Serum 14-3-3η as predictor of clinical remission and progression of structural damage in early rheumatoid arthritis following a treat-to-target strategy in a randomized controlled trial

Objective 14-3-3η is a proinflammatory mediator critical to joint destruction in rheumatoid arthritis (RA). We aimed to evaluate serum 14-3-3η for predicting disease activity and radiographic progression in patients with early RA in the double-blinded, randomized OPERA trial. Method 180 patients with early RA were randomized to receive methotrexate (MTX) + adalimumab or MTX + placebo in combination with glucocorticoid injections into swollen joints. Disease activity was measured using the 28-joint Disease Activity Score–C-reactive protein (DAS28-CRP). Clinical remission was defined as DAS28-CRP < 2.6. X-rays of hands and feet were evaluated by the Total Sharp van der Heijde score (TSS). Radiographic progression was defined as exceeding the smallest detectable change (1.8 TSS-units). Serum 14-3-3η was determined by enzyme-linked immunosorbent assay. Multivariate logistic regression models were used to identify predictors of DAS28-CRP remission at 6 months and radiographic progression at 12 months. Results Baseline 14-3-3η was a borderline significant independent predictor of radiographic progression at 12 months (odds radio = 1.02, 95% confidence interval 1.00–1.03, p = 0.05). In anti-cyclic citrullinated peptide antibody (ACPA)-negative patients, a moderate/high baseline 14-3-3η concentration increased the risk of radiographic progression at 12 months [4/51 (8%) vs 3/9 (33%), χ2 = 4.823, p = 0.028]. No value of 14-3-3η for predicting achievement of clinical remission was found. Conclusion Serum 14-3-3η was a borderline significant predictor of radiographic progression, particularly in ACPA-negative patients, but not of predicting achievement of clinical remission. Optimal cut-off levels of 14-3-3η for predicting radiographic progression in RA need further clarification.

Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disorder involving multiple, peripheral joints, often leading to joint destruction and thus giving rise to severe pain, loss of function, and reduced quality of life. Early diagnosis and treatment are crucial to prevent long-term disability. Current treatment options include early intervention with conventional diseasemodifying anti-rheumatic drugs (DMARDs), such as methotrexate (MTX), biological DMARDs, and intraarticular administration of glucocorticoids. The treatment goal is to achieve inflammatory control, including clinical remission and halted radiographic progression (1).
There is an ongoing interest in biomarkers involved in the pathogenesis of RA, and especially the theory that soluble biomarkers can serve as a guidance tool for clinical monitoring and therapeutic management of the disease (2,3).
The serological markers included in the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) 2010 classification criteria for RA are immunoglobulin M-rheumatoid factor (IgM-RF), anti-cyclic citrullinated peptide antibodies (ACPA), and C-reactive protein (CRP) (4). However, the predictive value of these biomarkers for the achievement of clinical remission or for structural joint damage progression is limited. Novel biomarkers with high sensitivity and specificity for such monitoring would therefore be clinically very useful (5).
14-3-3 is a family of regulatory proteins that normally exist as molecular chaperones interacting with a wide range of intracellular proteins and thereby regulating important biological functions. The isoform 14-3-3η has been found at significantly higher levels in serum and synovial fluid in patients with RA when compared with healthy individuals (6). Previous cell stimulation studies have shown that when 14-3-3η is present in the extracellular environment at concentrations reflecting the levels in serum from patients with RA, it stimulates cells of the innate immune system, resulting in an activation of cell signalling cascades that regulates proinflammatory cytokines such as interleukin-6 (IL-6) and IL-β, and factors involved in bone and cartilage metabolism, such as matrix metalloproteinase-9 (MMP-9) and receptor activator of nuclear factor kappa-B ligand (RANKL) (7). In addition, 14-3-3η is shown to be expressed at higher levels in serum from RA patients with radiographic progression, further supporting the theory that 14-3-3η is involved in the joint damage process (8). To determine the role of 14-3-3η in joint damage, a mechanistic study found that in fibroblast-like synoviocytes (FLSs), which are immature, transformed fibroblasts playing a key role in the pathological process of joint degradation because of their ability to degrade articular cartilage by the formation of actin-rich protrusions called invadosomes, high 14-3-3η mRNA levels were positively correlated with invadosome formation in FLS cultures, indicating that 14-3-3η is involved in the extracellular invadosomeinduced degradation of extracellular matrix (9). Considering this, 14-3-3η may have great potential as a biomarker in predicting the prognosis of the disease.
In this study, we aimed to investigate the biomarker serum 14-3-3η and early changes in this protein as a potential predictor of disease activity and radiographic progression in patients with early RA treated to target (in all patients and stratified by ACPA status). Blood samples and clinical data were taken from patients who participated in the double-blinded randomized controlled Optimized Treatment in Early Rheumatoid Arthritis (OPERA) trial (10,11).

