Introduction
Autoantibodies often characterize patients with distinct clinical features and often have prognostic relevance in different connective tissue diseases. Anti-PM/Scl antibodies, first described in patients with an overlap syndrome of polymyositis (PM) and scleroderma (systemic sclerosis [SSc]), seem to be rare antibodies, especially when SSc patients were studied [
1]. In what is currently the largest study on the prevalence of anti-PM/Scl antibodies using the Pittsburgh Scleroderma Databank, only 2.5% of the SSc patients exhibited anti-PM/Scl antibodies [
2]. The low number of anti-PM/Scl-positive patients did not allow conclusive analyses concerning associated clinical features, and the SSc patients were not classified according to their disease subsets. However, the descriptions of anti-PM/Scl-positive patients point to a higher prevalence of patients with muscular involvement, supporting other investigations using smaller populations or patients with myositis [
1,
3‐
6]. An association between the presence of anti-PM/Scl antibodies and Raynaud phenomenon (RP), arthritis, and interstitial lung disease was suggested as well [
5].
Anti-PM/Scl antibodies are a heterogeneous group of autoantibodies directed to several proteins of the nucleolar PM/Scl macromolecular complex. The two main autoantigenic protein components were identified and termed PM/Scl-75 and PM/Scl-100 based on their apparent molecular weights [
7,
8]. According to former studies indicating PM/Scl-100 as the main target of the autoimmune response to PM/Scl, the majority of commercially available assays use recombinant PM/Scl-100 protein [
3]. However, recent studies also suggest the diagnostic importance of anti-PM/Scl-75 antibodies, especially when the major isoform PM/Scl-75c is used as an antigen source [
9,
10]. The percentage of patients presenting anti-PM/Scl-75c antibodies is supposed to exceed that for anti-PM/Scl-100 antibodies [
9]. However, analyses of larger SSc cohorts to identify the prevalence and specificity of these antibodies are missing. Furthermore, it remains elusive whether the different antibodies reflect different SSc subsets and clinical features present in these patients.
Based on the growing knowledge about the anti-PM/Scl antibody targets, very sensitive methods such as an enzyme-linked immunosorbent assay (ELISA), which is based on a PM/Scl-100-derived peptide called PM1-alpha, have been developed [
11]. In recent years, line immunoblot assay (LIA) has become a popular technique for the simultaneous detection of several autoantibodies. As recently shown and exemplified for the determination of anti-topoisomerase I (anti-topo I) antibodies, LIA provides a valuable tool as an alternative to ELISA [
12].
In the present study, a large monocentric cohort of consecutive SSc patients was analyzed by LIA, allowing the simultaneous monospecific detection of both anti-PM/Scl-75 and anti-PM/Scl-100 antibodies. Clinical data were assessed simultaneously by a standardized procedure with only a limited number of investigators. For patient assessment, we applied criteria and strategies developed by the German Network of Systemic Scleroderma (DNSS) and the European Scleroderma Trials and Research (EUSTAR) network [
13‐
15]. By this approach, we identified several clinical features associated with the presence of either anti-PM/Scl antibody.
Discussion
Anti-PM/Scl antibodies are supposed to be a marker for overlap syndromes; however, the diagnostic impact of their major subspecificities, anti-PM/Scl-75 and anti-PM/Scl-100, as well as their prevalence in different SSc subsets are still not known. In the present study, a large well-characterized cohort was analyzed for the presence of the different anti-PM/Scl antibodies.
As shown here, anti-PM/Scl antibodies, in particular anti-PM/Scl-75, are more frequent than had previously been described in SSc cohorts. Reactivity to either PM/Scl-100 or PM/Scl-75 was found to depend on the underlying disease subset. Anti-PM/Scl-75 antibodies are found mostly in patients with dSSc and overlap syndromes, whereas anti-PM/Scl-100 antibodies are detected mainly in patients with overlap syndromes. Anti-PM/Scl antibodies were highly specific for SSc; however, our analyses did not include patients with primary PM, dermatomyositis, or inclusion body myositis, conditions that could influence the specificity of the assays.
When the clinical data of the patients were studied, anti-PM/Scl antibody-positive patients significantly more often showed muscle involvement and lung fibrosis, confirming other studies [
2,
24]. Furthermore, and not described before, digital ulcers were found to be associated with the presence of anti-PM/Scl antibodies. In contrast, PAH was less frequently detected in anti-PM/Scl-positive patients. Anti-PM/Scl-75 antibody-positive patients were younger at disease onset compared with the anti-PM/Scl-75-negative patients. In the presence of anti-PM/Scl-100 antibodies, fewer gastrointestinal symptoms were found. In view of these findings, detection and distinction of both antibody specificities appear to be important beyond the increase in sensitivity for SSc and overlap syndromes.
