Extended autoantibody panel in Turkish patients with early‐stage systemic sclerosis: Coexpressions and their influences on clinical phenotypes

Abstract Background/Aim To investigate the frequency and clinical relevance of an extended autoantibody profile in patients with systemic sclerosis (SSc). Materials and Methods In this cross‐sectional study, serum from 100 consecutive patients was subjected to indirect immunofluorescence (IIF) (HEp‐20‐10/primate liver mosaic) and Systemic Sclerosis Profile by EUROIMMUN to evaluate anti‐nuclear antibodies (ANA) and autoantibodies against 13 different autoantibodies in patients with SSc less than 3 years. Results Ninety‐three of 100 patients were positive for ANA by IIF. Fifty‐three patients showed single positivity, 26 anti‐topoisomerase antibodies (anti‐Scl70 ab), 16 anticentromere antibodies (ACAs), six anti‐RNA polymerase III antibodies (anti‐RNAPIII ab), one anti‐Ku antibody, one anti‐PM/Scl100 antibody, two anti‐PM/Scl75 antibodies, one anti‐Ro52 antibody, whereas 32 patients had multiple autoantibody positivities. Among classic SSc‐specific autoantibodies, anti‐Scl70 and anti‐RNAPIII abs showed the highest cooccurrence (n = 4). One patient was simultaneously positive for anti‐RNAPIII ab and ACA, and one was positive for ACA and anti‐Scl70 ab. The clinical features were not statistically different between single and multiple autoantibody‐positivity for classic SSc‐specific autoantibodies (ACA, anti‐Scl70 ab, and anti‐RNAPIII ab), except for digital ulcer in the multiantibody positive ACA group (p = .019). Conclusion Based on our results, coexpression of autoantibodies is not uncommon in SSc patients. Although autoantibodies specific to SSc in early disease show generally known clinical features, it remains to be investigated how the coexpression of autoantibodies will affect clinical presentation.


| INTRODUCTION
][5] The clinical course of SSc is not easily predictable because of the clinical and serologic heterogeneity.][11] The most known and widely used autoantibodies targeting topoisomerase, centromere proteins, and RNA polymerase III have been reported to be mutually exclusive and strongly associated with certain clinical phenotypes. 12However, there is also evidence of overlap between these autoantibodies. 3,13Although the relationship between the single positivity of some specific autoantibodies and the involvement of certain organs is well known today, the relationship between the positivity of compound autoantibodies and their clinical significance in SSc patients with short disease duration has yet to be investigated in detail. 14herefore, we aimed to investigate autoantibodies' frequency and clinical relevance in the early stage of SSc using an expanded panel of autoantibodies.We also aimed to follow these patients to investigate how the relationship between these autoantibodies and organ involvement progressed.Here we report our initial baseline data.

| Patient selection
One hundred patients with SSc were recruited consecutively from six tertiary centers in Turkey, specializing in the care of patients with SSc.We included limited or diffuse cutaneous SSc patients 10 in the early stage of the disease with a disease duration of <3 years from the first non-Raynaud symptom, 15 meeting the 2013 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) SSc classification criteria. 9e included the SSc patients with overlap syndromes, and the overlap syndromes were defined as cases meeting the classification criteria for one or more connective tissue diseases concurrent with SSc.Patients with other comorbidities that could lead to autoantibody positivity were excluded.
Demographic characteristics and clinical and laboratory findings of the patients were recorded.Disease duration was calculated as the time between the onset of the first non-Raynaud symptom and the enrollment date.Variables included Raynaud's phenomenon, skin and musculoskeletal involvement, pulmonary arterial hypertension (PAH), interstitial lung disease (ILD), renal crisis (ever), gastrointestinal symptoms (dysphagia, reflux, early satiety, constipation, diarrhea) and if recorded any malignancy.The extent of the skin involvement was assessed using the modified Rodnan skin score (mRSS). 16atients with a pulmonary artery pressure above 45 mmHg on echocardiography underwent right heart catheterization because of the strong correlation between this estimated cut-off level and right heart catheterization. 17Pulmonary hypertension was defined as a mean pulmonary arterial pressure of ≥20 mmHg, and precapillary pulmonary hypertension was defined as pulmonary vascular resistance of ≥2 wood units and a pulmonary capillary wedge pressure of ≤15 mmHg on right-sided heart catheterization. 18ILD was defined as the presence of any evidence of pulmonary fibrosis on lung imaging by high-resolution computed tomography scan.The renal crisis was described as an abrupt onset of severe hypertension (systolic blood pressure [BP] ≥ 180 mmHg and/or diastolic BP ≥ 100 mmHg) without an alternate etiology, with or without microangiopathic anemia or decline in renal function. 19his study complied with the Declaration of Helsinki and was approved by the Kocaeli University School of Medicine Ethics Committee, Kocaeli, Turkey, with study number KOU/GOKAEK 2017/347.