Study population and design
The study included 180 patients who participated in the OPERA study, an investigator-initiated, randomized, double-blinded, placebo-controlled, twoarmed, parallel-group, multicentre trial (10,11). The patients had early RA (disease duration < 6 months) and fulfilled the ACR 1987 revised criteria, and only patients with moderate to severe disease activity [28-joint Disease Activity Score with C-reactive protein (DAS28-CRP) ≥ 3.2] were included. Key exclusion criteria were treatment with glucocorticoids within the last 4 months, previous DMARD treatment, previous malignancy, or latent tuberculosis (10).
The patients were randomized into two treatment arms: one arm received 40 mg adalimumab subcutaneously every second week in combination with oral methotrexate, 7.5 mg/week at baseline, increasing to 15 mg/week after 2 months (the 'adalimumab group'); and the other arm received the same treatment except that they were given a placeboadalimumab (the 'placebo group'). Patients in both groups were treated with intra-articular injections of triamcinolone (40 mg/mL, 0.5-2 mL/joint) if any swollen joints were observed at baseline or at subsequent follow-up visits (at a maximum of 160 mg or four joints per visit). Add-on treatment with hydroxychloroquine (200 mg/day) and sulphasalazine (2 g/day) was offered to patients who had persisting unacceptable disease activity at the 3 month visit or later (i.e. either DAS28-CRP ≥ 3.2 and at least one swollen joint or intra-articular injections of 160 mg of triamcinolone given monthly for 3 consecutive months). If no acceptable effect on disease activity was observed after an additional 3 months, the treatment was discontinued and the patient was excluded from the study (10).

Outcomes of clinical measurements and imaging procedures
The primary outcomes of the present post-hoc analyses were clinical remission at 6 months and radiographic progression at 12 months. Disease activity was measured at baseline and after 3, 6, and 12 months using the DAS28-CRP, Clinical Disease Activity Index (CDAI), Simplified Disease Activity Index (SDAI), and CRP. At the same time-points, serum samples for biomarker analyses were collected. Clinical remission was defined as DAS28-CRP < 2.6.
Conventional X-rays of the hands and wrist (posteroanterior view) and forefeet (postero-anterior view) were obtained at 6, 12, and 24 months and evaluated by a trained reader, blinded to all additional patient data and image chronology. The images were scored according to the Sharp van der Heijde method (12). The smallest detectable change (SDC) in total Sharp van der Heijde score (TSS) in this study was 1.8 and radiographic progression at 12 months was defined as progression exceeding the SDC, that is ∆TSS 0-12m ≥ 2 (10,13).

