Anne Tebo, Ph.D.
Professor of Laboratory Medicine and Pathology
Division of Clinical Biochemistry
Mayo Clinic, Rochester, Minnesota
Hello and welcome. My name is Anne Tebo, and I am a professor of laboratory medicine and pathology in Mayo Clinic. I also co-direct the Antibody Immunology Laboratory at the Mayo Clinic in Rochester, Minnesota. I am delighted you can join me for this brief presentation in which I will be discussing recent updates in the testing for anti-SS-A/Ro antibodies in systemic autoimmune rheumatic diseases. I will be particularly focusing on the clinical significance for the separate determination and reporting of antibodies to Ro52 and Ro60 in the evolution of patients with this disorder.
I have no disclosures for this presentation.
The SS-A/Ro is part of the Ro/La heterogeneous antigenic complex which is made up of three unique proteins (52 kDa Ro, 60 kDa Ro and the La proteins as well as four small RNA particles) which is elegantly displayed in this figure here. Unlike antibodies to SS-B/La that are less common, reported separately and mainly associated with a diagnosis of primary Sjögren's syndrome, antibodies to Ro or SS-A are most prevalent among many autoimmune diseases such as primary Sjögren's syndrome, SLE, rheumatoid arthritis, systemic sclerosis, and inflammatory myopathies. Historically, Ro52 and Ro60 antibodies were considered as a uniform antibody-system. Therefore, when Ro52 or Ro60 antibody is positive, the result is typically reported as anti-SS-A/Ro antibody. However, recent studies have provided evidence that Ro60 and Ro52 are not part of a stable macromolecular complex and that these antibodies have separate clinical associations with implications for diagnosis, prognosis, and patient management.
It is recognized that the Ro52 and Ro60 antigens are encoded by separate genes that are found in distinct cellular compartments. The significance of antibodies to these proteins in the pathogenesis of systemic autoimmune diseases are not fully defined; however, both proteins have potential roles in loss of immune tolerance which is a breakpoint in the onset of many autoimmune diseases. Ro52 is an interferon (IFN)-inducible protein and can also be induced by viral infections or Toll-like receptor (TLR) engagement via type I IFN induction. It is therefore conceivable that it plays a role as a negative regulator for proinflammatory cytokine production. On the other hand, Ro60 is a DNA-binding protein with protective functions against the development of autoimmune responses as has been shown in some experimental model systems.
Antibodies to SS-A/Ro is one of the 2016 American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) classification criteria for primary Sjögren's syndrome. Sjögren's syndrome is one of the most common systemic autoimmune diseases with a prevalence of about 0.6% and affects about 1 to 4 million Americans. As a secondary disease, it may occur with pre-existing connective tissue diseases such as SLE, rheumatoid arthritis, systemic sclerosis or in the context of idiopathic inflammatory myositis (commonly referred as myositis), it may occur with one of the most specific markers for disease diagnosis. Of the five item domains in the classification criteria for primary Sjögren’s syndrome, the presence of SS-A or Ro antibodies is the only clinical laboratory test with a weighted score of three as shown in this table. All others relate to the dysfunction of the exocrine glands. Therefore, appropriate testing and reporting of these antibodies is essential in the diagnosis of primary Sjögren's syndrome.
As show in this slide, the clinical manifestations in systemic autoimmune diseases are commonly associated with antibodies to SS-A/Ro. Therefore, SS-A/Ro has been shown to have limited diagnostic utility in the evaluation of a number of systemic autoimmune diseases. Some of these clinical manifestations as shared amongst primary Sjögren's disease, SLE and RA. Of diagnostic importance, the features of gastrointestinal, hematologic, renal, skin and vasculitis. In addition to Raynaud’s phenomenon, muscular and pulmonary manifestations are quite common in these disorders.
As you would notice in this slide here, there is some plasticity of anti-Ro antibodies in the common systemic autoimmune rheumatic diseases. For example, although SS-A antibodies is a classification criteria for primary Sjögren's syndrome, it is commonly found in patients with SLE and rheumatoid arthritis and many other autoimmune diseases. Therefore, given the overlapping clinical features associated with these disorders, and the presence of SS-A antibodies, in these diseases, it is widely recognized that this autoantibody, if reported, without distinguishing between the presence of Ro52 and Ro60, lacks diagnostic specificity.
