Background/Purpose: Serum elevations of antibodies to citrullinated protein antigens (ACPA) and rheumatoid factor (RF) can identify individuals at risk for developing rheumatoid arthritis (RA). Prior studies have identified serum calprotectin (seCal) as a promising biomarker for active inflammation among RA patients (Hurnakova 2018). Importantly, identifying biomarkers to accurately predict RA onset within a defined time frame may be especially useful to help design clinical trials for prevention of RA. The goal of this project was to evaluate the role of ACPA, RF and the novel marker seCal in improving the prediction of onset and timing of RA.

Methods: From the Department of Defense Serum Repository we studied pre-RA diagnosis samples from 214 RA Cases (212 meeting 1987 Criteria, and 2 diagnosed with RA by a board-certified rheumatologist), and 1,287 Controls matched to Cases on age, gender, race and duration of sample storage. All Case samples were tested for anti-cyclic citrullinated peptide-3 (CCP3, Inova), RFIgM (Inova) and serum calprotectin (seCal, Inova, research only). All Controls were tested for CCP3, and a subset were tested for RFIgM and seCal to set cutoff levels and evaluate diagnostic accuracy. CCP3 positivity was based on the established range (≥20 Units), and 2x and 3x the standard cutoff; RFIgM positivity was based on a level present in <5% of a subset of Controls; seCal positivity was based on a level greater than the mean+2 standard deviations (SD) from a set of Controls. The diagnostic accuracy of CCP3 for Cases was compared to Controls. In Cases, the diagnostic accuracy for a sample being within ≤3 years before diagnosis was evaluated using positivity of CCP3, RFIgM and seCal in 2×2 table analyses. Across models the positive predictive values (PPV) were compared with chi-squared testing.

Results: CCP3 positivity was 98.5%, 99.2% and 99.8% specific for the Cases versus Controls at standard, 2x, and 3x cutoff values, respectively. Within Cases, CCP3 positivity in a sample was strongly associated with developing RA in ≤3 years at all cutoff values although the differences in PPVs across cutoffs were not significant (p=0.43) (Table). The addition of RFIgM to CCP3 significantly improved the PPV of an RA diagnosis in ≤3 years from 78.9% to 88.0% (p=0.01). The addition of seCal to RFIgM and CCP3 (standard cutoff) improved the PPV to 100% although this was not significant (p=0.37). In Cases, the mean seCal was 8.6 ng/mL (SD 7.5) and 13.3 ng/mL (SD 19.0) for >3 and ≤3 years to diagnosis, respectively (p<0.01).

Conclusion: These findings demonstrate that CCP3 at multiple cutoffs is highly predictive of a diagnosis of RA in ≤3 years, with improved prediction if RFIgM positivity is included. These results can be used in the development of clinical prevention trials for RA. SeCal also shows promise in improving prediction of RA although further evaluation is needed, including comparison to other measures of inflammation (e.g. C-reactive protein).