CONCENTRATION OF SURVIVIN IN CHILDREN WITH JUVENILE IDIOPATHIC ARTHRITIS (JIA): DIAGNOSTIC AND PROGNOSTIC VALUE – SINGLE CENTER STUDY.

: BACKGROUND: The goal of the study was to assess the diagnostic and prognostic value of survivin in Juvenile Idiopathic Arthritis (JIA). METHODS: Seventy children with JIA – 59 newly diagnosed and 11 biologically treated (46 girls and 17 boys) aged 1.5-18 years and 29 healthy children as a control group, appropriately matched in terms of sex and age, were included into the study. Concentration of survivin was assessed by an ELISA test in serum and also 18 matched synovial fluid samples collected from JIA patients. RESULTS: Children with JIA were divided according to the subtype of the JIA. In 65.7% of patients oligoarthritis was diagnosed. The disease activity was established on the basis of JADAS-27 criteria, distinguishing three levels of inflammatory process activity: low, medium and high. The largest group comprised children of low activity (62.9%) of the disease. The serum concentration of survivin was significantly higher in JIA compared to the controls (p<0.001). Higher concentration of survivin correlated positively with the presence of anti-cyclic citrullinated peptide autoantibodies (ACPA) (p=0.001). In all synovial fluid samples the concentration of survivin was higher than in matched serum (p=0.003). concentration did not statistically significantly change according to radiological damage status or active synovitis assessed by joint ultrasonography. Survivin level was independent of the disease duration time and treatment with TNF-α inhibitors in DMARD’s non-responders. CONCLUSIONS: On the basis of the conducted study, it could be concluded that survivin seem to be an independent biomarker that may be helpful in the diagnosis of JIA. the higher concentration of survivin is being associated with ACPA positivity, survivin can act as a unique biomarker that identifies an additional group of patients with JIA negative for autoantibodies, even in the early stage of the disease. We have failed to confirm survivin prognostic potential for the active and destructive course of the rheumatoid process, however it is worth to underline that children have better potential for bone regeneration than adults. Survivin measurement should be considered as aiding tool identifying DMARD’s non-responders.

patients. The prevalence of RF in patients with JIA is very low (<5%), it confers a worse prognosis. In particular, polyarticular patients with positive RF are at higher risk of a more aggressive disease course and bone erosion [3,4]. The identification of ACPA highly specific for adult RA was a milestone for adult rheumatology. ACPA have been shown to predict future risk for developing RA in otherwise healthy individuals.
However, as with RF, the sensitivity of ACPA for detecting JIA is low, but in patients with RF presence and polyarticular JIA, these autoantibodies are highly specific and predict a more severe disease course [6]. ACPA positive children with JIA are recommend earlier and more aggressive therapy. Nevertheless, the diagnosis of JIA still depends mainly on clinical characteristics, imaging examination and exclusion of other, more common causes of persistent arthritis and with low serological support [3][4][5][6]. Therefore, it is necessary to establish alternate methods or discover new autoantibodies to further improve precise diagnosis at the early stage of the disease.
Survivin is an anti-apoptotic protein, an oncoprotein, known as a tissue marker of cancer. During recent years, the role of survivin in non-malignant cells was intensively explored. Survivin has been shown essential for differentiation, growth, and regeneration of healthy tissues. Due to survivin roles in apoptosis and proliferation, it plays important roles in pathogenesis of autoimmune diseases [7]. At preclinical phase of RA , high levels of survivin correlate with cytokines assuring formation of aggressive Th1 and Th17 cells. In patients after RA diagnosis, survivin predicts joint destructive course of the disease and resistance to anti-rheumatic treatment [8][9][10]. Survivin has been suggested as a predictive marker of severe course of adult RA and could be used for preclinical recognition of the disease. Survivin-positive patients have poor outcomes if treated with methotrexate (MTX) monotherapy. Serum? survivin concentration decrease is associated with better clinical response to treatment [11].
With the continuous development of medicine, the inflammatory process in JIA patients can be alleviated by regular treatment but still cannot be completely cured.
Therefore, exploring the pathogenesis of rheumatoid process is of high importance for developing precise, personalized treatments, and new drug targets. The aetiology of RA is still not clear and JIA the more/especially because of the heterogeneity of the disease phenotype. Unfortunately, because there are far fewer patients with JIA than with RA, much of our knowledge about the role of RF, ACPA, and other autoantibodies in inflammatory arthritis is derived from the adult literature. This is clearly a limitation, since these studies only pertain to a small subset of JIA patients overall, specifically polyarticular children RF positive.
Taking advantage of the recent reports in adults with RA, we designed this study aiming to validate the diagnostic and prognostic utility of survivin in patients with JIA. healthy children as a control group, appropriately matched in terms of sex and age, took part in the analysis. Children with JIA were divided according to the subtype of the disease. In the majority of the study group -65.7% -oligoarthritis was diagnosed and 32.9% children have polyarticular subtype of the JIA. In the study group there was only one child with systemic disease. The type of onset was defined according to ILAR criteria (2001) [12]. The activity of the disease was established on the basis of 27-joint Juvenile Arthritis Disease Activity Score (JADAS-27) [13]. Low, intermediate, and high stages of the disease activity have been distinguished accordingly.

