Expert analysis: The importance of serological markers and serodiagnostics in RA

Rheumatoid arthritis (RA) is a prevalent chronic inflammatory autoimmune disease mainly characterised by joint swelling and pain, affecting approximately 0.5–1% of the population in industrialised countries.¹

Besides clinical features such as joint inflammation, duration of symptoms or, in advanced cases, radiographic changes, serological markers represent a hallmark in diagnostics. Among these, autoantibodies such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are the most specific markers for RA. They are included in the 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria of RA¹ and the presence of either of these in combination with clinical symptoms is highly indicative for the diagnosis of RA, particularly when occurring at high positive titers (Table 1).

Table 1. The 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for RA¹

Current state of the art serodiagnostics for RA

RF autoantibodies recognise the Fc-tail of immunoglobulin (Ig) G and are measured either by nephelometry or latex agglutination – which capture all classes of Igs but mainly large molecules such as IgM – or IgM-specific enzyme-linked immunosorbent assays (ELISAs). A meta-analysis comparing all three types of test systems revealed that ELISA had the highest positive likelihood ratio (6.13) for RA followed by latex agglutination (5.05) and nephelometry (4.15).² Although the sensitivity of RF is somewhat higher compared with ACPA, its specificity is lower, particularly the specificity of low-titer IgM-RF, owing to its occurrence in other inflammatory diseases.³ ACPA recognise post-translationally modified proteins in which the amino acid arginine has been converted into a citrulline. They are predominantly of the IgG isotype and are commonly measured by assays employing a cyclic citrullinated peptide (CCP) as antigen. Different generations of CCPs (CCP1–3) show different diagnostic performance – an issue that has been evaluated in several studies. The anti-CCP2 assay was found to be the most stable and most widely used assay worldwide.^4–6 However, diagnostic performance might differ due to different cut-off values recommended by the manufacturers.⁷ This is even more true for RF, and highlights the need for harmonisation of assays for established biomarkers.⁸ Supported by the IUIS/WHO/AF/CDC Committee for the Standardization of Autoantibodies in Rheumatic and Related Diseases, reference material for ACPA has been developed to fulfil this need of critical assay validation and harmonisation.^3,9,10

Novel serological markers with diagnostic potential for RA

Seronegativity, that is, the absence of pathological levels of RF and ACPA, which occurs in 20–30% of RA patients, remains a major limitation of the two routinely used serological markers. This underlines the importance of finding new complementary markers that could improve the diagnostic sensitivity of autoantibody testing. Thus, the detection of RA33 IgG antibodies – which are directed to the nuclear antigen hnRNP-A2 – has been repeatedly described as contributing an additional diagnostic value, since they are also found in seronegative patients.^11,12

Lately we showed that the determination of other isotypes of RF, ACPA and RA33 antibodies in addition to those determined by routine diagnostics might provide an added diagnostic value as RA patients were found having multiple reactivities compared with disease controls who usually showed only one or two reactivities (Figure 1). Thus, the presence of three or more reactivities proved highly specific for RA.¹³

Figure 1. Antibody positivities in RA patients (n=290) and disease controls (n=261) tested for the presence of IgA, IgG, and IgM isotypes of RF, ACPA and RA33 antibodies. Number of positivities was significantly higher in RA patients compared with disease controls. Adapted from Sieghart et al 2018.

Besides ACPA, autoantibodies recognising other post-translational modifications have gained attention.¹⁴ Among these, anti-carbamylated protein (anti-CarP) antibodies have been analysed intensively for their additional diagnostic value in RA. Although they also occur in seronegative patients, they appear to be of only limited usefulness for differentiation between seronegative RA and other arthritic disorders or connective tissue diseases.^15,16

Acetylation represents one of the most common and most important post-translational modifications, and autoantibodies to acetylated vimentin have been described.^17,18 They have been termed anti-acetylated peptide antibodies (AAPA) and appear to be less specific than ACPA. Their value for RA diagnostics has not yet been clarified, but they might assist in identifying RA patients who are negative for RF and ACPA as determined by standard routine diagnostics.¹⁹

The search for disease markers is not limited to autoantibodies. The 14-3-3η protein was found to be hyper-expressed in the synovial joints of patients with RA^20,21 and elevated serum levels have been observed in patients with RA. Remarkably, RA patients might also develop autoantibodies to 14-3-3η, which can increase the sensitivity of ACPA and RF testing in early RA patients by 20%.²²

In addition, serum levels of the caspase inhibitor survivin were associated with the development of RA in an arthralgia cohort,²³ and even interleukins might represent potential biomarkers for RA, as recently suggested for interleukin-7.²⁴

Antibodies against protein-arginine deiminases 3 and 4 have been described as additional diagnostic antibodies, showing very good discriminatory capability but poor sensitivity (around 13%).²⁵

Although all these novel markers appear promising candidates for RA diagnostics, larger studies are needed to fully elucidate their diagnostic potential.

Autoantibodies as predictors of disease development, progression and therapy response

Biomarkers identifying individuals at risk for developing RA have been studied intensively but the sensitivity of these markers is relatively low. Thus autoantibodies can be detected several years before onset of RA and some autoantibodies might even represent risk factors for disease development and play an important role in its pathogenesis, as suggested for ACPA, RF or 14-3-3η.²⁶ Interestingly, in a recent study RF isotypes – and particularly IgA-RF – were the earliest antibodies detectable, being present in some individuals for more than 15 years before onset of RA, preceding the earliest ACPA specificities.²⁷

Regarding progression of disease, ACPA positivity was associated with a more severe, erosive phenotype and a higher mortality rate compared with seronegative RA (reviewed in Alivernini et al, 2017²⁸) while the contribution of RF in relation to ACPA has diminished.²⁹ Similar to ACPA and RF, anti-CarP antibodies have also been shown to be associated with radiographic progression. Because this was also seen in ACPA-negative individuals, anti-CarP antibodies might have some prognostic value.²⁷

Finding predictive tools for treatment response to methotrexate (MTX) – the first-line therapy in RA – is a major goal of biomarker research because effective early intervention is decisive for clinical outcome. Recently, it was reported that patients with a broad autoantibody profile at baseline had a significantly better treatment response than seronegative patients or patients presenting a more restricted profile.^30,31 Some interesting findings have been reported regarding anti-tumour necrosis factor alpha (anti-TNF) therapy, which is used in MTX non-responders. The IgA RF isotype was associated with reduced response to anti-TNF treatment³² whereas the presence of AAPA might be linked to a more favourable response.³³ Furthermore, ACPA status was associated with favourable response to biologics targeting pathways involving autoantibody-producing cells such as B lymphocytes.²⁸ However, all these data should be considered preliminary in nature and need to be confirmed in larger studies.

Conclusions

There are increasing efforts by laboratories and manufacturers to harmonise currently available assays of the two established serological markers for RA (RF and ACPA) to make them more comparable and stable. Improving the diagnostic sensitivity of RA serodiagnostics is an intensively studied field in which some progress has been made in recent years. Generating a panel of diagnostic markers – autoantibodies and other serum proteins – to diminish or even close the serological gap left by RF and ACPA will be the major goal of ongoing, and future, biomarker research. In the last couple of years, it has been demonstrated that serological markers have the potential to identify patients at risk for developing RA, patients who will have a more erosive phenotype or even patients who will or will not respond to certain therapies. Increasing the accuracy of prediction by combining the existing markers of routine diagnostics with novel markers might have an impact on treatment guidelines and clinical decision making.

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