Although there is established evidence that Ro52 and Ro60 are different and have unique clinical attributes, which may allow for prognosis and stratification of patients, testing and reporting of these antibodies are yet to be harmonized. This figure clearly shows the different ways in which antibodies are being tested and reported in the clinical laboratories, not only in the United States but worldwide. For example, some laboratories may test Ro52 and Ro60 singly with singleplex assays or using multiplex assays but report these as SS-A positive in a traditional way. Alternatively, they can use a combination of analytes for Ro60 and Ro52 in single while testing but reports the presence of these antibodies while not differentiating which specificity is present. In another scenario, labs may report either Ro52 or Ro60 as being positive for SS-A antibodies. And while the importance of negative results are not largely impacted, stratification based on single positivity for Ro52 or single positivity for Ro60, or dual positivity for Ro52 and Ro60, it’s very important in patient’s evaluation and stratification.
In an elegant study that was published in 2019 by a French group, they conducted a retrospective, observational study which included every adult patient with positive antinuclear antibodies (ANA) tested in their immunology laboratory and that was associated with Ro52 and/or Ro60 antibodies, between 2011 and 2014. In all, they identified about 399 patients out of 1,300 patients with positive results for Ro52 and/or Ro60 antibodies. The summary of their findings is shown in this figure and the table here. Based on their findings, single positivity of Ro52 was more common in the general population than when you have single positivity for Ro60 or dual positivity for Ro52 and Ro62. In the stratification of patients based on the presence of autoimmune diseases, combined positivity for Ro52 and Ro60 shows higher prevalence in autoimmune diseases than in nonautoimmune diseases whereas single positivity for Ro52 was highly prevalent in the nonautoimmune diseases compared to patients with the other combination. Of diagnostic significance, for example in the stratification of patients based on systemic lupus, primary Sjögren's, systemic sclerosis, inflammatory myositis or inflammatory rheumatism, it was the demonstrated that dual positivity for Ro60 and Ro52 versus single positivity for Ro52 was significantly associated with a diagnosis of systemic sclerosis, primary Sjögren's, inflammatory myopathies, and inflammatory rheumatism. In contrast, the presence of Ro60 versus combination of Ro52 and Ro60 was highly indicative of a diagnosis of Sjögren's syndrome. This points to a significant role for separate detection and reporting of these antibodies, not only for the general population, but also in the stratification into specific rheumatic autoimmune disorders.
In a subsequent study that was conducted in Belgium, these investigators expanded the repertoire of autoantibody tests to include the SS-B/La antibodies which is associated with Sjögren's to further assess the role of Ro52 and Ro60 in the diagnosis and re-stratification of patients with primary Sjögren's syndrome.
Overall, the principal component analysis of the data point to a stepwise model in which mono-reactivity against Ro60 displayed the least objective and subjective glandular primary Sjögren's syndrome features, whereas glandular abnormalities and signs of B-cell hyperactivity were most present in patients showing triple reactivity against Ro60, Ro52 and SSB/La. In this context, the authors suggested that separate determination of these antibodies is recommended in the context of primary Sjögren's diagnosis and disease phenotyping.
In addition to the diagnostic and prognostic roles for the differential expression of Ro60 and Ro52 antibodies in primary Sjögren's, there are evidence for their relevance as prognostic markers for interstitial lung disease in patients with interstitial pneumonia with autoimmune features (IPAF), systemic sclerosis, SLE, and inflammatory myopathies. In the reference study presented here, the investigators sought to evaluate the probable role of anti-Ro52 antibodies which coexist with anti-MDA5 or anti-Jo1 antibodies as risk indicators for inflammatory myositis that is associated with rapidly progressive interstitial lung disease as well as predictors of inflammatory myositis associated with interstitial lung disease related survival in the Chinese patient cohort.
In figure A, the Kaplan–Meier curve demonstrated that survival was lower in patients with inflammatory myositis associated with interstitial lung disease who tested positive for anti-Ro antibodies than those without the antibodies. In figure B, the survival was lower in patients with inflammatory myopathies who tested positive for both Ro52 and MDA5 antibodies than those with anti-MDA5 alone by log-rank test. All this suggests that when patients are diagnosed with myositis or at risk for myositis, the role of this Ro52 antibodies may have additional prognostic value in patient management.