MATERIAL AND METHODS
Patient characteristics was presented in Table 1. Main group of children with JIA (59/70 -84.3%) was biologically naïve and had not been treated with disease modifying anti-rheumatic drugs (DMARDs) yet. However, most of them were occasionally taking non-steroid anti-inflammatory drugs (40/59 -67,7%). Additionally, a group of 11/70 (15.7%) patients with JIA had been already treated at the time of inclusion into the study were recruited. All of them (11) were taking DMARDs (8 -methotrexate, 3 -methotrexate with sulfasalazine) and TNF-α inhibitors were occasionally taking non-steroid anti-inflammatory drugs.
The study protocol was approved by regional Medical University of Lodz, Poland At enrollment, all patients and their parents gave their written informed consent to participate in the study.

METHODS:
Serum samples were obtained simultaneously with routine laboratory examinations, including red blood cells (RBC), white blood cells (WBC), and platelets (PLT) counts, as well as erythrocyte sedimentation ratio (ESR) (cut off value <12mm/h) or C-reactive protein (CRP) (cut off value < 5mg/L). ESR was assessed by Westergren method and CRP level using the immunoturbidimetric method. Additionally, ACPA, ANA and RF were routinely assessed using standard methods.
Synovial fluid, if available, was obtained during diagnostic or/and therapeutic puncture of the swollen joint.

Measurements of serum and synovial fluid survivin:
Blood and synovial fluid samples were centrifuged and stored at -80 ºC. Concentration of survivin was assessed by a sandwich enzyme-linked immunoassay (ELISA test) in blood and 18 matched synovial fluid samples collected from swollen knees of JIA patients and in sera of children from control group by commercially available Kit (rabbit anti-human survivin; R&D, no DSV00, Lille, France). The sensitivity of the assay was 4.45 pg/ml, with the cut-off at 9.96 pg/ml. The intra-assay precision for serum -4.5-5.5% and inter-assay precision -5.7-9.5%. All serum and synovial fluid samples were tested twice.

Joint ultrasonography and hands' X-ray:
Affected joint ultrasonography assessment was performed at the time of JIA diagnosis, by a clinician experienced in musculoskeletal ultrasonography. Synovitis detected by ultrasonography was graded as mild, moderate, or severe (score from 0 to 3). Power Doppler ultrasonographic signal (PDUS) was scored on semiquantitative four-grade scale: 0 no signs of vascularization, 1 = mild (presence of single/vessel dots), 2 = moderate (presence of confluent vessel dots in less than half of the synovial area), and 3 = marked (presence of confluent vessel dots in more than half of the synovial area) (Table 1.) [14].
A Philips CX50 CompactXtreme ultrasound system and a 5-12 MHz linear transducer were used in this study.
Conventional plain-film radiographs of both hands and wrists of all children with JIA included into the study were obtained. The radiographs were scored using the Steinbrocker assessment method, with a global damage score to hands and wrists on a four-point scale from I (minimal damage) to IV (severe damage) as previously described (Table 1.) [15].

