Novel diagnostic approaches and markers for follow-up in systemic sclerosis

Fibrosis is a leading cause of morbidity and mortality in the Western World.
The hallmark of fibrosis is abnormal and exaggerated fibroblast proliferation
and deposition of extracellular matrix components leading to irreversible organ
damage and dysfunction. Inflammatory diseases such as systemic sclerosis,
pulmonary fibrosis, but also liver cirrhosis and myocardial infarction end up
with fibrosis. This concerns also involvement of the pulmonary arterial
vessels, leading to pulmonary arterial hypertension (PAH). PAH and pulmonary
fibrosis have a very bad prognosis . Together responsible for 60% of the
mortality in systemic sclerosis.
Systemic sclerosis is a complex multisystem autoimmune disease with a wide
heterogeneity in phenotype and outcome. Apart from progressive fibrosis,
pathologic processes in systemic sclerosis include immune system activation and
vasculopathy.
Although recent ethiopathogenic advances have been made, systemic sclerosis
remains one of the most complex autoimmune diseases in terms of identifying
patients at risk of developing adverse outcomes, therapeutic management and
establishing the therapeutic effects. Further insight in factors predicting
disease severity and organ involvement may lead to earlier and organ-specific
treatment and improves diagnostic follow-up.
The focus of the present study will be to determine factors that predict
disease activity, severity and progression. This will be done through combining
evaluation of laboratory parameters (including inflammatory markers) and
estimation of vasculopathy by nailfold capillaroscopy. Moreover, we will
evaluate blood flow and changes in flow in the hands of patients with systemic
sclerosis as well as effects of treatment on this blood flow. Finally, we will
identify factors that evaluate therapeutic efficacy and may be used as markers
for follow-up.
By determining factors that predict disease severity and give insight in
(future) organ involvement follow-up of patients with systemic sclerosis could
be further individualized and future treatment may be initiated in earlier
stages based on the risk of development of certain organ damage.

The main objective of the current study is to determine factors that are
markers for disease activity, future progression and may be indicative for
specific organ involvement. In the current project we will focus on the
following objectives:

I) Determination of factors (blood abnormalities) that predict systemic
sclerosis disease severity, organ involvement, and progression

II)Evaluation of correlations between disease severity, laboratory findings and
nailfold capillaroscopy findings as well as blood flow analysis

III) Determination of factors that represent or evaluate therapy efficacy

Objective I (approx. 6 months):
When patients are diagnosed with systemic sclerosis it is of importance to
determine which patients may develop early progressive fibrosis or are at risk
for future pulmonary arterial hypertension. Currently available biomarkers show
insufficient sensitivity and specificity in patient risk stratification. The
ideal biomarkers predict future developments of relevant outcomes, are easily
measurable and change with effective therapy. In the present study we will
determine potential novel biomarkers for systemic sclerosis. Various
biomarkers, as mentioned below, will be evaluated:

Connective tissue growth factor (CTGF)
TGF-beta responsive gene signature
N-terminal pro-peptide of type I (PINP) and type III (PIIINP) collagen
C-terminal pro-peptide of type I collagen markers of collagen formation
cross-linked carboxyterminal telopeptide of type I collagen
C-terminal telopeptide of type I collagen
soluble intercellular adhesion molecule-1 (sICAM-1)
soluble vascular adhesion molecule 1 (sVCAM-1) markers of vasculopathy
E-selectin
NT-proBNP
Uric acid markers of PAH
Auto-antibodies associated with systemic sclerosis as autoantibodies against
Scl-70, CENP A, CENP B, RP11, RP155, Fibrillarin, NOR90, Th/To, PM-Scl100,
PM-Scl75, Ku, PDGFR, Ro-52
inflammatory parameters (including cell biology) and interleukins

In preliminary studies it was described that the above mentioned biomarkers
were elevated in systemic sclerosis patients. Studies were all performed in
small patient groups and the correlation with disease severity or progression
has not been studied.
In the proposed study, we have the opportunity to include a large number of
patients and their data. Due to the large study population (Dept. of Internal
Medicine, Erasmus MC 60 patients, Dept. of Dermatology, Erasmus MC 20 patients)
we might gain a significant insight into the relationship between expression of
above mentioned markers and disease activity/severity.
Informed consent will be obtained from all patients included in our study and
disease activity/severity will be well documented.
Disease activity will be evaluated and scored in all cases by estimation of the
degree of skin fibrosis (performed by a dermatologist) scoring, pulmonary
fibrosis (determined by computed tomography), pulmonary arterial hypertension
(determined by echocardiography), kidney function (determined by creatinin
levels in serum and urinary protein loss), diffusing capacity of the lung for
carbonmonoxide (DCLO, evaluated by lung function tests) and using the EUSTAR
Systemic Sclerosis Activity Score.

During follow-up of our patients determination of kidney function, DCLO and
degree of skin fibrosis will be performed every 6 months.
Echocardiography will be repeated every 12 months after inclusion in our study.
Clinical findings at baseline and after 5 years will be correlated to the
results obtained in our biomarker analysis in order to gain insight in the
potential relation between biomarker profiles and disease severity.

