Rheumatoid factor and anti-citrullinated protein antibody positivity, but not level, are associated with increased mortality in patients with rheumatoid arthritis: results from two large independent cohorts

In patients with inflammatory arthritis, the autoantibodies rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) have been associated with poor outcomes, such as increased disease activity, radiographic progression and disability [1]-[5]. However, the utility of antibody level in predicting the prognosis of inflammatory arthritis, in particular rheumatoid arthritis (RA), has not been clearly established. In a recent multicentre prospective study of patients with early inflammatory arthritis (EIA), the presence of RF and/or ACPA was a significant predictor of RA diagnosis within two years, but level did not appear to be important [6]. In contrast, in a study of patients with EIA from Norway in 2010, Mjaavatten et al. found that increasing levels of RF and ACPA were associated with persistent joint inflammation [7]. Other studies have failed to show consistently that either RF or ACPA antibody level is important in predicting poor outcome in patients with EIA and RA [8]-[10]. In addition, recent data from a subset of the Leiden Early Arthritis Clinic have shown that the avidity of ACPA may be prognostically more important than the level itself [11].

Nevertheless, antibody level is included in the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria for RA [12], which aim to identify those patients with EIA with poor prognosis sufficient to require intervention with disease modifying therapy. The presence of RF and ACPA are weighted as part of the total score according to their level; patients are said to be low positive if their level is greater than the upper limit of normal (ULN) but less than three times the ULN, and high positive if their level is at least three times the ULN. Thus, patients with high antibody levels are more likely to fulfil the criteria, and it would be interesting to investigate whether these cut-offs are appropriate in predicting other adverse outcomes, such as mortality.

The increased mortality in patients with RA has been long established [13]. It is also well recognised that the presence of RF in sera of patients with inflammatory arthritis (whether or not they meet formal classification criteria for RA) is associated with an increased risk of premature death [14]-[16]. In fact, this association has been demonstrated even in subjects without symptoms of arthritis [17]. ACPA positivity has also been shown to predict premature mortality in the Norfolk Arthritis Register [18]; however this association has yet to be confirmed in other cohorts.

The aims of this study were to investigate the association between mortality and RF and/or ACPA positivity and level in patients with EIA. The term EIA includes all patients with RA early in the disease process, and studying these patients allows additional inclusion of those patients who may later go on to meet formal classification criteria for RA. It has been recognised that significant variability in antibody testing can occur between laboratories [19]. Thus, to strengthen the external validity of the study results, we investigated these questions in two large prospective cohorts of patients with EIA: the Norfolk Arthritis Register (NOAR) in the UK and the Leiden Early Arthritis Clinic (EAC) in the Netherlands.

A total of 4,962 patients with EIA were included in the study (3,053 from NOAR, 1,909 from Leiden EAC). The cohorts had similar age and gender distributions, 65% (1,970) female in NOAR, 63% (1,205) female in the EAC, respective median (interquartile range) age at symptom onset 56 (44 to 68) and age at inclusion 54 (42 to 67) years. In NOAR, 63% of patients fulfilled the 2010 ACR/EULAR classification criteria for RA, in the EAC this proportion was 57% of patients. Baseline characteristics of patients from the two cohorts are shown in Table 1. The mean (standard deviation) follow up in each study was 11.8 (5.8) years in NOAR and 8.5 (5.2) years in EAC. There were 787 deaths during 36,109 person years follow up in NOAR, and 275 deaths during 16,187 person years follow up in the EAC; this resulted in crude death rates of 21.8 and 17.0 deaths per 1,000 person years in each cohort, respectively. The number of deaths in each of the antibody subgroups are shown in Table 2.

In two well established observational cohorts of EIA and its sub-population of patients with RA, we have shown that RF and ACPA positivity are predictors of excess mortality, and that the presence of both antibodies was a stronger predictor of mortality than single antibody positivity. However, in this first large study to investigate the association between antibody levels and mortality, the influence of increasing antibody level was not consistent between the two cohorts.

Our data have once again demonstrated the known relationship between RF positivity and early mortality [14], and confirmed that a similar association exists in patients who are ACPA positive. This has previously been described in NOAR [18] but only reported elsewhere by two other groups of investigators. The first study was in a subset of 299 patients in the Rochester epidemiology project [22], half of whom had RA. The second small study, by Sihvonen et al. [23] used logistic regression (which does not allow for censoring) rather than Cox models to analyse the data. It was, therefore, important to corroborate this association in another large EIA cohort, such as the Leiden EAC.

The results of our study are concordant with the findings of Ursum et al., who studied 545 patients with early arthritis in the Netherlands [10]. They found no association after two years between antibody levels and early disease outcomes, including disease activity measured by DAS28, functional status measured by the Health Assessment Questionnaire (HAQ) and radiographic progression. Similarly, a number of other small studies have reinforced the association between ACPA positivity and other poor outcomes, such as increased disease activity and radiographic damage, but have failed to identify an association with increasing ACPA levels [8],[24]. By contrast, Syversen et al. conducted a study of 125 patients who met the 1987 ACR classification criteria for RA [25] in a subpopulation of the European Research on Incapacitating Disease and Social Support (EURODISS) project [26]. They found that 10 year radiographic progression was increased in patients with low-moderate ACPA levels (>ULN and ≤8 times ULN), but this appeared to be further increased in patients with very high levels of ACPA (>8 times the ULN). However, they also demonstrated that the highest probability of radiographic progression occurred in patients who were positive for both RF and ACPA. A recent study in Italy examined progression from EIA to RA in 192 patients [6]. In accordance with our findings, they demonstrated the presence of both antibodies predicted RA, but antibody high or low positivity had no influence. In the Norwegian Very Early Arthritis Clinic (NOR-VEAC) study, Mjaavatten et al. showed additive value in testing for both antibodies in order to predict disease persistence [7]. They also demonstrated an association between antibody level and persistent arthritis, however the number of patients per group was small (<30). In addition, their analysis employed last observation carried forward to account for patients who did not have complete follow up. It is possible, therefore, that their results were influenced by attrition bias; that is, patients whose arthritis resolved may not have attended further follow up, and at their last recorded visit, their arthritis appeared to be persistent even though it subsequently resolved. It is possible that the different characteristics and follow up of these cohorts account for the different findings; in addition the different cut-offs of the commercially available assays may not correspond. Nevertheless, this emphasises that the role of antibody levels in predicting outcomes for patients with inflammatory arthritis has not been robustly established.

There are limitations to our study. We decided not to perform a pooled analysis of data from both cohorts because the different inclusion criteria of the two cohorts could potentially produce misleading conclusions. We did not aim to develop a full predictive model for mortality in RA, but focussed specifically on the association between antibody status and level, and mortality. Therefore, the number of confounders included in the multivariate model was small, and the final model does not account for all predictors of mortality in RA. As in all observational studies, there remains potential for residual confounding for which we have not adjusted. Further, in our analyses we did make the assumption that antibody status is fixed. This assumption seemed reasonable as the majority of studies have shown for both RF and, particularly, ACPA, that few patients convert from seropositive to negative over time [27]-[29], and when this does occur, risk of poor outcome may be maintained [30].

In conclusion, in this large study investigating the relationship between antibody levels and mortality in EIA, we have shown that patients with both RF and ACPA, rather than the higher levels of the antibodies, had increased rates of early death. We have also confirmed the association between ACPA positivity and excess mortality in a second large EIA cohort. Therefore, in patients presenting with early rheumatoid arthritis, the number of positive antibodies may be more important than the antibody levels in assessing the mortality risk in clinical practice.