Beyond systemic autoimmune rheumatic diseases, the prevalence of Ro52 antibodies have been reported in a number of autoimmune liver diseases and this association seems to have prognostic significance especially when additional autoantibodies are present. In one early study dating back to 2007, patients with primary biliary cholangitis who tested positive for Ro52 and centromere antibodies were reported to be at increased risk for more advanced histological stage of disease. A similar role for the use of anti-Ro52 in disease prognosis was reported in autoimmune hepatitis when present with the soluble liver antibodies. In autoimmune hepatitis therefore, both autoantibodies were shown to be independently associated with the development of cirrhosis, hepatic death or liver transplantation. It does appear that in patients with autoimmune liver disease, testing for Ro52 would be implicated for disease prognosis.
In a Mayo study, we compared the performance of two solid-phase immunoassay for the separate detection and reporting of anti-Ro52 antibodies in a consecutive cohort of patients undergoing evaluation for connective tissue disease. The multiplex bead assay and the chemiluminescence bead assays overall showed comparable performance when using the kit-specific cut-off for positivity. When the antibody positivity was stratified based on single or dual autoantibody positivity, we recognized that the positive correlations for Ro52 was higher than for Ro60 antibodies. However, overall, the positive agreement was acceptable with the higher correlation for Ro52 as denoted by the Kappa and the chi-square values. The lower correlation between the Ro52 assays could be explained by how these antibodies are recognized or the prevalence of the specific connective tissue diseases in the cohorts. Antibody reactivity to Ro60 is largely dependent on conformational epitopes whereas that for Ro52 is dependent on linear epitopes. Further analysis are being done to determine the prevalence of these biomarkers in the different clinical subsets in this cohort.
So who should be tested for anti-Ro52 and anti-Ro60 antibodies? To address this question, it’s important to first indicate that with increase interests in early diagnosis and stratification of patients with autoimmune diseases when the disease process or burden may be modulated, delayed, or halted with targeted therapies, appropriate testing is very important. As a criterion laboratory test for primary Sjögren's syndrome, Ro52 and Ro60 should be considered as first line tests. Separate determination of Ro52 and Ro60 antibodies would also be recommended when suspicion of systemic autoimmune rheumatic disease is high. For example, in patients with overlap Sjögren's syndrome, patients with SLE systemic sclerosis and inflammatory myopathies. In addition, both tests should be considered in the evaluation of autoimmune liver diseases particularly when overlapping features of connective tissue diseases is present or in patients who have those diseases are at a risk for systemic sclerosis or secondary Sjögren's syndrome.
With that background to who should be tested for anti-Ro52 and anti-Ro60 antibodies, I would just like to use the last few minutes to talk about how should testing be done for these antibodies. Antibodies to Ro52 and Ro60 are associated with antinuclear antibody nuclear fine speckled pattern which is generally referred to as AC-4, using the HEp-2 substrate by indirect immunofluorescence assay (IFA). Recently, Ro60 antibodies have further been characterized as a variant of this AC-4 type with distinctive myriad discrete nuclear speckles. Although the nuclear speckled pattern is associated with diverse analytes, testing for Ro52 and Ro60 is generally recommended due to their prevalence in autoimmune diseases. It is also recognized that the ANA IFA may be negative using setting substrates for testing antinuclear antibodies recommended testing for Ro52 and Ro60 antibodies should be sought if suspicion for Sjögren's syndrome is high.
In Mayo Clinic Laboratories, the SS-A antibody testing is offered in several ways. This can be tested followed by a positive ANA by either the solid phase immunoassay or the HEp-2 substrate for determining specific patterns. In the context of specific testing for SS-A, there are several strategies by which clients can test for these autoantibodies. The SS-A antibodies is included in the connective tissue cascade test as well as it is offered in a multiplex bead assay in which there is no differentiation between the Ro52 and Ro60 antibodies. However, positive results from this system can be confirmed for Ro52 or Ro60 using a chemiluminescence based assay which offers separate detection and reporting of these autoantibodies.
Thank you for your time and I’m happy that you could join me today for this presentation that highlights the importance of testing for Ro52 and Ro60 antibodies, not only for diagnosis but for re-stratification for patients with connective tissue diseases and in some cases in autoimmune liver diseases. Thank you.
For this presentation, I’ve used a couple of references which I’ve presented in this slide and can be used for your reference.
Contact us: email@example.com
Image credit: NA