been in used as a
diagnostic marker in combination with other modalities in-
cluding TTE and PFT. This approac h, as r eported b y the
DETECT study and others, gave an improved sensitivity and
reduced missed diagnosis for early SSc-PAH as compared to
the ESC/ERS guidelines [125, 127, 128]. However, it is im-
portant to note that this marker is not specific to PAH, as it
may results from pulmonary venous hypertension or other
cardiac dysfunction.
In search for surrogate marker for SSc-PAH, many inves-
tigators have looked into the molecules produced by or acting
on the endothelium and have attempted to correlate them to
hemodynamic and pulmonary function parameters. Markers
of vascular injury such as vascular endothelial growth factor
(VEGF), endothelin-1 (ET-1), and von Willebrand factor
(vWF), as well as the soluble adhesion molecules ICAM-1
andVCAM-1havebeenshowedtobeelevatedinSScsera
and associated with PAH [129, 130]. Both circulating VEGF
and ET-1 levels were found higher in SSc patients with PAH
compared to those without and their level correlated positively
with pulmonary arterial pressure [129, 131]. However, ET-1
associat ion with pulmona ry pressure or function was not
Table 5 Biomarker in SSc-ILD
Biomarker Source Association Reference
SP-D and KL-6 Serum HRCT, FVC, DLCO [53, 92, 93, 118, 119]
CCL18 Serum, BAL, lung HRCT, FVC, TLC, DLCO [95, 96, 98–100]
CXCL4 Plasma, BAL HRCT, FVC, DLCO [81, 101]
CXCL8 Serum, BAL HRCT, FVC, TLC, DLCO [62, 92, 104–106]
S100A8/9 Serum, plasma, BAL HRCT [92, 109, 114]
COMP Serum HRCT [92]
GDF-15 Serum DLCO, FVC [64, 65]
Semin Immunopathol (2015) 37:475–487 481
found in another study [132]. Serum VEGF levels also corre-
lated with decline of DLCO in cohorts of SSc patients without
ILD [131] and in limited subtype [56]. The level of vWF in
plasma of SSc patients was found to correlate with PAP, based
on Doppler cardiography [133]. In a substudy from QUINs
randomized placebo-controlled trial, baseline serum vWF an-
tigen concentrations significantly related to disease activity,
inversely correlated to %FVC and %DLCO at baseline, and
were able to predict elevated PAP of >40 mmHg after 3 years,
based on TTE [52]. However, another study did not find cor-
relation between serum vWF and PFT measurements, perhaps
due to a smaller cohort and a difference in statistical analysis
[134]. In an 18-month prospective cohort, plasma vWF did
not correlate with future changes in DLCO or skin score [81].
Furthermore, the use of vWF as a biomarker can be challeng-
ing since there is already a large variation in healthy individ-
uals: ABO blood group, genetic polymorphism, and age are
among the determinants [135, 136].
In addition to its association with skin and lung fibrosis,
circulating CXCL4 levels were elevated in SSc patients who
had evidence of PAH compared to those without, as deter-
mined on RHC [81]. In this study, high CXCL4 levels were
also associated with an earlier development of PAH. In a tran-
scriptome analysis of lung biopsies from idiopathic PAH
patients, CXCL4 was one of the most highly and differentially
expressed genes as compared to healthy controls [137]. As
CXCL4 exerts angiostatic properties on pulmonary arterial endo-
thelial cells [138], this suggests that CXCL4 might be involved in
the pathophysiology of PAH in SSc.