| Autoantibody analysis
Sera from all patients were tested using a commercially available indirect immunofluorescence (IIF) (ANA, Mosaic Hep-20-10/Liver; Euroimmun) assay and line immunoblot assay (Systemic Sclerosis [Nucleoli] Profile EuroLine [IgG]; Euroimmun) simultaneously in a single central laboratory.Serum aliquots were stored at −80°C until the time of testing.The assays were performed according to the manufacturer's instructions.For ANA, results above the dilution of 1:100 were considered positive.The Systemic Sclerosis [Nucleoli] Profile kit contained 13 recombinant antigens: those expressed in Escherichia coli (RNA polymerase III [RNAPIII; subunits RP11 and RP155], fibrillarin, the 90-kd nucleolar protein NOR-90, and Th/To) or insect cells using the baculovirus system (CENP-A, CENP-B, PM/Scl-100, PM/Scl-75, Ku, and tripartite motif-containing protein 21 [TRIM-21]/Ro52) plus platelet-derived growth factor receptor expressed in mammalian cells and native topo I (Scl70) isolated from calf and rabbit thymus.Sera were analyzed at a dilution of 1:101, and autoantibodies were detected using alkaline phosphatase-labeled anti-human IgG.The EuroLine flatbed scanner was used to provide semiquantitative results.Readings obtained with a signal intensity of 0−5, 6−10, 11−25, 26−50, and >50 were defined as negative, borderline, medium, strong, and very strong bands and were given equivalent scores of negative, (+), 1+, 2+, and 3+, respectively.We classified autoantibodies into two groups: classic SSc-specific autoantibodies-anti-Scl70, anti-ACA and anti-RNAPIII abs, and SSc-associated autoantibodies-all others autoantibodies.

| Statistical analysis
Descriptive statistics for clinical and demographic characteristics of the patients are presented as frequency and percentage (%) for categorical variables and mean with standard deviation (mean ± SD) or median with interquartile range (median [Q3−Q1]) according to the distribution of the continuous variables.The distribution normality was assessed visually and through the Shapiro−Wilk test.An independent sample T-test was used to analyze how specific autoantibodies affected clinical outcomes in positive and negative groups and cases of single and multiple positivity.A χ 2 test was also performed for categorical variables.
In evaluating semiquantitative results, we considered a score of ≥+1 for each autoantibody to be positive.In addition, in statistical analyses, we assumed CENPA and/or CENPB positivity as ACA positive and, similarly, RNAP11 and/or RNA 155 positivity as RNAPIII positive.
Statistical analyses of further demographic and phenotypic data were performed using SPSS, version 20.0 (IBM Inc.).Two-sided p values less than 0.05 were considered statistically significant (p < .05).

| Characteristics of the study population and frequency of autoantibodies
Demographic, clinical, and serologic characteristics of the 100 SSc patients are presented in Table 1.Most patients had lcSSc (63%), whereas 36 had diffuse involvement, and only one had sine scleroderma.Ninety-three out of 100 patients were positive for ANA by IIF.Anti-topoisomerase antibodies (Anti-Scl70 ab) was the most frequent (41%) autoantibody, followed by ACA with a rate of 27%.The anti-RNAPIII ab positivity was 15%.Except for classic SSc-specific autoantibodies (ACA, anti-Scl70, or anti-RNAPIII), anti-Ro52 was the most common SSc-associated autoantibody (22%).None of the patients were positive for either anti-fibrillarin or anti-PDGF abs.
The majority of the patients exhibited single positivity for analyzed autoantibodies.Of 100 patients, 53 were single positivity for any autoantibodies and 48 of which were classic SSc-specific autoantibodies (26 with anti-Scl70 ab, 16 with ACA, 6 with anti-RNAPIII ab), and 5 were SSc-associated autoantibodies (1 with anti-Ku ab, 1 with anti-PM/Scl100 ab, 2 with anti-PM/ Scl75 ab, and 1 with anti-Ro52 ab).The distribution, copositivity of autoantibodies, and the IFA patterns determined by the International Consensus on Autoantibody Patterns (ICAP: anapatterns.org)are shown in Figure 1 with an emphasis on the SSc-specific autoantibodies.
There were 32 patients with multiple antibody positivity.Amongst the classic SSc-specific autoantibodies, anti-Scl70 and anti-RNAPIII antibodies showed the highest cooccurrence and were simultaneously positive in 4 patients.One patient was positive for anti-RNAPIII ab and ACA, and another for ACA and anti-Scl70 ab simultaneously.All patients with anti-Ro52 ab except one were also positive for the other antibodies.Eight patients (8%) were positive for SSc-associated autoantibodies with single or multiple staining.No specific autoantibodies were detected in 15 patients (15%), and ANA was positive in 11 of them.

