The value of coronary artery calcium (CAC) scoring versus multiple biomarkers in increasing risk prediction for cardiovascular disease (CVD) remains unknown. The study group consisted of 1,286 asymptomatic participants (mean ± SD 59 ± 8 years old) with no known coronary heart disease. Mean follow-up time was 4.1 ± 0.4 years with the primary outcome of combined CVD (cardiac death, myocardial infarction, stroke, and late target vessel revascularization). CAC was calculated by the method of Agatston. Biomarkers measured were C-reactive protein, interleukin-6, myeloperoxidase, B-type natriuretic peptide, and plasminogen activator-1. During follow-up 35 participants developed CVD events including cardiac deaths (6%), myocardial infarction (23%), strokes (17%), and late revascularizations (54%). In Cox proportional-hazards models adjusted for Framingham Risk Score (FRS), presence of log CAC beyond FRS was associated with increased hazards for CVD events (hazard ratio 1.7, 95% confidence interval [CI] 1.4 to 2.0, p <0.001). Multiple biomarkers score was also associated with increased risk beyond FRS (hazard ratio 2.1, p = 0.02) per 1-U increase in score; however, the c-statistic did not increase significantly (0.75, 95% CI 0.68 to 0.84, p = 0.32). The c-statistic increased when log CAC was incorporated into FRS without or with multiple biomarkers score (c-statistic 0.84, p = 0.003 and p = 0.008 respectively). Addition of CAC to risk factors showed significant reclassification (net reclassification improvement 0.35 (95% CI 0.11 to 0.58, p = 0.007; integrated discrimination index 0.076, p = 0.0001), whereas addition of multiple biomarkers score did not show significant reclassification. In conclusion, in this study of asymptomatic subjects without known CVD, addition of CAC but not biomarkers substantially improved risk reclassification for future CVD events beyond traditional risk factors.
In population-based studies, coronary artery calcium (CAC) scoring has emerged as a robust predictor of incident cardiovascular disease (CVD) events and provides incremental predictive information beyond that yielded by standard risk factors. In this regard, the most recent American College of Cardiology/American Heart Association guideline for assessment of cardiovascular risk in asymptomatic adults recommends use of CAC scanning as a class IIa indication for risk stratification of asymptomatic patients with an intermediate risk of events. In several recent studies, use of multiple blood biomarkers has also shown promise for the prediction of future CVD events. Given the potential utility of other blood biomarkers for risk assessment and the absence of available data directly comparing CAC to multiple biomarkers, we evaluated asymptomatic subjects within the Early Identification of Subclinical Atherosclerosis by Noninvasive Imaging Research (EISNER) prospective randomized trial who underwent evaluation by CAC and measure of multiple biomarkers.
Methods
The study population represented a subset of 1,286 adults (mean 58.6 ± 8.5 years old, 47% women) without known CVD or symptoms within the EISNER research study, as we have previously reported. The present study participants represent those for whom consecutive CAC and biomarker data were available. The present study was approved by the Cedars-Sinai Medical Center institutional review board.
A dedicated medical history was taken to assess previous cardiac disease, diabetes, and medication use. Collection of fasting lipid profile and measurements of blood pressure and body mass index has been detailed previously. Subjects were scanned using electron beam computed tomography (General Electric/Imatron, South San Francisco, California) or a multidetector scanner (Siemens, Munich, Germany). Foci of CAC were identified and scored by an experienced technician using semiautomatic commercial software (ScImage, Inc., Los Altos, California) and verified by a board-certified cardiologist.
The following biomarkers were studied: C-reactive protein (CRP), interleukin-6, myeloperoxidase, B-type natriuretic peptide, and plasminogen activator-1, representing distinct biological pathways for inflammation, left ventricular dysfunction, and fibrinolysis. Serum and ethylenediaminetetraacetic acid plasma samples for all study participants were stored at −70°C for up to 4 years until all baseline samples were accumulated. All biomarker assays were performed by Alere, Inc. (San Diego, California). CRP (range 0.065 to 35 μg/ml) was measured by the Luminex Competitive method (Alere, Inc.). Interleukin-6 (range 16 to 60,000 pg/ml) was measured using a microtiter immunoassay. Myeloperoxidase (range 2 to 1,200 ng/ml) was measured by the Luminex Sandwich immunoassay (Alere, Inc., protocol CLN0002, internal Alere myeloperoxidase project documentation). Plasma B-type natriuretic peptide (range 6.6 to 2,600 pg/ml) was analyzed in the blood sample anticoagulated with ethylenediaminetetraacetic acid using the quantitative immunofluorescence assay. Plasminogen activator-1 (range 0.1 to 55 ng/ml) was measured using the microtiter immunoassay.
Follow-up for CVD events consisted of administering patient questionnaires, patient interviews, and/or using hospital records to obtain outcome data on consented patients. All deaths were verified by the National Death Index and/or independent review of death reports by 2 physicians. Primary end point of the present study was time to first CVD event defined by cardiac death, myocardial infarction, stroke, and late revascularizations (>90 days after CAC scanning). Mean follow-up time of study for CVD events was 4.1 ± 0.4 years.