As mentioned above, serum GDF-15 was associated with
pulmonary fibrosis and impaired lung function. In SSc pa-
tients with PAH, the plasma level of GDF-15 was significantly
higher as compared to those without [139]. Plasma concentra-
tions of GDF-15 showed strong correlation with right ventricular
systolic pressure (RVSP) on echocardiography, NT-proBNP
plasma levels, and negative correlation with DLCO, but no
Table 6 Biomarker in SSc-PAH
Biomarker Source Association Reference
NT-proBNP Serum PAP, PVR, DLCO [121–126]
VEGF Serum PAP, DLCO [56, 131]
ET-1 Plasma PAP [130, 132]
vWF Serum, plasma PAP, FVC, DLCO [52, 81, 133, 134]
Anti-AT1R and anti-ETAR Serum PAH development [33, 34]
CXCL4 Plasma PAH development [81]
GDF-15 Plasma RVSP, DLCO [64, 65, 139]
Macrophage
Monocyte
IFI44
Siglec1
IL-10
IL-6
CXCL4
S100A8/9
Type-I IFN
IL-6
CXCL8
pDC
Endothelial
cell
ET-1
vWF
ICAM-1
Ang-2
VEGF
Epithelial
cell
KL-6
SP-D
CXCL8
B cell
Plasma
cell
Muscle cell
mDC
Fibroblast
ATA
ACA
Anti-Th/To
Anti-U3RNP
Anti-AT
1
R
Anti ET
A
R
Others Abs
NT-proBNP
VEGF
GDF-15
Undefined
sources
miR-30b
miR-92
miR-142-3p
miR-150
miR-196a
COMP
TSP-1
TIMP-1
Inflammatory
mediators
Vascular injury
Pro-fibrotic
factors
Myofibroblast
Fibrosis
ECM
Vasculopathy
Tissue remodelling
Other
cells
let-7a
HA
Loss of organ function
MMPs
LH2
LOX
VEGF
GDF-15
CCL-18
Fig. 1 A schematic depiction of biomarkers in systemic sclerosis and
their production by different cell types. Immune cells produce a large
number of biomarkers that have been investigated in SSc, and their
interaction with endothelial cells, fibroblasts, and other cell types may
eventually lead to extracellular matrix (ECM) deposition and the
progression of disease
482 Semin Immunopathol (2015) 37:475–487
correlation with any RHC-based hemodynamics [139]. Im-
portantly, a ROC curve analysis showed that a plasma GDF-15
cut-off level at 125 pg/ml was able to identify SSc-PAH better
than NT-proBNP at 473 pmol/L (93 % sensitivity and 88 %
specificity vs 86 % sensitivity and 30 % specificity) and was able
to predict mortality in SSc patients [139]. Later studies measuring
serum GDF-15 in SSc patients had very few patients with PAH
in their cohorts that make it difficult to interpret [64, 65]. In a
cohort of patients with idiopathic PAH, elevated serum GDF-15
level was associated with right atrial pressure, wedge pressure,
and serum NT-proBNP level [140]. They also showed potential
prognostic value of serum GDF-15 as it was related to changes of
serum NT-proBN P and venous oxygen saturation in their
follow-up cohort [140](Table6).
Novel approaches to identify biomarkers in SSc
The fast advancement of current molecular biology and bio-
chemical techniques has moved research from the reductionist
approach of studying one individual component at a time,
towards a more holistic approach where multiple high-
throughput omics layers—so called systems medicine—can
be determined in clinically well-defined patient groups.
Genomic-wide association studies, whole transcriptome, and
proteome analysis have been performed in recent studies and
yielded novel candidate biomarkers for SSc [141–143]. The
use of more recent state-of-the-art technologies such as mass
cytometry, that would enable us to phenotype immune com-
partments or other cells of interest in a great detail, are cur-
rently underway. The challenge of systemic multilayered
large-data gathering approach is the complexity of big-data
management, analysis, and interpretation. Computational
models and computer learning algorithms are essential to an-
swer specific research questions that hopefully lead investiga-
tors to the discovery of new biomarkers and understanding
pathways.
Conclusions
Discoveries of new biomarkers and composite scores in SSc
have supported the more conventional approaches in patients’
evaluation including mRSS, PFTs, RHC, and HRCT. For ex-
ample, incorporating NT-proBNP to TTE and PFT measures
has improved diagnosis of SSc-PAH significantly. Several
biomarkers with clinically important multipurpose utility can
give an added value, for example, both KL-6 and CXCL4
showed correlation with skin and lung involvement and pre-
dictive of future disease course. Many of these new promising
biomarkers (see Fig. 1), however, still require validation and
assessment in longitudinal cohorts or in clinical trials. More
investigations in prognostic markers that can predict patients’
disease trajectory or differences in response to therapy are of
urgent need. In the near future, systems medicine approaches
including the true integration of multilayered data may pro-
vide more complete assessment of patients, novel biomarkers,
understanding of disease, or even drug discovery and person-
alized therapy.
Acknowledgments This study was supported by the Dutch Arthritis
Association (T.R.D.J. Radstake), the Netherlands Organization for Scien-
tific Research (VIDI grant to T.R.D.J. Radstake and Mosaic grant to A.J.
Affandi), ERC starting grant (to T.R.D.J. Radstake), and Marie Curie
Intra-European Fellowship (to W. Marut).
Conflict of interest The authors declare that they have no competing
interests.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use, dis-
tribution, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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