Serum 14-3-3η measurements
Quantitative measurements of human 14-3-3η were performed post hoc on banked serum samples from the OPERA study at baseline, 3 months, 6 months, 12 months, and 24 months. Serum had been separated with centrifugation and stored in a biobank freezer at −80º C until the time of analysis. The 14-3-3η concentrations were measured in the research laboratory of Copenhagen Center for Arthritis Research (COPE-CARE), Rigshospitalet, Glostrup, using the JOINTstat TM enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's protocol (Augurex Life Sciences Corp, Vancouver, BC, Canada). In brief, the serum samples were diluted 1:20 using the assay buffer, and 100 µL samples were pipetted into the wells of a microtitre plate coated with a 14-3-3η monoclonal antibody. The plates were incubated at 27°C for 2 h at 500 rpm. Following incubation, the plates were washed thoroughly with wash buffer and a horseradish peroxidase (HRP)-conjugated 14-3-3η monoclonal antibody was added. The plates were again incubated for 1 h at room temperature and washed, before adding an HRP substrate, reacting to the bound 14-3-3η conjugate antibody. After incubation for 30 min at room temperature in the dark, the reaction was stopped using a 1 N hydrochloric acid solution. The intensity of generated colour was detected in a microtitre plate reader measuring 450 nm wavelength, and 14-3-3η concentration was calculated using the plate reader's built-in software. To eliminate interassay variation, serial samples from each patient were analysed on the same ELISA plate using the same batch number. The intra-assay and interassay coefficients of variance were 4% and 13%, respectively. Dilution linearity was assessed by serially diluting three serum samples with known 14-3-3η concentration, and recovery was between 86% and 105%. Freeze-thaw experiments with up to five cycles revealed no impact on 14-3-3η concentration, and neither did storage at room temperature from 30 min to 4 h. The 14-3-3η concentration cut-offs were predefined by the manufacturer as negative in samples with levels < 0.19 ng/mL and as positive in samples with levels ≥ 0.19 ng/mL. Furthermore, based on cut-offs suggested by the manufacturer, 14-3-3η levels were categorized into negative (< 0.19 ng/mL), low (0.19-0.39 ng/mL), moderate (0.40-0.79 ng/mL), and high (≥ 0.80 ng/mL). These cut-off values have previously been described by Maksymowych et al using the same ELISA kit (14).

Statistical analyses
Descriptive statistics were used to describe demographics and patient characteristics, presented as mean ± sd or median [interquartile range (IQR)], depending on data distribution.
Baseline predictors of outcome were tested using univariate logistic regression analysis and the corresponding odds ratios (ORs) with 95% confidence intervals (CIs). Variables with p-values < 0.1 as well as a priori defined covariates (age and gender) were subsequently included in multivariable logistic analyses. Separate analyses were conducted for serum 14-3-3η as a continuous variable and serum 14-3-3η divided into categories (negative, low, moderate, and high).
Correlations between 14-3-3η and clinical disease activity measures or radiographic progression were determined using Spearman's correlation coefficient. Fisher's exact test was used to determine the risk of radiographic progression or the likelihood of achieving DAS28-CRP remission based on 14-3-3η categories. Pearson's chi-squared test was used to determine the difference in risk of radiographic progression and likelihood of achieving DAS28-CRP remission, based on stratification of baseline 14-3-3η by ACPA status.
All statistical analyses were performed based on a predefined statistical analysis plan using IBM SPSS statistics version 25.0 (IBM Corp, Armonk, NY, USA). All p-values below 0.05 were considered statistically significant.

Ethics
All included patients had given written informed consent to participate in the OPERA study, including contributing blood samples to a biobank (VEK-20070008).
The current study was approved by the data authorities (journal number 2019-522-0141) and the ethics committee in the Capital Region of Denmark (journal number 2012-58-0004).
The fraction of patients with moderate/high serum 14-3-3η concentration at baseline was higher in Table 1. Baseline characteristics of patients in the OPERA trial overall and by treatment arm, by radiographic progression at 12 months, and by clinical remission at 6 months.    (7) for details.
Correlation between 14-3-3η and radiographic damage or clinical disease activity measures We found a weak, but statistically significant correlation between 14-3-3η concentration at baseline and changes in TSS from baseline to 12 months (rho = 0.22, p = 0.005) 300 ( Table 2). The raw data is also illustrated in Figure 1A.
*p-Values < 0.05 were considered statistically significant. p-Values were not corrected for multiple testing, and the results should be seen in that light.
Similar analyses of 14-3-3η categories as a predictor of clinical remission (DAS28-CRP < 2.6) at 6 months showed no statistically significant associations (data not shown).