For all analyses, continuous biomarker variables were standardized after being log-transformed toward normality, resulting in mean ± SD of 0 ± 1. CAC was log-transformed (log [CAC + 1]) toward normality in all the models. We performed multivariable Cox proportional hazards models to examine the association between biomarkers and incident events after verifying that the assumption of proportional hazards was met using Schoenfeld residuals. All models were adjusted for Framingham Risk Score (FRS). Hazard ratios (HRs) were expressed per 1 SD in the log increment in the respective biomarker. Each biomarker was individually tested in models for CVD events with adjustment for conventional risk factors. These analyses included only biomarkers known to have a clinical interest (5 biomarkers for cardiovascular events). In addition, a risk score was developed using the linear prediction from a fitted Cox model consisting solely of these 5 biologically relevant biomarkers, taking the form of a risk score = (β1 × CRP) + (β2 × interleukin-6) + (β3 × myeloperoxidase) + (β4 × B-type natriuretic peptide) + (β5 × plasminogen activator-1), where β1 to β5 denote estimates of beta coefficients associated with each standardized log-transformed biomarker.
We constructed a Harrell c-statistic and area under the receiver operating characteristics curves for predicting CVD events at 4 years and compared them for CAC and biomarkers. We pursued further discrimination based on combined biomarkers risk score achieving a p value <0.05. We also evaluated the ability of multiple biomarkers score and CAC to reclassify risk, as we previously described. Using a Cox model consisting of FRS components, participants were initially classified as low, intermediate, or high risk if their predicted 4-year risk of CVD was <2.4%, 2.4% to 8%, or >8%, respectively. These correspond to FRS cutpoints <6%, 6% to 20%, or >20% for 10-year risk adjusted to 4 years. Cross tabulations of risk categories based on models with and without multiple biomarkers risk score or CAC were performed to describe the number and percentage of participants who were reclassified appropriately (i.e., to a lower risk group for nonevents or to a higher risk group for events) and inappropriately (i.e., to a lower risk group for events or to a higher risk group for nonevents). We calculated the net reclassification improvement and integrated discrimination index. A p value <0.05 was considered statistically significant. All statistical analyses were performed with STATA 11 (STATA Corp., College Station, Texas).
Results
Baseline characteristics of the 1,286 asymptomatic subjects comprising the study group are presented in Table 1 . Thirty-five (2.7%) CVD events occurred, consisting of 2 cardiac deaths (6%), 8 myocardial infarctions (23%), 6 strokes (17%), and 19 late revascularizations (54%). Age, systolic blood pressure, hypertension, log CAC, diabetes, and multiple biomarkers score (p values not shown) were significantly different between the event and nonevent groups.
| Characteristics | Total Population (n = 1,286) | Events | |
|---|---|---|---|
| No (n = 1,251) | Yes (n = 35) | ||
| Age (years) | 58.6 ± 8.5 | 58.5 ± 8.5 | 61.5 ± 8.0 |
| Men | 679 (52.8%) | 655 (52.4%) | 24 (68.6%) |
| Systolic blood pressure (mm Hg) | 133.1 ± 17.5 | 132.8 ± 17.2 | 144.5 ± 22.2 |
| Diastolic blood pressure (mm Hg) | 82.0 ± 10.7 | 81.9 ± 10.7 | 85.1 ± 12.0 |
| Hypertension | 744 (57.9%) | 715 (57.2%) | 29 (82.9%) |
| Body mass index (kg/m 2 ) | 27.5 ± 5.2 | 27.4 ± 5.2 | 28.7 ± 5.8 |
| Total cholesterol (mg/dl) | 215.8 ± 41.4 | 215.6 ± 41.5 | 224.6 ± 37.1 |
| Low-density lipoprotein cholesterol (mg/dl) | 136.1 ± 37.6 | 135.8 ± 37.6 | 145.7 ± 37.3 |
| High-density lipoprotein cholesterol (mg/dl) | 54.2 ± 16.5 | 54.3 ± 16.6 | 50.6 ± 13.8 |
| Diabetes mellitus | 104 (8.1%) | 97 (7.8%) | 7 (20.0%) |
| Current smoking | 70 (5.4%) | 67 (5.4%) | 3 (8.6%) |
| C-reactive protein (μg/ml) | 5.0 ± 7.6 | 4.9 ± 7.5 | 7.1 ± 10.7 |
| Log-transformed | 0.8 ± 1.3 | 0.8 ± 1.3 | 1.1 ± 1.3 |
| Interleukin-6 (pg/ml) | 92.7 ± 785.0 | 93.2 ± 795.3 | 74.4 ± 188.6 |
| Log-transformed | 3.4 ± 1.0 | 3.4 ± 1.0 | 3.4 ± 1.0 |
| Myeloperoxidase (ng/ml) | 15.2 ± 34.8 | 15.3 ± 35.3 | 13.8 ± 9.2 |
| Log-transformed | 2.5 ± 0.5 | 2.5 ± 0.5 | 2.5 ± 0.5 |
| B-type natriuretic peptide (pg/ml) | 49.3 ± 104.7 | 48.9 ± 105.4 | 63.6 ± 75.9 |
| Log-transformed | 3.4 ± 0.9 | 3.4 ± 0.9 | 3.6 ± 1.0 |
| Plasminogen activator-1 (ng/ml) | 15.7 ± 10.2 | 15.6 ± 10.2 | 19.0 ± 9.7 |
| Log-transformed | 2.5 ± 0.8 | 2.5 ± 0.8 | 2.8 ± 0.5 |
| Multiple biomarker score ⁎ | −0.00 ± 0.6 | −0.01 ± 0.6 | 0.34 ± 0.6 |
| Coronary artery calcium | 116.3 ± 347.3 | 98.6 ± 279.4 | 747.7 ± 1,125.2 |
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