An Aggregate Biomarker Risk Score Predicts High Risk of Near‐Term Myocardial Infarction and Death: Findings From BARI 2D (Bypass Angioplasty Revascularization Investigation 2 Diabetes)
Background In a previous study, we found that a biomarker risk score (BRS) comprised of C‐reactive protein, fibrin‐degradation products, and heat shock protein‐70 predicts risk of myocardial infarction and death in coronary artery disease patients. We sought to: (1) validate the BRS in the independent BARI 2D (Bypass Angioplasty Revascularization Investigation 2 Diabetes) cohort, (2) investigate whether 1 year of intensive medical therapy is associated with improved BRS, and (3) elucidate whether an altered BRS parallels altered risk.
Methods and Results Two thousand thirty‐two subjects with coronary artery disease were followed for 5.3±1.1 years for cardiovascular events. Biomarkers were measured at baseline and retested in 1304 subjects at 1 year. BRS was determined as the biomarker number above previously defined cut‐off values (C‐reactive protein >3 mg/L, heat shock protein‐70 >0.313 ng/mL, and fibrin‐degradation products >1 μg/mL). After adjustment for covariates, those with a BRS of 3 had a 4‐fold increased risk of all‐cause death and a 6.8‐fold increased risk of cardiac death compared with those with a BRS of 0 (95% CI, 2.9–16.0; P<0.0001). All individual biomarkers decreased by 1 year, with ≈80% of patients decreasing their BRS. BRS recalibrated at 1 year also predicted risk. Those with 1‐year BRS of 2 to 3 had a 4‐year mortality rate of 21.1% versus 7.4% for those with BRS of 0 to 1 (P<0.0001).
Conclusions Our results validate the ability of the BRS to identify coronary artery disease patients at very high near‐term risk of myocardial infarction/death. After 1 year of intensive medical therapy, the BRS decreased significantly, and the reclassified BRS continued to track with risk. Our results suggest that repeated BRS measurements might be used to assess risk and recalibrate therapy.
Coronary artery disease (CAD) remains the predominant cause of death worldwide.1 Clinical tools, such as the Framingham risk score, predict risk of cardiovascular disease (CVD) and its adverse outcomes in community‐based populations free of known CVD, but fail to reliably predict risk in patients with established CAD.2, 3, 4 We have recently identified a pathway‐specific biomarker risk score (BRS) comprised of C‐reactive protein (CRP) representing inflammation, fibrin degradation products (FDP) representing the thrombosis pathway, and heat shock protein‐70 (HSP‐70) representing cell stress, that significantly predicts risk of MI and death in patients with suspected or established CAD and is independent of multiple clinical risk measures.5
BARI 2D (Bypass Angioplasty Revascularization Investigation 2 Diabetes) determined whether type II diabetics with complex CAD would benefit from early revascularization (coronary artery bypass grafting or percutaneous coronary intervention) and from insulin‐sensitizing (IS) medications. All recruited subjects received intensive medical therapy targeting individual cardiovascular risk factors with a goal for glycated hemoglobin (HbA1C) of <7%, blood pressure (BP) ≤130/80 mm Hg, low‐density lipoprotein level of <100 mg/dL, smoking cessation counseling, and other lifestyle modifications.
By analyzing samples drawn at baseline and after 1 year in the BARI 2D study, we aimed to: (1) validate the BRS in the BARI 2D cohort; (2) investigate whether 1 year of intensive medical therapy is associated with improved BRS; and (3) whether altered BRS is associated with altered risk.
Study Population and Design
Details of the study design were previously published.6 Briefly, 2368 patients with type II diabetes mellitus and CAD were enrolled from 49 clinical sites between 2001 and 2005 and randomized to 2 treatment strategies in a 2‐by‐2 factorial design. In the first strategy, patients were assigned to undergo prompt coronary revascularization (coronary artery bypass grafting or percutaneous coronary intervention) plus optimal medical therapy, or optimal medical therapy alone. In the second strategy, patients were assigned to undergo either IS or insulin‐providing therapy to achieve a target HbA1C level <7.0%. The study protocol was approved by the institutional review board at the University of Pittsburgh (Pittsburgh, PA), and all patients had provided written informed consent before enrollment in the BARI 2D study.
