A Flare Risk Index Informed by Select Immune Mediators in Systemic Lupus Erythematosus

Abstract

Objective: Systemic lupus erythematosus (SLE) is marked by immune dysregulation linked to varied clinical disease activity. Using a unique longitudinal cohort of SLE patients, this study sought to identify optimal immune mediators informing an empirically refined flare risk index (FRI) reflecting altered immunity prior to clinical disease flare.

Methods: Thirty-seven SLE-associated plasma mediators were evaluated by microfluidic immunoassay in 46 samples obtained in SLE patients with an imminent clinical disease flare (preflare) and 53 samples obtained in SLE patients without a flare over a corresponding period (pre-nonflare). SLE patients were selected from a unique longitudinal cohort of 106 patients with classified SLE (meeting the American College of Rheumatology 1997 revised criteria for SLE or the Systemic Lupus International Collaborating Clinics 2012 revised criteria for SLE). Autoantibody specificities, hybrid SLE Disease Activity Index (hSLEDAI) scores, clinical features, and medication usage were also compared at preflare (mean ± SD 111 ± 47 days prior to flare) versus pre-nonflare (99 ± 21 days prior to nonflare) time points. Variable importance was determined by random forest analysis with logistic regression subsequently applied to determine the optimal number and type of analytes informing a refined FRI.

Results: Preflare versus pre-nonflare differences were not associated with demographics, autoantibody specificities, hSLEDAI scores, clinical features, nor medication usage. Forward selection and backward elimination of mediators ranked by variable importance resulted in 17 plasma mediator candidates differentiating preflare from pre-nonflare visits. A final combination of 11 mediators best informed a newly refined FRI, which achieved a maximum sensitivity of 97% and maximum specificity of 98% after applying decision curve analysis to define low, medium, and high FRI scores.

Conclusion: We verified altered immune mediators associated with imminent disease flare, and a subset of these mediators improved the FRI to identify SLE patients at risk of imminent flare. This molecularly informed, proactive management approach could be critical in prospective clinical trials and the clinical management of lupus.

Figure 1.. Immune mediators informing a Flare Risk Index (FRI).

(A) Variable Importance of top 17 immune mediators differentiating Pre-Flare from Pre-Nonflare as determined by random forest (top 12 mediators in blue and remaining top 5 mediators in red as determined by forward selection and backward elimination, respectively); ranking of all immune mediators and demographic/clinical features can be found in Table S2); (B) FRI (Mean ± 95% CI) in Pre-Flare (top panel) vs. Pre-Nonflare (bottom panel) samples informed by top number of mediators in variable importance order from (A); (C) Performance of FRI informed by top 12 (FRI-12) vs 17 (FRI-17) mediators listed in (A); graphs presented as box plots ± maximum and minimum scores. **p<0.01; ***p<0.001, ****p<0.0001 Mann-Whitney test

Figure 2.. Flare Risk Index (FRI) designed for clinical/commercial use.

(A) Immune mediators (n=11) informing FRI-11 (bold mediators determined by 1:10 sample dilution, all others determined by 1:2 dilution on a separate Ella microfluidic immunoassay cartridge); (B) FRI-11 differentiation of Pre-Flare vs. Pre-Nonflare samples from Fig. 1 (****p<0.0001 Mann-Whitney test, graph presented as box plot ± maximum, minimum); (C) FRI-11 differentiation of Pre-Flare (Severe vs. Mild/Moderate [Mild/Mod]) vs. Pre-Nonflare samples in (B) (**p<0.01, ****p<0.0001 Kruskal-Wallis test with Dunn’s multiple comparison, graph presented as mean ± 95% CI); (D) Decision Curve Analysis plotting Threshold Probability vs. calculated Net Benefit; Low (24%) and High (76%) Probability marked with dashed lines; overlaid decision curves: black = Pre-Flare + Pre-Nonflare, blue = Pre-Nonflare, red = Pre-Flare (E) Predictive probability of future disease flare determined by logistic regression; Low (−6.4) and High (9.0) FRI-11 cut-off levels (associated with 24% and 76% Threshold Probability, respectively) marked with dashed lines; (F) Performance characteristics of FRI-11 (AUC = Area Under the Curve [receiver operating characteristic curve analysis]), CI = confidence interval, PPV = positive predictive value, NPV = negative predictive value

Figure 3.. Flare Risk Index (FRI) at time of concurrent Flare vs. Nonflare by organ system manifestations.

(A) FRI-11 differentiation of Flare (Severe vs. Mild/Moderate [Mild/Mod]) vs. Nonflare samples; (B-G) FRI-11 differentiation of Flare vs. Nonflare (NF) by Arthritis (B), Renal (C), mucocutaneous (Mucocut, D), Serositis (E), Immunologic (F), or Hematologic (G) organ system manifestations (*p≤0.05, **p<0.01, ***p<0.001, ****p<0.0001 Kruskal-Wallis test with Dunn’s multiple comparison, graphs presented as mean ± 95% CI).