| Associations of autoantibodies with clinical features
The association between the classic SSc-specific autoantibodies (ACA, Scl70, and RNAPIII) and clinical features was evaluated by comparing the antibody-positive patients (regardless of being single or multiple positive) with the rest of the study population (Table 2).Overlap syndrome was more common in patients with ACA, and only 1 (3.7%) patient with ACA had ILD.Anti-RNAPIII ab was associated with a common disease subtype, ILD, and the highest mRSS among the three groups.Among all SSc patients, two out of 3 patients with malignancy were anti-RNAP ab positive.
When we compared the clinical features of the patients in terms of single and multiple ab positivity for each of the classic SSc-specific autoantibodies (ACA, Scl70, and RNAPIII), the clinical features were not different between the subgroups (Table 3).However, the digital ulcers were more frequent in the multiple abpositive ACA group than single positives.
Clinical features of the patients who were positive only for SSc-associated autoantibodies are shown in Table 4.All the patients except one with anti-Ku (dcSSc) and another with anti-PM/Scl100 (sine scleroderma) had limited lcSSc.Only one patient with anti-Ro52 had overlap syndrome (Sjogren's syndrome [SS]).None of these patients had either ILD, PAH, or malignancy.
When we compared the anti-Ro52 positive and negative patients, we found that DUs and ACA positivity were more common in anti-Ro52 positive patients compared to negative ones (27.3% vs. 9%, p = .035and 45.5% vs. 21.8%,p = .027,respectively).Anti-Ro52 positive patients also showed more NOR90 positivity simultaneously (9.1% vs. 0%, p = .047).There was no difference between anti-Ro52 positive and negative patients regarding ILD, PAH, GIS involvement, disease duration, or other autoantibody positivities.In this study, we investigated an extended autoantibody profile and its association with the clinical manifestations in a group of patients with early-stage SSc.
Consistent with the general knowledge, classic SScautoantibodies (anti-Scl70 ab, ACA, anti-RNAPIII ab) were more frequent among all tested autoantibodies and exhibited the expected clinical features.However, our results revealed that a substantial proportion of patients were positive for more than one autoantibody, including classic SSc-specific autoantibodies known as mutually exclusive.Another result that should be considered is that in patients negative for classic SSc-specific autoantibodies (8%), an extended test profile showed the presence of another autoantibody.
The prevalence of anti-Scl70, ACA, and anti-RNAPIII abs in our patients was 41%, 27%, and 15%, respectively.1][22] However, the frequency of autoantibodies may vary by ethnicity, and data on SSc-specific autoantibodies from Turkey are limited to the frequencies of anti-Scl70 and anti-centromere abs. 23In addition, in only one study, the frequency of anti-RNAPIII antibodies was reported as 2.2%, which was lower than our results. 24The difference in results may be because the previous study was conducted in patients with extensive SSc and long disease duration or because the two studies' antibody analysis methods were different.
Our results revealed that a substantial proportion of patients were positive for more than one autoantibody, including classic SSc-specific autoantibodies.Although classic SSc-specific autoantibodies (ACA, topo I, and RNAPIII) are thought to be mutually exclusive and do not change from one to another during the disease, there is evidence that they may occur together. 3,4,25With the recent advent of multiplexed immunoassays, the notion that these autoantibodies are mutually exclusive is slowly disappearing. 26Consistent with these we detected the coexpression of classic SSc-specific autoantibodies in some patients: anti-Scl70 ab and anti-RNAPIII Ab positivity in 4, ACA and anti-RNAPIII ab in 1, and anti-Scl70 ab and ACA in 1. 27,28 Regardless of being single or multiple positive, comparison of classic SScspecific autoantibody-positive patients with the rest of the study population showed expected clinical associations with these abs (ACA with IcSSc, anti-Scl ab with ILD, and anti-RNAPIII ab with dcSSc and malignancy, etc.).When we compared the single and multiple positivities for each of classic SSc-specific autoantibodies in terms of clinical involvements, there were no significant differences between subgroups, except for the higher occurrence of DUs in patients who were also positive to ACA.There are uncertainties about the clinical features of multiple antibody-positive SSc patients in the studies reported to date.Unexpectedly, in 7 dcSSc patients with anti-RNAPIII ab and ACA, reported by Satoh et al., none had significant organ  involvement, such as renal crisis during the disease course. 29imilarly, in our study, none of the patients with multiple ab positivity (1 with anti-Scl70 ab and ACA, and 2 with anti-RNAPIII ab and ACA positivity) had severe internal organ involvement; interestingly, all had lcSSc.It was difficult to comment the clinical significance of these multiple autoantibody positivities because the number of patients with SSc-associated autoantibodies needed to be higher to make detailed comparisons.Although in daily clinical practice, we only evaluate a limited number of autoantibodies in SSc patients, in multiple previous studies, agonistic and classical diagnostic autoantibodies have also been reported.This indicates the importance of poly autoimmunity in the SSc pathogenesis, and significant percentage of multiple autoantibody positivity in our patients also supports this hypothesis. 30ur results showed that, in patients with more than one autoantibody, HEp-2 IFA specified by the ICAP patterns were consistent with the LIA results to a certain extent.This was especially found for ACA, which is expressed as AC-3 (centromere), anti-Scl70 ab as AC-29 (topo I like), anti-RNAPIII ab as AC-5 (nuclear large/ coarse speckled), and anti-PmScl as AC-8 (homogeneous nucleolar).One of our observations regarding the results of this study was that in patients with multiple autoantibodies, we could not detect all associated patterns.In most situations, HEp-2 IFA patterns reflect the staining of a single autoantibody in a single cell compartment and conserve basic morphological characteristics that allow their identification according to the ICAP guidelines.However, in daily practice, some situations may not fit this simple rule, and morphological characteristics do not allow the classification as elementary patterns.For example, in the presence of different autoantibodies that react with the same cell compartment, a single pattern may be detected in IFA, and unseen patterns may remain hidden under this pattern. 31nti-Ro52 was positive in 22% of the patients in our study.Anti-Ro52 can be found in a variety of autoimmune conditions, including SS, systemic lupus erythematosus, SSc, and inflammatory myositis. 32Results from two large cohorts of the German network for systemic Scleroderma and the Canadian Scleroderma Research Group demonstrated that anti-Ro52 was the second most common autoantibody in patients with SSc. 12,33It was our study's third most common autoantibody following anti-Scl70 and ACA antibodies.Anti-Ro52-positive SSc patients were more likely to be older, to have ILD, and to have overlap syndrome compared with anti-Ro52-negative patients. 34Unlike these results, we found that anti-Ro52 was more prevalent in ACA-ab-T A B L E 4 Clinical features of the patients who were positive only for scleroderma-associated autoantibodies.positive patients and more associated with DUs.Regarding the aforementioned clinical conditions, it seems essential to re-evaluate the patients during the follow-up period of the disease.Although anti-RNAPIII ab (15%) positivity was not uncommon in our study patients, none had SRC.In a large SSc cohort of 1325 patients, than 90% developed SRC within 5 years of SSc onset. 11The occurrence estimates of SRC were 6.5%, 7.1%, and 7.6% at 5, 10, and 15 years, respectively.SRC prevalence is lower in North America than in Europe. 35SRC is considered less frequent in the Turkish SSc population, with a reported frequency of 3% in a cross-sectional study from Turkey. 20Therefore, the absence of SRC in our study can be partly explained by genetic and geographical differences and the short disease duration of the patients in our study population.