CAD was defined as ≥50% stenosis of a major epicardial coronary artery. Myocardial jeopardy index was used to assess CAD severity by calculating the percentage of myocardium jeopardized by significant lesions (≥50%) at a core laboratory.7, 8 Type II diabetes mellitus diagnosis was based on need for oral hypoglycemic or insulin therapy, or a confirmed elevated blood glucose level. Patients with left main disease, those with history of revascularization within the previous 12 months, or in need of urgent revascularization were excluded. Patients with elevated serum creatinine (≥2.0 mg/dL), HbA1C of ≥13%, or class III or IV heart failure were also excluded. Patients were followed monthly for 6 months and then every 3 months for a mean of 5.3 years.
Venous blood was drawn at baseline and at 1 year, and stored serum was available in a subset of 2032 subjects at baseline and 1304 subjects at 1‐year (Figure 1). Serum was stored at −70°C. Details of the biomarker assays were previously described5: serum high‐sensitivity CRP and FDP measurements were determined using a sandwich immunoassay by FirstMark, Inc (San Diego, CA). Serum HSP‐70 was measured by sandwich ELISA (R&D Systems, Minneapolis, MN) optimized by FirstMark. Minimum detectable CRP, FDP, and HSP‐70 were 0.1 mg/L, 0.06 μg/mL, and 0.313 ng/mL, respectively.
We used the same discriminatory cutoffs for each biomarker reported previously5 (9): Cut points for CRP, FDP, and HSP‐70 were 3 mg/L, 1.0 μg/mL, and 0.313 ng/mL, respectively.5 The BRS was computed by counting the number of biomarkers above their respective cutoffs.
Evaluation of Outcomes
The primary end point was all‐cause death; the principal secondary end point was a composite of death and myocardial infarction (MI).6 Other secondary end points were cardiac death and composite all‐cause death/MI/revascularization.
Diagnosis of MI was based on a doubling of cardiac enzymes and evidence of ischemia based on symptoms, ECG, or imaging. Periprocedural MI was defined as a 3‐ and a 10‐times increase in creatinine kinase MB after percutaneous coronary intervention and coronary artery bypass grafting, respectively. Silent MI was defined as a 2‐grade change of a Q‐wave on annual routine ECGs.6 Data on cause of death were adjudicated by personnel blinded to the group allocation, and cardiac death was defined as sudden death, fatal MI, congestive heart failure death, and presumed cardiac death. A subsequent revascularization procedure is defined as the first nonprotocol revascularization procedure for patients in the optimal medical treatment alone arm or a second revascularization procedure for patients in the prompt revascularization arm.
Means and SDs are presented for continuous normally distributed variables. One‐way ANOVA and Wilcoxon rank‐sum tests were used, as appropriate, to determine the differences between BRS groups. Kaplan–Meier cumulative incidence estimates were calculated 5 years after randomization and 4‐years after the 1‐year blood draw, and differences among BRS groups were detected using the log‐rank statistic. For the survival end points, patients without events were censored at the last time they were known to be alive. For the other outcomes, patients not experiencing the events were censored at the time of their last protocol follow‐up visit. Unadjusted and adjusted Cox proportional hazard models were used to estimate the relationship between the BRS and incident cardiovascular events. The adjusted models accounted for clinically relevant risk factors for CVD outcomes (age at baseline, sex, race, body mass index, history of hypertension, dyslipidemia, history of MI, history of coronary artery bypass grafting, smoking status, use of statins, aspirin, clopidogrel/ticlopidine, estimated glomerular filtration rate (calculated using the Modification of Diet in Renal Disease equation), myocardial jeopardy index, and left ventricular ejection fraction). Statistical interaction between BRS and each covariate was tested individually by adding an interaction term to the multivariable Cox model. Additionally, we analyzed the cardiac death end point using Fine–Gray competing risk models where noncardiac death events were handled as competing risks rather than censored values. Harrell's c‐statistics for discrimination, continuous net reclassification improvement, and integrated discrimination improvement metrics were calculated based on these multivariable Cox models. In addition, calibration was internally validated using 500 bootstrap samples for each of the multivariable Cox models and creating calibration plots based on the 4‐year predicted probability of an event. Landmark analyses censored individuals who did not survive or contribute a 1‐year serum sample at the 1‐year time point. Spearman correlations are presented for the 3 biomarkers. Values of P<0.05 from 2‐sided tests were considered to indicate statistical significance. SAS software (version 9.3; SAS Institute Inc, Cary, NC) was used for all analyses, except for the c‐statistics and calibration plots, where the Regression Modeling Strategies R package (http://biostat.mc.vanderbilt.edu/rms; January 1, 2017) was used.
