0531: Evaluating the Diagnostic Utility of 14-3-3 eta Autoantibodies in Axial Spondyloarthritis: A Multiplex Assay Approach

Background/Purpose: Novel biomarkers for axSpA are urgently needed to enable earlier diagnosis and improve patient outcomes. An assay for quantifying 14-3-3η autoantibodies (AAbs) has been developed, utilizing peptides derived from the 14-3-3η protein with distinct specificities. This study aims to establish cut-off values in a training cohort and apply them to an evaluation cohort to assess the diagnostic utility of the 14-3-3η multiplex AAb assay (MAAA) alone and with HLA-B27 and C-reactive protein (CRP).

Methods: The performance of the 14-3-3η MAAA was evaluated using a training cohort of 105 axSpA patients and 74 healthy controls (HC); N=179. ROC AUCs, sensitivity, and specificity for each peptide were generated. Positivity cut-offs for each of the five peptides were defined using the Youden Index, and these were summed to yield a composite score. The strength of the association for axSpA diagnosis was assessed by comparing the composite positivity status of axSpA patients to healthy controls (HC) using Fisher's Exact test. Diagnostic utility was further assessed in an evaluation cohort of 101 axSpA patients. CRP positivity was defined as >5 mg/L. Statistical significance was set at p < 0.05.

Results: All five 14-3-3η peptides exhibited significant areas under the curve (AUC) in distinguishing axSpA patients from HC in the training cohort (Table 1). The assay showed high precision, with intra-laboratory precision %CVs ranging from 8.3% to 14%. The assay showed minimal interference from normal serum elements (hemoglobin, bilirubin, triglycerides, cholesterol, and albumin), non-target antibodies (HAMA, heterophile, and infliximab), and organic chemicals (ibuprofen, sulfasalazine, and dexamethasone).

In the training cohort, the composite score yielded 67% TP and 69% TN with 33% FN and 31% FP. Fisher’s Exact test produced a Chi-Square value of 31.8, with a p-value of less than 0.0001, and an Odds Ratio of 8.7 (95% CI: 3.9 - 19.6). Table 2 shows that patients with composite positivity have a significantly higher relative risk of axSpA compared to healthy controls.

Table 3 shows that adding 14-3-3η AAb composite positivity to HLA-B27 and CRP increases axSpA detection from 93.3% to 99.0% in the training cohort and from 81.0% to 91.4% in the evaluation cohort. In HLA-B27 negative patients, 14-3-3η AAb improves detection to 70.6% in the training cohort and 55.0% in the evaluation cohort. Combined with CRP, detection increases to 88.2%, highlighting its potential for early diagnosis.

Conclusion: The 14-3-3η MAAA significantly enhances the early diagnosis of axSpA, especially when combined with HLA-B27 and CRP, demonstrating its potential as a valuable diagnostic tool. This combined approach could lead to better patient outcomes through earlier and more accurate detection. Future research should focus on validating the 14-3-3η multiplex autoantibody assay in larger, diverse cohorts to confirm its diagnostic utility across different populations.