Only a few studies have analyzed the sensitivity and specificity of anti-PM/Scl antibodies in large SSc cohorts. One of the first studies from the Pittsburgh group identified 23 (4%) of 617 patients with connective tissue diseases as being positive for anti-PM/Scl; this cohort included 314 patients with SSc, 89 patients with overlap syndromes, and 106 patients with pure dermatomyositis/PM. For the identification of the anti-PM/Scl antibodies, immunoprecipitation and immunodiffusion were used. The description of anti-PM/Scl-positive patients revealed a higher frequency of myositis and a lower incidence of kidney involvement. There were no differences in the frequency of pulmonary diseases [
4]. However, the number of anti-PM/Scl-positive patients was too small to evaluate significant differences compared with the antibody-negative group. Other studies suggested a higher incidence of muscle involvement, confirming studies analyzing myositis patients with or without SSc overlap [
2,
7,
24,
26‐
28]. Here, we showed the association between the presence of anti-PM/Scl antibodies in SSc patients with myositis and with muscle atrophy. In addition, anti-PM/Scl antibodies are a marker of lung and skin fibrosis and of active disease, as previously described also for SSc patients with anti-topo I antibodies in the same cohort [
5,
12]. Indeed, a significant proportion of the anti-PM/Scl-positive patients also exhibited reactivity to the topo I autoantigen (35.1%). This subgroup of double-positive patients exhibited higher frequencies of restrictive lung disease (91% versus 46%;
P = 0.02) and of contractures (92% versus 59%;
P = 0.049) when compared with the anti-PM/Scl single-positive patients. In this double-positive group, a lower percentage of patients showed CK elevation (23% versus 36%;
P = 0.02) compared with single-positive patients. In contrast to anti-topo I antibodies, and probably due to the low number of anti-PM/Scl-positive patients, there was no increased mortality related to the signal strengths of the anti-PM/Scl antibodies. However, 4 out of 29 anti-PM/Scl-75-positive patients (13.8%) died within 3 years after antibody detection (3 of them were also positive for anti-topo I antibodies and 2 also exhibited anti-PM/Scl-100 reactivity) compared with 6.1% in our whole SSc cohort. One further patient received autologous stem cell transplantation. This mortality, the co-incidence with anti-topo I antibodies, the disease characteristics with a high frequency of lung fibrosis, and the increased disease activity score especially in anti-PM/Scl-75-positive patients do not support former studies claming a milder disease with a favourable prognosis and response to immunosuppression [
5,
6].
Here, we could demonstrate for the first time that the reactivity to PM/Scl depends on the underlying disease and furthermore on the clinical symptoms. Interestingly, the majority of patients showed reactivity to only one of the two major autoantigens. Only 37.1% of anti-PM/Scl-positive patients were double-positive for both subsets of antibodies. Nevertheless, the higher prevalence of anti-PM/Scl-75 antibodies and the higher rate of clinical associations indicate that PM/Scl-75 is the main autoantigen in SSc patients. Therefore, when tests are used based on PM/Scl-100 as an antigen source, as most ELISA and LIA techniques are [
3], reactivity to the anti-PM/Scl-75 antigen can be missed, especially in dSSc patients. According to our results, both specificities should be determined.
Results concerning the main antigenic targets of anti-PM/Scl antibodies are controversial. Studies analyzing sera from large myositis cohorts ascribed the highest reactivity to the PM/Scl-100 autoantigen [
26]. However, this finding might be representative for myositis patients. In an analysis of sera from different disciplines including SSc patients, the PM/Scl-75, especially the major isoform PM/Scl-75c, was considered the main epitope of anti-PM/Scl antibodies [
9,
10]. Just recently, the PM/Scl-100 epitope-based ELISA (PM1-alpha) was compared with recombinant PM/Scl-100 and PM/Scl-75c [
29]. Thus, further studies are mandatory to address diagnostic accuracy of the individual PM/Scl antigens.
Furthermore, reactivity to the PM/Scl antigens seems to be influenced by the system applied for antigen expression. For the PM/Scl-100 antigen, the baculovirus expression system did not provide additional benefit when compared with the
E. coli expression system. Therefore, post-translational modifications made by eucaryotic cells do not appear to play a role in anti-PM/Scl-100 antibody binding. Consequently, the PM/Scl-100 antigen can be produced using the
E. coli expression system as an easy and cost-effective method [
30,
31].
In summary, this is the first report about the prevalence of different anti-PM/Scl antibodies in SSc patients classified and assessed by the commonly used standards of the DNSS and EUSTAR network. Antibodies against PM/Scl-75 and PM/Scl-100 can be considered independent markers for different SSc subsets and show partial differences with respect to associated clinical manifestations, substantiating the diagnostic relevance of their parallel determination.
Competing interests
GR has received fees from EUROIMMUN AG for lectures on these data at the 'Eurodoctor' meeting in Brussels, Belgium. KH was invited by EUROIMMUN AG to participate in a national meeting to show the results of the study. After finishing the study, she received a grant from EUROIMMUN AG for additional scientific work. The other authors declare that they have no competing interests.
Authors' contributions
KH and AK helped to provide preclinical analyses, statistics, and graphics and to write the manuscript. CD, AJ, LK, and WM helped to develop the LIA and to perform the tests. M Backhaus provided access to the patients in her outpatient department. CSB helped to provide clinical data. M Becker corrected and helped to write the manuscript. DH supervised statistical analyses. KE participated in discussions of the data with GR, made intellectual contributions, helped to prepare the manuscript, and provided sera for the analyses. GRB and FH participated in discussions of the data with GR, made intellectual contributions, and helped to prepare the manuscript. WS organized all of the cooperation with EUROIMMUN AG and made intellectual contributions. GR, as the author responsible for this report, initiated this study and controlled the work. She collected and assessed the patients, helped to provide clinical data, and wrote and reviewed the manuscript. All authors read and approved the final manuscript.