The study population of BARI 2D patients with a baseline serum sample was aged 62.4±8.9 years, 70% male, 66% white (Table 1). Patients with baseline serum samples were comparable with those who had missing samples (data not shown).
Participants were separated based on the serum levels of CRP, HSP‐70, and FDP at the baseline visit into 4 BRS categories as described above. Thus, 18.3%, 45.2%, 30.3%, and 6.2% of patients had a BRS of 0, 1, 2, and 3, respectively, based on the number of biomarkers above the cut‐off threshold value. Those with a higher BRS were older, more likely to be female, obese, hypertensive, and more likely to have severe angina, a higher HbA1C, higher low‐density lipoprotein level, a history of MI, and less likely to be white or to be on statins and aspirin (Table 1). There were no differences in the myocardial jeopardy index or left ventricular ejection fraction between categories of the BRS. Importantly, a similar proportion of subjects in each BRS category were randomized to either the revascularization versus medical therapy or insulin‐providing versus IS treatments.
Relationship Between the Individual Biomarkers and Incident CVD Events
There were modest, but significant, correlations among the 3 individual biomarkers (Table 2). All 3 biomarkers (continuous variables) were associated with incident adverse events in both unadjusted and multivariable models adjusted for aforementioned covariates (Table 3). Elevated levels (above cut points) of CRP, HSP‐70, and FDP were all associated with increased risk of all‐cause death, the composite of all‐cause death and MI, cardiac death, and composite of all‐cause death/MI/revascularization in unadjusted and adjusted models (Table 3).
Relationship Between the BRS and Incident CVD Events
There was a significant association between the BRS and all incident CVD events in both unadjusted and adjusted models (Table 3). Each unit increase in the BRS corresponded to a 54% increase in risk of future all‐cause death and a 75% increase in risk of cardiac death after adjustment for the aforementioned variables (P<0.0001). Figure 2 demonstrates Kaplan–Meier survival curves for the outcomes of death, cardiac death, death/MI, and death/MI/revascularization. Compared with those with a BRS of 0, adjusted hazard ratios for incident cardiac death for patients with a BRS of 1, 2, and 3 were 2.4, 3.4, and 6.8, for all‐cause death were 1.75, 2.49, and 3.99, and for death/MI were 1.77, 2.38, and 3.53. Five‐year Kaplan–Meier event probabilities for all‐cause death for a BRS of 0, 1, 2, and 3 were 6.2%, 10.4%, 15.8%, and 26.4%, respectively (P<0.0001; Figure 2). Treating the noncardiac deaths as competing risks rather than censored events, the Fine–Gray models for cardiac death yielded comparable results to the Cox model results shown in Table 3.
To evaluate whether the association of the BRS with adverse outcomes was modified by demographic and clinical risk factor covariates or the assigned treatment strategies, interaction analyses were performed and revealed no significant effect modification (Figure 3). Importantly, there were no interactions between randomization assignments or the assigned strata with the death/MI outcome.
Discrimination and Calibration Analyses
Addition of the BRS to a baseline model consisting of the aforementioned demographic and clinical variables was associated with significant improvements in the c‐statistic for all outcome measures, including all‐cause death (area under the curve [AUC], 0.700–0.715; Δ AUC=0.015; P=0.044), all‐cause death/MI (AUC, 0.634–0.655; Δ AUC=0.021; P=0.002), all‐cause death/MI/revascularization (AUC, 0.574–0.589; Δ AUC=0.015; P=0.015), and for cardiac death (AUC, 0.740–0.760; Δ AUC=0.020; P=0.053) with a trend toward significance. Similarly, the addition of the BRS to the baseline model was associated with improvements in both net reclassification improvement and integrated discrimination improvement for each outcome measure (Table 4). The continuous net reclassification improvement for the composite outcome of all‐cause death/MI was 15%, which corresponded to 76% and 61% improvements in reclassification of risk in event‐ and nonevent groups, respectively. Calibration plots from each of the 4 models (corresponding to the adjusted model 3 for each outcome in Table 3) indicate that the risk models are fairly well calibrated (Figure 4).
