Personalized medicine in rheumatology: the paradigm of serum autoantibodies

ANA are the most frequently (and often inappropriately) prescribed autoantibody test and they are virtually always positive in connective tissue diseases, i.e., systemic lupus erythematosus (SLE), Sjögren’s syndrome (SjS), scleroderma (SSc), polymyositis and dermatomyositis (PM/DM). The ANA interpretation must thus be driven by clinical suspicion, but their interpretation also depends on both titer and pattern. Different staining patterns, classified into three major groups, nuclear, cytoplasmic and mitotic, give indication on the significance of ANA and type of rheumatic disease (Table 1), and the recent work from the autoantibody standardization group (http://​www.​ANApatterns.​org) provided a long overdue description of the possible profiles. In fact, the gold standard for detecting ANA remains indirect immunofluorescence test (IIF) on HEp-2 cells; however, IIF is time consuming and requires skilled operators to avoid variability. So, new ANA testing strategies have emerged in the last decades, particularly in terms of automation and multiplex analyses. The expected advantages of automated IIF are the reduction of false-negative and false-positive results, the improvement of the uniformity between different readers and laboratories and the efficiency of the evaluation procedure [23]; but unfortunately the sensitivity for rare patterns is not yet adequate and can lead to false-negative results [24, 25].

It is still unclear what significance ANA may have in asymptomatic patients, since they are not specific markers of connective tissue diseases and can be falsely positive in healthy subjects (the prevalence of ANA in the general population is 13.8%) [26], especially in seniors, as well as in patients with other chronic inflammatory or infectious diseases; otherwise, they can precede clinical manifestations and diagnosis in SSc [27] and SLE [28]. The current recommendations state that ANA-positive subjects should be tested for antibodies to extractable nuclear antigens (anti-ENA) and anti-double-stranded DNA antibodies (anti-dsDNA) [29, 30]. The presence of multiple antibodies thus becomes specific for systemic rheumatic disorder and helps the diagnostic process. To improve the appropriateness of the immunological diagnosis of systemic autoimmune diseases, to accelerate time for completing diagnostic process and to avoid waste of money, the introduction of ANA reflex test has been proposed [31]. The diagnostic algorithm suggested by Tonutti and colleagues begins with a first-line high sensitivity test (i.e., ANA IIF on HEp-2 cells) to allow antibody positivity recognition and the definition of pattern and titer. The second-line tests (high specificity) are done for ANA titers ≥1:160 and include, as mentioned, anti-dsDNA and anti-ENA (by ELISA) to evaluate specific antigenic expression. Figure 1 shows the second-line tests based on the ANA patterns found in IIF.

The predictive significance of ANA has been clearly demonstrated in the seminal work by Arbuckle and colleagues [28]. They studied 130 patients with SLE, whose serum had been collected many years before the diagnosis. Most patients harbored at least one autoantibody up to 9 years before the development of clinical manifestations and therefore diagnosis of SLE, in particular ANA and also antiphospholipid, anti-Ro and anti-La antibodies [32]. The mean time to diagnosis for these autoantibodies was about 3.4 years, while for anti-double-stranded DNA autoantibodies 2.2 years. Later predictors of disease were anti-Sm and anti-nuclear ribonucleoprotein antibodies, which tended to coincide with the onset of signs and symptoms. Another interesting observation is that new types of autoantibodies gradually accumulated before the diagnosis and reached a plateau at the diagnosis. Considering that while ANA, anti-Ro, anti-La and anti-phospholipid antibodies may also be present in healthy subjects, anti-dsDNA, anti-Sm and anti-nuclear ribonucleoprotein antibodies are very rare in the general population. Accordingly, the positivity of these aforementioned autoantibodies should lead to close monitoring.

The value of ANA is not limited to diagnosis, but may have a prognostic role in specific clinical settings such as SSc, where the nucleolar pattern has been associated with a more rapid progression to late scleroderma pattern at nailfold capillaroscopy [33]. Moreover, the presence of abnormal nailfold capillaries with ANA positivity is associated with an increase of mortality in patients with Raynaud’s phenomenon without previously known connective tissue disease exclusively in women [34]. The ANA prognostic value is also reported in juvenile idiopathic arthritis, being associated with the predisposition to uveitis [35].

Finally, ANA and anti-ENA, in particular anti-Scl-70 antibodies, may be associated with malignancies, especially in patients with SSc and inflammatory myositis [36]. Another correlation has been reported with lymphomas, as serum ANA were significantly higher in patients with disease than in controls. In addition, it has been found that the level of lactate dehydrogenase in ANA-positive patients was lower, thus suggesting that these patients have a better prognosis than ANA-negative patients, probably demonstrating the existence of an anti-tumor response that can be associated with a better prognosis [37].