Discussion
In this post-hoc study from the OPERA trial, we investigated the predictive value of adding serum 14-3-3η measurements to IgM-RF and ACPA, i.e. the existing panel of RA-associated antibodies, aiming to identify patients with early RA who were more likely to achieve clinical remission after 6 months or radiographic progression after 12 months. We found that serum 14-3-3η was an independent predictor of radiographic progression at 12 months (borderline significant). We also found that in ACPA-negative patients, baseline 14-3-3η identified patients who were at increased risk of radiographic progression. No predictive value of 14-3-3η for the achievement of clinical remission was found.
The correlation between inflammatory markers and radiographic progression has been studied extensively during the past few decades. The traditional marker of inflammation is CRP, and elevated levels are associated with poor radiographic outcome (15). However, CRP is not specific for joint inflammation and, paradoxically, radiographic progression may be seen in patients with few clinical signs of inflammation and a decreased or normal level of CRP (15). In recent years, other protein biomarkers involved in the pathogenesis of joint destruction, such as MMP-3, have been proven to be associated with disease activity. A multi-biomarker disease activity (MBDA) score combining 12 biomarkers has been developed in an attempt to cover multiple aspects of the disease (16). The MBDA score has also been investigated in post-hoc analyses of the OPERA trial, and baseline MBDA score was an independent predictor of radiographic progression (17). We found that ACPA-negative patients who had a moderate/high 14-3-3η concentration at baseline were at increased risk of radiographic progression at 12 months. This suggests that adding 14-3-3η measurements for ACPA-negative patients is helpful in identifying patients at risk of progressing radiographically. This is clinically relevant, since being ACPA negative is normally considered to be associated with a low risk of progression (18). However, this association needs to be validated and further explored in additional studies with a larger population of ACPAnegative patients.
The association of RA and 14-3-3η was first reported in 2007 by Kilani et al (6). Since their discovery, several studies have investigated the diagnostic and prognostic value of 14-3-3η. Of relevance to our study, Maksymowych et al investigated the relationship with radiographic progression in two cohorts of patients with early RA and established RA (total of 73 patients). They found that in both cohorts serum 14-3-3η was expressed more in patients who progressed radiographically (8). In another study, Carrier et al also found that over a 5 year period, a 14-3-3η concentration ≥ 0.50 ng/ mL at baseline was associated with radiographic progression [∆TSS ≥ 5; relative risk (RR) = 1.60 (95% CI 1.28-2.00), p < 0.001, and ∆Erosion (progression in the erosion component of the Sharp score modified by van der Heijde) ≥ 5; RR = 2.04 (95% CI 1.53-2.70), p < 0.001] (19). In agreement with these results, our study found that the 14-3-3η concentration at baseline was higher in the group that had radiographic progression after 1 year compared to those who did not progress. Furthermore, multivariate regression analysis demonstrated that baseline 14-3-3η was an independent predictor of radiographic progression (borderline significant).
In this study, we were not able to confirm previously published findings that 14-3-3η positivity is correlated with a higher disease activity or that it may act as an independent predictor of clinical remission (19)(20)(21), as we did not find any association between 14-3-3η values and clinical remission according to any of the composite disease activity score systems. Nor did we find that DAS28-CRP had a predictive value for radiographic progression. This may, partly, be explained by the fact that the OPERA trial included only patients with moderately to severely active RA, defined as DAS28-CRP ≥ 3.2.
The strength of the current study is that the data originated from a strictly designed, double-blinded, 14-3-3η and rheumatoid arthritis placebo-controlled study with a well-characterized population and a low rate of loss to follow-up. The population comprised treatment-naïve, early RA patients, and they were treated according to a modern treat-to-target strategy. A limitation of the study is that radiographic progression was observed in only 42/164 (23%) of the patients after 12 months and, furthermore, only a limited percentage of the patients (35%) was    ACPA negative at baseline, making the statistical analyses less powerful.

Conclusion
The present study showed that in early RA 14-3-3η positivity was a borderline significant independent predictor of radiographic progression at 12 months, particularly in ACPA-negative patients. No value of 14-3-3η for predicting clinical remission was found. This study encourages further studies on identifying the optimal cut-offs for 14-3-3η for the prediction of structural damage progression in patients with RA, particularly in ACPA-negative patients.