STATISTICAL ANALYSIS:
Continuous data were presented as median with interquartile range and categorical data were presented as number with respective percentage. The differences for continuous variables were tested with the Kruskal-Wallis ANOVA, Wilcoxon signedrank test or U Mann-Whitney rank sum test. Nominal variables were analyzed using Chi 2 test or Fisher test when appropriate. Correlations analyses were performed with Spearman rank test. Additionally, receiving operating characteristic (ROC) curve was used for determining best cut-off value for the survivin concentration in JIA diagnosis.   Additionally, the survivin concentration did not statistically significantly change according to radiological damage status based on hands X-ray or active synovitis grade of the affected joints assessed by joint ultrasonography (Table 2).     There was no statistically significant difference in survivin positivity according to: gender, age, JIA onset subtype, disease duration time, disease activity, radiological damage or ultrasonographic synovitis grade (Table 3). Table 3. Clinical characteristics of children with JIA divided by survivin status.
Nineteen out of 59 survivin positive children with JIA (32.2%) were also positive for RF and/or anti-CCP. Only 2 patients with positive survivin -2 (3.4%) was recognized also by simultaneous presence of RF and anti-CCP antibodies (ACPA).

DISSCUSION:
Suppression of apoptosis has been suggested as a key mechanism supporting selection and accumulation of distinct lymphocyte subsets in chronically inflamed joints  [2,4,6]. Thus, we failed to confirm survivin prognostic potential for the active and destructive course of the rheumatoid process [7,11,[21][22][23]. Surprisingly, we do not support the prior correlation of high survivin level with higher disease activity found in adults with RA and by Galeotti et al. in children with JIA [8,11,16,17,19].
Almost 63% of our study group consisted of children with oligoarticular JIA, mostly with low disease activity and short duration time of JIA symptoms. On the other side, it is confirmed that survivin level is irrespective of disease duration time. It has been postulated, that rheumatoid process starts years before clinical symptoms and may be identified by autoantibody measurement. Previous studies advocated survivin to provide insight in the pre-antibody process. Some authors suggest that survivin occurs at the earlier phase of disease development followed by autoantibody production [8,17]. Our results indicate that survivin concentration is independent of the disease duration timecould be increased in sera of newly diagnosed children at the beginning of the disease and after years, even in patients who had been biologically treated.
However, some authors noticed significant decrease in survivin levels from baseline over 24 th months [11]. supports previous studies in adults with RA [8]. However, other authors noticed no dependence of survivin presence due to ACPA or RF positivity in RA patients [11].
They also underlined that neither the presence of RF or ACPA, nor the combined multibiomarker disease activity score supported discrimination in the disease outcome achieved by survivin measurements. Surprisingly, we found no significant dependence Nevertheless, the observations assessing the influence of treatment on survivin release are conflicting. In the previous studies in adults with RA it was indicated that in survivin positive methotrexate non-responders, anti-TNF treatment appeared to be less successful than combination of synthetic disease-modifying drugs [11]. The authors speculated that poor response to anti-TNF treatment and lack of a direct correlation between serum survivin and inflammatory markers or disease activity suggested independent mechanism of survivin release. The process triggering and abrogating survivin release in RA could therefore pave a way to efficient therapeutic control of the disease [11]. On the other hand, some researchers proved that survivin concentration is decreased in TNF-α treatment responders. Some authors showed that  [11]. Therefore, measuring survivin adds new dimension to early recognition and monitoring of patients at risk of developing rheumatoid process [17].
We are aware of the limitations of our work. First of all, small group of children with JIA biologically treated -only with TNF-α inhibitors. These study group patients had long disease duration time, with DMARDs treatment failure before implementation of TNF-α inhibitors. Additionally, only two subtypes of JIA onset are properly represented, and there is small number of joint fluid samples available.

CONCLUSIONS:
On the basis of the conducted study, it could be concluded that survivin seem to be an independent biomarker, irrespective of disease duration time, that may be helpful in the diagnosis of JIA. Nevertheless, the higher concentration of survivin is being associated with ACPA positivity, survivin can act as a unique biomarker that identifies an additional group of patients with JIA negative for autoantibodies even in the early stage of the disease. We have failed to confirm survivin prognostic potential for the active and destructive course of the rheumatoid process, however it is worth to underline that children have better potential for bone regeneration than adults. Survivin measurement should be considered as aiding tool identifying DMARDs nonresponders and biomarker probably not dependent of treatment with TNF-α inhibitors. At enrollment, all the patients and their parents gave their written informed consent to participate in the study.

CONSENT FOR PUBLICATION:
Not applicable.

AVAILABILITY OF DATA AND MATERIALS:
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.