Change in Biomarker Levels at 1 Year
Biomarker measurements were available in 1304 subjects at 1 year largely attributed to a protocol‐related delay in storing follow‐up samples. Minor differences were noted between patients with 1‐year serum samples and those who had missing samples (Table 5). There were significant reductions in all 3 biomarker levels at 1 year compared with baseline (Δ CRP=−1.2±3.5 mg/L, Δ HSP‐70=−0.36±1.2 ng/mL, and Δ FDP=−0.24±1.45 μg/mL; P=0.0001 for all). There were also substantial improvements in cardiovascular risk factors between baseline and 1 year (Δ systolic BP=−5.1±21.9 mm Hg, Δ diastolic BP=−2.6±11.6 mm Hg, Δ low‐density lipoprotein=−12.4±37.5 mg/dL, Δ high‐density lipoprotein=2.5±8.2 mg/dL, Δ HbA1c=−0.6±1.5%, and Δ glomerular filtration rate=−3.4±30.3). There were significant correlations between the change in HbA1c, low‐density lipoprotein, and high‐density lipoprotein levels and the change in CRP level (r=0.14, P=0.0001; r=0.07, P=0.01; and r=−0.18, P=0.0001, respectively). Similarly, there were significant correlations between the change in HSP‐70 and the change in systolic and diastolic BPs (r=0.09, P=0.002 and r=0.07, P=0.008; Table 6). Importantly, the significant reduction in the biomarkers levels at 1 year occurred irrespective of the randomization strategy for revascularization or insulin‐producing or IS assignment (data not shown).
Change in the BRS at 1 Year Compared to Baseline
Among patients with a BRS of 2 and 3 at baseline, the BRS fell in 81% and 68% of patients, respectively, in the first year of the study to levels of 0 or 1. A change from low levels to higher levels was less common; only 6% and 13% of patients with a baseline BRS of 0 and 1, respectively, increased to a score of 2 at 1 year (Table 7). The change in the BRS from baseline to 1 year was not associated with the revascularization versus medical therapy randomization, but its association with glycemic control randomization showed statistically significant difference (Tables 8 and 9).
Association Between the BRS at 1 Year and CVD Events
The reclassified BRS derived at 1 year after randomization among the 1304 survivors significantly predicted risk of all‐cause mortality. Those who had a BRS of 2 to 3 at 1 year had a 4‐year mortality rate of 21.1% compared with a 7.7% in those with a BRS of 1 and 7.1% with a BRS of 0 (P<0.001). Similarly, the 4‐year death/MI event rate was 30.3% in those with a BRS of 2 to 3 at 1 year as compared with 14.4% for those with a BRS of 1 and 14.1% with a BRS of 0 (P<0.0001; Figure 5).
Association of the Change in BRS at 1 Year With Risk of Incident Outcomes
Among patients with a baseline BRS of 2 to 3, 10.1% suffered death or MI at 1 year. In the survivors at 1 year, those who had a lowering of their BRS from 2 to 3 to 0 to 1 had significantly lower 4‐year death/MI rate compared with those whose risk score remained unchanged (19% versus 34.2%; P=0.0025; Figure 6).
In patients with diabetes mellitus and stable CAD enrolled in the BARI 2D study, a BRS comprised of markers of inflammation (CRP), thrombosis (FDP), and cell stress (HSP‐70) identifies a subgroup of CAD patients at very high near‐term risk of incident MI and death. The BRS was additive to the clinical, demographic, and angiographic features. Thus, when BRS was added to the baseline model, it was associated with significant improvement in c‐statistic and risk reclassification metrics. These results confirm those previously obtained in a totally independent cohort of CAD patients.5
Additionally, we found that following aggressive medical and lifestyle intervention, all biomarkers and the BRS were modifiable, such that 78% of subjects with a BRS of 2 or 3 demonstrated a decrease to 0 to 1 after 1 year. These reductions were associated with improvements in CVD risk factors, including the lipid profile, BP, and HbA1C, but not associated with either assignment to revascularization or insulin‐providing compared to IS strategies.