If necessary skin biopsies will be taken by experienced dermatologists in order
to obtain skin biopsies of good quality and from standardized sites of the
human body
ELISA assays will be performed according to manufacturer*s protocol using the
following kits: Human CTGF ELISA construction kit, Antigenix America Inc., RIA
Kit for Human PINP, RIA Kit for Human PIIINP, ELISA Kit for human PICP, ELISA
Kit for Human CTXI (all by Uscn Life Science Inc.), RayBio® sICAM-1 ELISA Kit,
RayBiotech Inc., Quantikine® Human SVCAM-1 Immunoassay, R&D Systems Inc., Human
sE-selectin ELISA Kit, Abnova Corporation.
Detection of autoantibodies using the EuroImmun Systemic Sclerosis Blot
(detecting the above mentioned autoantibodies)
Cytokine analysis will be performed using a Cytokine Blot
TGF-beta responsive gene expression profile will be evaluated using RT-PCR
after design of appropriate primers and probes

Objective II (total time span 6-9 months):
Nailfold capillaroscopy has recently been proven to be of significance in
determining vasculopathy in systemic sclerosis. A typical systemic sclerosis
associated pattern is characterized by microhemorrhages, capillary loss, giant
capillaries and ramifications. Until now, based on these observations, three
patterns have been described, that enable stratification between *early*,
*active* and *late* disease. These patterns have been found to correlate with
disease severity. Using the capillaroscopic skin ulcer risk index (CSURI score)
the risk of developing digital ulcers in systemic sclerosis patients can be
predicted.
By performing nailfold capillaroscopy at different time points and correlate
these outcomes with factors as described under objective I, we will try to
characterize specific disease severity patterns in more detail.
Moreover we will incorporate questionnaires (Scleroderma Health Assessment
Questionnaire (sHAQ), Cochin Scale determining hand disability and SF-36
determining patient health). We expect that we will also identify additional
specific nailfold capillary patterns that enable improved patient risk
stratification. Potentially, based on these scores, interventional approaches
can be adjusted.

Set up:
1. Nailfold capillaroscopy will be performed on all systemic sclerosis patients
from the department of Internal Medicine, Erasmus MC included in our study at
different time points. First evaluation will be on study entrance and nailfold
capillaroscopy will be repeated every three months. We will evaluate and score
all single capillaries of digits two to four of both hands in order to generate
a more accurate description of the nailfold capillary abnormalities.
2. Correlate findings under Objective I to findings obtained with nailfold
capillaroscopy in order to more accurately estimate disease activity and
predict progression.
3. Evaluate nailfold capillaroscopy findings before and after start of
treatment to determine potential differences in capillary profiles (in relation
to potential differences in biomarkers), that may be predictive for treatment
responses. Final evaluation will be 12 months after initial start of
immunosuppressive therapy

Laser Doppler imaging has recently been shown to effectively demonstrate blood
flow restrictions in the hands of systemic sclerosis patients. In our
outpatient clinics we will use this technique to monitor blood flow gradients
in hands of patients with systemic sclerosis before and during treatment and
evaluate potential improvement in this gradient. We will correlate the findings
obtained under Objective I with Laser Doppler images in order to more
accurately estimate disease activity.

Objective III (total time span of 18 months):
We will evaluate the correlation between the effects of treatment, as initiated
based on disease severity and organ involvement, on the disease severity and
serum levels of markers of collagen synthesis, vasculopathy, systemic sclerosis
associated autoantibodies and other possible biomarkers that we identified
under Objective I.
We will include the data on serum biomarker levels throughout disease
progression or regression under therapy as identified under Objective I.

Set up:
1. Evaluation of therapy efficacy by determining the above mentioned disease
activity parameters by experienced physicians
2. Evaluation of biomarker expression in patients sera
3. Correlations between improvement of disease activity with serum biomarker
levels in stored patients sera collected from the different departments to
estimate which biomarker in systemic sclerosis patients may be useful in
follow-up of disease activity
4. Estimation of nailfold capillaroscopy and blood flow changes after treatment
to provide a non-invasive tool for determination of therapy efficacy
5. Implementation of measurements of biomarkers in follow-up of systemic
sclerosis patients, next to currently running measurements of for instance
auto-antibodies

In a 5 years period patients with established systemic sclerosis are followed
at the outpatient clinics of the department of Immunology accorinding to
current follow-up protocols at our department for patients with systemic
sclerosis.
During this periode, patients are asked to complete a set of 3 questionnaires
on 3 different occassions, taking approximately 30 minutes per set of
questionnaires.
Secondly, at regular blood drawing occassions an additional number of 4 tubes
(34 ml in total) will be taken from these patients, which menas that an extra
amount of 34 ml of blood will be drawn at 10 regular visits.
Based on these two interventions in the current study the burden for the
included patients is very low, meaning a visit taking 30 minutes longer for 3
times in 5 years years. As the blood drawings are part of regular blood
drawings no extra interventions are needed.
Additional risks therefore seem to be neglectable.

At current, there is no direct benefit for the included patients in the study.
Based on the results of this study our patients may benefit in the future of
optimized follow-up protocols and potentiaaly earlier start of organ-specifixc
treatment, however at the moment this is just speculative.