Biomarkers in plasma and extracellular vesicles for early detection of asymptomatic ischemic heart disease: a retrospective case-control study

Background: Asymptomatic ischemic heart disease (aIHD) often precedes acute coronary syndrome (ACS). Early detection of aIHD with evidence-based treatment may reduce the risk of ACS and sudden cardiac death. Current risk prediction modalities may not fully capture at-risk patients. We aimed to explore whether plasma and extracellular vesicle (EV) proteins could help predict aIHD in at-risk individuals. Methods: We performed a pilot case-control study in asymptomatic individuals with a coronary artery calcium (CAC) score of >300 who underwent stress-perfusion cardiac magnetic resonance (CMR) imaging in three Dutch hospitals between May 2019 and September 2020. In total, 44 participants demonstrated the presence of aIHD (myocardial ischemia or infarction), and 43 did not. Plasma and EV protein concentrations were measured using the Olink Cardiovascular III panel (92 proteins). Differential expression was used to identify potential biomarkers with correction for multiple testing using the Benjamini-Hochberg method. Forward and backward logistic regression analysis was performed to identify independent clinical and proteomic predictors for aIHD. The trial was registered at ClinicalTrials.gov (NCT04680338; date registered 2020-12-22). Results: Baseline patient characteristics between patients with and without aIHD were similar; only hypertension was more prevalent in patients with aIHD (P=0.02). After Benjamini-Hochberg correction for multiple testing, no proteins were differentially expressed in groups with and without ischemia. However, in logistic regression models, higher plasma aminopeptidase-N [AP-N, odds ratio (OR) 8.74, 1.12-68.37, P=0.04], retinoic acid receptor responder protein 2 (RARRES2, OR 12.64, 1.88-85.15, P=0.009), and chitinase-3-like protein (CHI3L1, OR 0.54, 0.30-0.96, P=0.04) were independently associated with aIHD. After determining threshold values with the Youden index, the sensitivity for aIHD was 0.84 for AP-N, 0.93 for RARRES2, and 0.93 for CHI3L1. Positive predictive value was 0.62, 0.62, and 0.52, respectively. In an EV model, AP-N was able to predict the presence of aIHD (OR 7.24, 1.34-39.14, P=0.02). In a combined plasma and EV model, RARRES2 in plasma (OR 12.77,1.76-92.73, P=0.01) and AP-N in EVs (OR 13.27, 2.04-86.37, P=0.007) remained independently associated with aIHD. Established clinical risk scores [Systematic Coronary Risk Evaluation (SCORE), Framingham Heart Study (FHS)] did not discriminate aIHD (area under the curve <0.60). Conclusions: In this exploratory study, selected protein biomarkers showed potential to help identify aIHD in asymptomatic individuals with a high CAC score. These preliminary findings support further validation of AP-N and RARRES2 as gatekeepers for non-invasive risk stratification.