ACPA, together with rheumatoid factor (RF), are the characteristic antibodies of rheumatoid arthritis (RA), manifesting higher specificity and lower sensitivity compared to RF for RA. ACPA can divide patients with RA into two subgroups that differ in prognosis and response to treatment [38, 39] (Fig. 2). Patients with ACPA-positive RA differ from seronegative ones in genetics and environmental risk factors. The presence of ACPA is associated with genetic interaction between HLA-DRB1 shared epitope (SE) and PTPN22 risk allele [40], and this points out how MHC class II-dependent T cell activation plays a central pathogenic role in the development of ACPA-positive RA. Furthermore, other gene interactions associated with ACPA-positive RA have been confirmed in several studies [4, 38]. ACPA are highly prevalent not only in patients with HLA-DRB1 SE, but also in those with HLA-DRB1*15 non-SE, proposing that this latter allele can act as a second trigger that may intensify autoimmune response in predisposed subjects [41]. Even gene–environment interaction can determine ACPA-positive RA, particularly in association with smoking and HLA-DRB1 SE, or with smoking and PTPN22 [38]. It is interesting to note that SE and smoking are associated with the presence of all three autoantibodies specifically related with RA [ACPA, RF and anti-carbamylated protein antibodies (anti-CarP)] [42].

The influence of environmental factors, particularly smoking, has been shown to be a risk factor for the development of ACPA-positive RA; other environmental risk factors associated with ACPA are excessive coffee consumption, use of oral contraceptives and periodontitis caused by Porphyromonas gingivalis, while moderate alcohol consumption may be protective against this RA subtype [43, 44].

As previously discussed for ANA, also ACPA has a predictive role in autoimmune diseases, as well as RF and anti-RA-33 autoantibodies. In fact, they may occur early in the course of RA and are often present several years before the first symptoms [45, 46].

Numerous studies have shown that the presence of ACPA and their concentration are associated with the development of a more aggressive disease. Positive serum ACPA at baseline leads to a progressing and destructive form of RA, and they are the strongest independent predictor of radiographic damage [47‐49]. ACPA have also been correlated with increased inflammation and extra-articular manifestations, high rates of disability and cardiovascular disease, with the latter most responsible for the premature mortality in RA [38].

The distinction between ACPA-positive and ACPA-negative patients can also help us in the therapeutic choice; in fact, the superior efficacy of agents such as rituximab and abatacept in ACPA-positive patients has been demonstrated [50, 51]. This can occur because in these patients, the contribution of B cells to RA pathogenesis is more pronounced, and they might be more responsive to B cell-directed treatments than other patients [52]. Also, methotrexate seems to be more effective in ACPA-positive subjects, in those patients in whom the arthritis stage is not fully defined by the ACR criteria, and it may delay the onset of RA [38]. Conversely, ACPA positivity is correlated with a reduced response to all anti-tumor necrosis factor biologics [53].

Rare autoantibodies characterize nearly all rheumatic diseases, despite not being included in any classification criteria set. Due to their low penetrance, they are not used as first-line test or generally available in routine laboratory, but they may help the clinician in making more accurate diagnoses and better management of patients, because they are associated with different disease manifestations and also with a greater incidence of cancer [54]. This is particularly relevant for SSc, inflammatory myositis and SLE, for which we should note that the proposed clinical significance is derived from small cross-sectional studies.

The most significant autoantibodies in SSc are anti-centromere, anti-RNA polymerase III (anti-RNAP) and anti-Scl70, while anti-U3RNP is rarely found. Other autoantibodies are listed in Table 2 with their proposed clinical associations. Anti-RNAP antibodies are related to diffuse cutaneous SSc [55], and they are the most consistent predictors of scleroderma renal crisis [55]. It has also been shown that patients with anti-RNAP antibodies present more solid tumors and that these are temporally associated with the onset of SSc [36, 56]. Anti-Scl-70 antibodies are associated with a higher skin score, disease severity and activity [57], and they are also strong predictors of the development of pulmonary fibrosis [58, 59], digital ulcers [60] and malignancies [61]. Finally, anti-U3RNP antibodies are strongly associated with muscle involvement and with an increased risk of pulmonary arterial hypertension [59, 62, 63].