Staining intensity
Anti-PM/Scl antibodies were positive in 10% of the patients in our study.They have been reported to be associated with inflammatory myositis and calcinosis. 36n a multinational cohort of SSc, myositis was seen only in subjects positive for both anti-PM/Scl75 and anti-PM/ Scl100 antibodies. 37From this perspective, the three patients with both anti-PM/Scl75 and anti-PM/Scl100 antibodies appeared at higher risk for myositis in our study.However, none had myositis or calcinosis at baseline clinical evaluation at enrollment.Despite all these results, continuous monitoring of serum creatine kinase levels in anti-PM/Scl ab positive patients may be helpful in the further diagnosis of myositis.
One of the limitations of our study was the small sample size.The most important reason for this was to include patients who met the 2013 ACR/EULAR classification criteria and had as short a disease duration.Including only patients with short disease duration might have affected the frequencies of the disease manifestations and prevented reaching statistically significant results in some subgroup analyses, such as associations between disease manifestations and autoantibodies.Another limitation of our study was that the study kits did not contain all SSc-specific autoantibodies, such as anti-U1 RNP and anti-U11/U12 RNP.In addition, the low anti-Th/To frequency may also be related to the antigen (hPOP1) contained in the kit we used. 38n conclusion, our results revealed that among classic SSc-specific autoantibodies, anti-Scl70, ACA, and anti-RNAPIII abs are more common in patients with early SSc, and coexpression of autoantibodies is not infrequent.Testing a broad panel of autoantibodies yielded diagnostic support in 8% of the patients who were negative for classic SSc-specific autoantibodies.Although autoantibodies specific to SSc in early disease show generally known clinical features, it remains to be investigated how coexpression autoantibodies will affect clinical presentation.

T A B L E 3
Clinical features of single and multiple antibody positive patient subgroups within each classic SSc-specific autoantibody groups.
Demographic, clinical, and laboratory characteristics of the SSc patients.
T A B L E 1