Atherosclerosis Progression in the APPLE Trial Can Be Predicted in Young People With Juvenile-Onset Systemic Lupus Erythematosus Using a Novel Lipid Metabolomic Signature

Abstract

Objective: Patients with juvenile-onset systemic lupus erythematosus (JSLE) have increased atherosclerosis risk. This study investigated novel atherosclerosis progression biomarkers in the Atherosclerosis Prevention in Pediatric Lupus Erythematosus (APPLE) trial, the largest investigator-led randomized control trial of atorvastatin versus placebo for atherosclerosis progression in JSLE, using carotid intima-media thickness (CIMT) as the primary outcome.

Methods: Unsupervised clustering of baseline CIMT and CIMT progression over 36 months was used to stratify patients with JSLE. Disease characteristics, cardiovascular risk scores, and baseline serum metabolome were investigated in CIMT-stratified patients. Machine learning techniques were used to identify and validate a serum metabolomic signature of CIMT progression.

Results: Baseline CIMT stratified patients with JSLE (N = 151) into three groups with distinct high, intermediate, and low CIMT trajectories irrespective of treatment allocation, despite most patients having low cardiovascular disease risk based on recommended assessment criteria. In the placebo group (n = 60), patients with high versus low CIMT progression had higher total (P = 0.001) and low-density lipoprotein (LDL) (P = 0.002) cholesterol levels, although within the reference range. Furthermore, a robust baseline metabolomic signature predictive of high CIMT progression was identified in the placebo arm (area under the curve, 80.7%). Patients treated with atorvastatin (n = 61) had reduced LDL cholesterol levels after 36 months, as expected; however, despite this, 36% still had high atherosclerosis progression, which was not predicted by metabolomic biomarkers, suggesting nonlipid drivers of atherosclerosis in JSLE with management implications for this subset of patients.

Conclusion: Significant baseline heterogeneity and distinct subclinical atherosclerosis progression trajectories exist in JSLE. Metabolomic signatures can predict atherosclerosis progression in some patients with JSLE with relevance for clinical trial stratification.

Figure 1:

JSLE stratification (all APPLE patients with complete baseline data, N=151) by baseline CIMT (12 measures). A) Baseline CIMT measures of patients with juvenile-onset SLE were stratified using unsupervised hierarchical clustering. All 12 CIMT measures were standardised within each row by Z score and plotted as a heat map, representing the relationship to the mean of the group (red represents relatively high CIMT measures and blue represents relatively low CIMT measures). Each column represents a patient with JSLE. Three groups of patients with distinct baseline CIMT profiles were identified. B-C) Box and whisker plots show baseline and 36-month MMeanIMT measurements (APPLE primary outcome) in the identified high, intermediate and low baseline CIMT groups. Comparisons between groups were performed using Wilcoxon signed-rank test. D) Distinct longitudinal MMeanIMT progression from baseline to 36 months of the high, intermediate, and low CIMT progression groups (Mean, 95% CI), irrespective of treatment allocation. (Only JSLE patients with completed CIMT data at 36 months were included in the panel C-D, N=121). Abbreviations: CIMT- carotid intima-media thickness; MMeanIMT - Mean-Mean IMT common carotid artery measurement.

Figure 2.

JSLE stratification by delta(Δ)CIMT (12 measurements) at baseline vs 36 months in the placebo and atorvastatin arms. A) Heatmap displaying ΔCIMT (z scored) from JSLE patients from the placebo arm (full CIMT dataset, N=60) stratified by unsupervised hierarchical clustering. Each column represents a patient with JSLE. A high and low ΔCIMT progression group were discovered over 36 months. B) Box and whisker plots showing comparisons of MMeanIMT between groups from ‘A’ at baseline and 36 months. C) Heatmap displaying ΔCIMT (z scored) from JSLE patients from the atorvastatin arm (full CIMT dataset, N=61) stratified by unsupervised hierarchical clustering. Each column represents a patient with JSLE. A high, intermediate, and low ΔCIMT progression group were discovered over 36 months. D) Box and whisker plots showing comparison of MMeanIMT between groups from ‘C’ at baseline and 36 months. E) Box and whisker plots showing comparison of MMeanIMT between high/low CIMT progression groups between placebo and atorvastatin arm patients. Wilcoxon signed-rank test or t-test. F) Box and whisker plots showing comparisons of PDAY scores between baseline groups, placebo and atorvastatin progression groups. Abbreviations: CIMT- carotid intima-media thickness; MMeanIMT- Mean-Mean IMT common carotid artery measurement. PDAY- Pathobiological Determinants of Atherosclerosis in Youth.

Figure 3.

Baseline serum metabolomics (N=245 after data cleaning) comparisons between CIMT progression groups - placebo and atorvastatin arms. A) Volcano plot displaying fold change of all metabolites and Log10 p values comparing high (N=35) and low (N=25) CIMT progression groups - placebo arm (p<0.01; log2(fold change)>0.2). Top six significant metabolites (Bonferroni correction) highlighted in red. B) Box and whisker plots showing the top six metabolite levels of the high vs low CIMT progression groups -placebo arm. Unpaired t-test. C) ROC analysis for discriminating high vs low CIMT progression groups using the top six metabolites combined and separately by AUC. D) Volcano plot displaying fold change of all metabolites and Log10 p values comparing high (N=22) and low (N=15) CIMT progression groups -atorvastatin arm (p<0.05; log2(fold change)>0.2). Top two significant metabolites (Bonferroni correction) highlighted in red. E) Box and whisker plots showing the top two metabolite levels of the high vs low CIMT progression groups -atorvastatin arm. Unpaired t-test. F) ROC analysis for discriminating high vs low CIMT progression groups using DHA% and Isoleucine by AUC. Abbreviations: AUC - area under the curve; CIMT- carotid intima-media thickness; ROC- Receiver Operator Curve; Abbreviations/full names of all metabolites are listed in the Supplementary Appendix.