In the case of PM/DM, serum autoantibodies can be divided into myositis-specific antibodies (MSAs) (anti-Jo1, anti-PL-7, anti-PL-12, anti-EJ, anti-OJ, anti-Mi-2, anti-SRP, anti-KS, anti-TIF1γ/α (anti-155/140), anti-TIF1β, anti-MJ/NXP-2, anti-MDA5/CADM-140, anti-SAE) and myositis-associated antibodies (MAAs) (anti-PM-Scl, anti-Ku, anti-U1-RNP, anti-U1/U2-RNP, anti-U3-RNP) [64]. The most noteworthy clinical associations are reported below, while others are reported in Table 3. In general terms, rare autoantibodies in myositis are used to predict more severe forms, including those associated with cancer as many studies support the possibility that DM and PM are paraneoplastic, particularly DM [65]. Some autoantibodies are associated with major recurrence of neoplasms, and these are anti-TIF1γ/α (anti-155-140) and anti NXP2 antibodies [66, 67]. Jo-1 and other anti-aminoacyl tRNA synthetases (anti-ARS) antibodies (PL-7, PL-12, EJ, OJ, KS) are associated with the so-called anti-synthetase syndrome, characterized by myositis, interstitial lung disease, arthritis, mechanic’s hands and Raynaud’s phenomenon [68, 69]. Anti-Mi-2 antibodies are related to DM with its typical skin manifestation, to good steroid response and good prognosis [70]. Anti-SRP is specific for PM and correlates with a form of necrotizing myopathy, associated with a worse therapeutic response and poor prognosis [64, 71]. Rare autoantibodies detectable in SLE are listed in Table 4; among these, anti-Sm antibodies are associated with lupus nephritis and neuropsychiatric complications, and renal disease association is stronger when they are found together with anti-dsDNA [72]. These antibodies are also associated with discoid lupus and photosensitivity [73]. Anti-ribosomal P antibodies are detected in the cerebrospinal fluid of patients with neuropsychiatric SLE [74], indicating blood–brain barrier permeation, while they are also related to renal [75] and hepatic damages [76], malar rash, oral ulcers and photosensitivity [77].

Since the introduction of biological drugs, the treatment of immune-mediated inflammatory diseases has witnessed an enormous improvement in the clinical armamentarium. Indeed, tumor necrosis factor α (TNF-α) inhibitors neutralizes the cytokine pro-inflammatory effect. Infliximab (IFX), adalimumab (ADL), golimumab (GOL), etanercept (ETA) and certolizumab (CTZ) can induce an immune response with the formation of autoantibodies against the drug, i.e., elicit immunogenicity, particularly after their prolonged use [78, 79].

ADA formation is caused by the recognition of drugs as non-self substances by the immune system. ADA ultimately act by causing the formation of complexes that block the binding between the drug and the target and also enhance the drug’s clearance [78]. Antibodies thus lead to a reduction in the serum levels of the drug to sub-therapeutic levels, resulting in loss of clinical efficacy [79].

Therapeutic drug monitoring, done by measuring the TNF-α inhibitors serum levels and ADA, is considered an important factor for personalized TNF-α inhibitor treatment in chronic autoimmune diseases [80]. Nevertheless, the mere presence of ADA does not directly correlate to drug loss of response and clinical consequences, since a significant amount of ADA is needed to neutralize much of the serum drug, which is usually given in high doses. More importantly, there is no agreement on the laboratory methods to be used for ADA detection in clinical practice. The significance of ADA largely depends on the quantity of antibodies produced and on the amount of drug not bound to them; if the quantity of ADA-free drug is substantial, the clinical impact will be minimal [81]. Several studies have shown that ADA become detectable especially within the first year of treatment with TNF-α inhibitors, while their immunogenicity is very low after the first year of therapy [82, 83]. ADA levels are diminished using maintenance therapy rather than episodic therapy, since prolonged periods in which the drug is sub-dosed are avoided [80]. Moreover, patients treated concomitantly with MTX have a lower risk of developing such antibodies, similarly to those responding to therapy [78]. Overall, the determination of ADA levels and anti-TNF-α drug may be useful in patients where the clinical efficacy of TNF-α inhibitor has dropped. Pecoraro and colleagues suggest that in the presence of high ADA levels, either with optimal or suboptimal drug levels, the anti-TNF-α should be changed, while, in the case of low ADA levels, if drug levels are not adequate, the dosage or frequency of drug administration should be increased. Ultimately, however, the secondary loss of efficacy of an anti-TNF-α drug will lead to a treatment switch regardless of the mechanisms responsible for such loss. However others, in particular Steenholdt, suggest a different approach as shown in Fig. 3.