Intriguingly, the decline in the BRS was associated with improved future prognosis. Thus, the observed 4‐year death/MI rates were 45% lower in those who started with a high BRS and had a reduction in their BRS, compared with those in whom the BRS remained high. This suggests that repeat measurements of the BRS accurately reflect time‐related alteration of risk and might be used to calibrate therapy. Furthermore, the almost one quarter of subjects with low BRS have a very low risk of adverse CVD events and a very low probability of increasing their BRS over the first year of participation in the BARI 2D study. The findings of this investigation lend major support to the importance of a strategy for identifying the “vulnerable patient.”9
Vulnerability of atherosclerotic plaques involves activation of several pathways that synergistically promote phenotypic changes that precipitate plaque rupture and consequent acute coronary events. Traditional risk models, such as the Framingham risk score, fail to predict risk in those with established CVD.4 We found that the aggregate of 3 biomarkers—CRP, FDP, and HSP‐70—each individually representing different biologic pathways (but not exclusively), independently predict risk of incident MI/death in CAD patients both at baseline and after 1 year.5 Although studies have identified high‐risk subgroups using single biomarkers, they have failed to identify patients with the extremely high or low near‐term risk as can be achieved using our aggregate biomarker strategy.10, 11
Elevated CRP levels, reflecting activation of the inflammatory pathway, has been shown to predict incident CVD events.10, 11 HSP‐70, a marker of cell stress, is an intracellular protein within the family of heat shock proteins that aid in the cellular response to acute stress.12 However, its relationship with CAD has been controversial.13 Whereas lower levels are associated with long‐term development of CAD,13 higher levels are associated with higher risk of plaque rupture and incident future outcomes.5 The association of HSP‐70 with future outcomes we found in this study is novel and validates our previous findings.5 Ongoing fibrin/fibrinogen degradation increases FDP levels, which measure D‐dimer, fragments D and E, and additional intermediate products of fibrin degradation. Elevation of FDP is indicative of a prothrombotic milieu and predicts incident CVD events in patients with peripheral vascular disease.14
A previous BARI 2D analysis demonstrated reductions in CRP, fibrinogen, and fibrinopeptide‐A after 1 year.15 Other studies have also demonstrated that inflammatory markers are modifiable with aggressive medical therapy, including statin regimens,16, 17 and that the magnitude of benefit from statin therapy is associated with the magnitude of reduction of CRP levels.18, 19, 20, 21, 22 In the BARI 2D trial, baseline levels of CRP, d‐dimer, but not fibrinogen, predicted risk of death.15 The present study extends the previous observations for CRP by incorporating FDP and HSP‐70 measurements and, most important, by calculating an aggregate of these 3 biomarkers into a BRS.
We cannot definitely conclude that the improved 1‐year BRS was caused by the aggressive medical and lifestyle interventions in the BARI 2D study. Also, we were able to measure biomarkers on fewer subjects after 1 year because of missing sample collection and thus cannot be certain that patient selection factors played a role in our results.
An aggregate risk score based on serum levels of CRP, FDP, and HSP‐70 significantly predicts the risk of death and MI in patients with diabetes mellitus and stable CAD and improves risk reclassification. Importantly, the BRS is modifiable and is significantly reduced with aggressive medical and lifestyle intervention. A decline in the BRS is associated with a significant improvement in risk, whereas a persistent elevation is associated with persistent high risk. Thus, serial measurements of the BRS may be used by physicians to titrate therapy and provide continuing information on changing risk in an individual patient, thereby permitting individualized tailoring of therapeutic strategies on an ongoing basis.
Sources of Funding
Ghasemzadeh's research fellowship in preventive cardiology was supported by the Katz Family Foundation Preventive Cardiology Grant, Atlanta, GA. Sample measurements were conducted by FirstMark, Division of GenWay Biotech Inc (San Diego, CA). This study was funded by GenWay Biotech Inc. The Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) is funded by the National Heart, Lung, and Blood Institute and the National Institute of Diabetes and Digestive and Kidney Diseases (U01 HL061744, U01 HL061746, U01 HL061748, U01 HL063804, and R21 HL121495). BARI 2D receives significant supplemental funding provided by GlaxoSmithKline (Collegeville, PA), Lantheus Medical Imaging, Inc (formerly Bristol‐Myers Squibb Medical Imaging, Inc; North Billerica, MA), Astellas Pharma US, Inc (Deerfield, IL), Merck & Co, Inc (Whitehouse Station, NJ), Abbott Laboratories, Inc (Abbott Park, IL), and Pfizer Inc (New York, NY). Generous support is given by Abbott Laboratories Ltd, MediSense Products (Mississauga, Ontario, Canada), Bayer Diagnostics (Tarrytown, NY), Becton, Dickinson and Company (Franklin Lakes, NJ), J. R. Carlson Labs (Arlington Heights, IL), Centocor, Inc (Malvern, PA), Eli Lilly and Company (Indianapolis, IN), LipoScience, Inc (Raleigh, NC), Merck Sante (Lyon, France), Novartis Pharmaceuticals Corporation (East Hanover, NJ), and Novo Nordisk, Inc (Princeton, NJ).
Epstein and Quyyumi are equity holders in Firstmark, GenWay Biotech and receive consulting fees. Sikora is an equity holder in Firstmark, GenWay Biotech. Brooks reports significant research grants from Firstmark and Genway Biotech, Inc. The remaining authors have no disclosures to report.
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