Arrhythmia/Electrophysiology

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1 Arrhythmia/Electrophysiology Cardiac Biomarkers Are Associated With an Increased Risk of Stroke and Death in Patients With Atrial Fibrillation A Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY) Substudy Ziad Hijazi, MD; Jonas Oldgren, MD, PhD; Ulrika Andersson, MSc; Stuart J. Connolly, MD; Michael D. Ezekowitz, MB, ChB; Stefan H. Hohnloser, MD; Paul A. Reilly, PhD; Dragos Vinereanu, MD, PhD; Agneta Siegbahn, MD, PhD; Salim Yusuf, MD, PhD; Lars Wallentin, MD, PhD Background Cardiac biomarkers are strong predictors of adverse outcomes in several patient populations. We evaluated the prevalence of elevated troponin I and N-terminal pro-b-type natriuretic peptide (NT-proBNP) and their association to cardiovascular events in atrial fibrillation (AF) patients in the Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY) trial. Methods and Results Biomarkers at randomization were analyzed in 6189 patients. Outcomes were evaluated by Cox proportional hazards models adjusting for established cardiovascular risk factors and the CHADS 2 and CHA 2 DS 2 -VASc risk scores. Patients were stratified based on troponin I concentrations: g/l, n 2663; to g/l, n 2006; to g/l, n 1023; g/l, n 497; and on NT-proBNP concentration quartiles: 387; 387 to 800; 801 to 1402; 1402 ng/l. Rates of stroke were independently related to levels of troponin I with 2.09%/year in the highest and 0.84%/year in the lowest troponin I group (hazard ratio [HR], 1.99 [95% CI, ]; P ), and to NT-proBNP with 2.30%/year versus 0.92% in the highest versus lowest NT-proBNP quartile groups, (HR, 2.40 [95% CI, ]; P ). Vascular mortality was also independently related to biomarker levels with 6.56%/year in the highest and 1.04%/year the lowest troponin I group (HR, 4.38 [95% CI, ]; P ), and 5.00%/year in the highest and 0.61%/year in the lowest NT-proBNP quartile groups (HR, 6.73 [ ]; P ). Biomarkers increased the C-statistic from 0.68 to 0.72, P , for a composite of thromboembolic events. Conclusions Elevations of troponin I and NT-proBNP are common in patients with AF and independently related to increased risks of stroke and mortality. Cardiac biomarkers seem useful for improving risk prediction in AF beyond currently used clinical variables. Clinical Trial Registration URL: Unique identifier: NCT (Circulation. 2012;125: ) Key Words: atrial fibrillation cardiac biomarkers natriuretic peptide risk prediction troponin The prevalence of atrial fibrillation (AF) is increasing and is projected to reach epidemic proportions in coming decades. 1 AF is associated with a 5-fold increase in the rate of ischemic stroke and doubled total mortality. 2 Strategies for identifying patients at risk for thromboembolism are commonly based on clinical variables, eg, congestive heart failure, hypertension, age, diabetes mellitus, and prior stroke or transient ischemic attack (TIA) in the widely used CHADS 2 risk score. 3 So far, no biochemical marker has been shown to provide incremental information. Cardiac troponin, an intracellular protein involved in heart muscle contraction, is an established biochemical marker of myocardial cell Received April 20, 2011; accepted February 16, From the Uppsala Clinical Research Center and Department of Medical Sciences, Cardiology, Uppsala University, Uppsala, Sweden (Z.H., J.O., L.W.); Uppsala Clinical Research Center, Uppsala University, Uppsala, Sweden (U.A.); Population Health Research Institute, Hamilton, Canada (S.J.C., S.Y.); Lankenau Institute for Medical Research and the Heart Center, Wynnewood, PA (M.D.E.); Department of Cardiology, J.W. Goethe University, Frankfurt, Germany (S.H.H.); Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT (P.A.R.); Department of Cardiology, University Hospital of Bucharest, Bucharest, Romania (D.V.); and Uppsala Clinical Research Center and Department of Medical Sciences, Clinical Chemistry, Uppsala University, Uppsala, Sweden (A.S.). The online-only Data Supplement is available with this article at /-/DC1. Correspondence to Ziad Hijazi, MD, Uppsala Clinical Research Center, Dag Hammarskjölds väg 14B, 1st floor, SE Uppsala, Sweden. Ziad.hijazi@ucr.uu.se 2012 American Heart Association, Inc. Circulation is available at DOI: /CIRCULATIONAHA

2 1606 Circulation April 3, 2012 damage. B-type natriuretic peptide (BNP), a neurohormone secreted from the cardiac ventricles, is a recognized marker of myocardial wall tension, and its inactive part, N-terminal fragment (N-terminal pro-b-type natriuretic peptide [NTproBNP]]), as well. 4 Elevated levels of troponin and NTproBNP have repeatedly been demonstrated as important markers of increased mortality and morbidity in acute coronary syndromes, 5,6 stable coronary artery disease, 7,8, congestive heart failure, 9,10 and even in general community-based populations. 11,12 The prevalence and clinical significance of elevated cardiac troponin I and NT-proBNP in nonvalvular AF patients at risk for stroke is unknown. Clinical Perspective on p 1616 The Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY) trial recently demonstrated the superiority of dabigatran versus warfarin for stroke prevention in AF patients. 13 In this prospectively designed biomarker substudy, we investigated the prevalence of elevated troponin I and NT-proBNP and their association to cardiovascular events in a representative subgroup constituting one-third of the RE-LY cohort. Methods Study Population and Trial Design The study organization, trial design, patient characteristics and outcomes of the RE-LY study have been published previously. 13 In brief, RE-LY was a prospective, multicenter, randomized trial comparing 2 blinded doses of dabigatran with open-label warfarin for a minimum of 12 months in patients. Inclusion criteria were documented AF and at least one of the following risk factors for stroke: previous stroke or TIA; congestive heart failure or reduced left ventricular ejection fraction ( 40%); at least 75 years of age; or at least 65 years of age with diabetes mellitus, hypertension, or coronary artery disease. Exclusion criteria included severe heart valve disorder, recent stroke, increased risk of hemorrhage, creatinine clearance 30 ml/min, or active liver disease. The 6189 (of 18113) patients in the present study represented 446 of 951 sites in 38 of 44 countries in the RE-LY trial. The primary efficacy outcome in the study was fatal and nonfatal stroke (ischemic, hemorrhagic, or unspecified) or systemic embolism, and the secondary outcomes were total mortality, vascular (including hemorrhagic) mortality, nonvascular mortality, and a composite thromboembolic end point consisting of ischemic stroke, systemic embolism, myocardial infarction, pulmonary embolism, and vascular mortality (excluding hemorrhagic death). The primary safety outcome was major bleeding. Median follow-up was 2.0 years for the main trial and 2.2 years for the substudy population. Outcomes were assessed by study visits scheduled at 3-month intervals during the first year and 4-month intervals thereafter. Each event was classified by 2 independent adjudicators from an international team, blinded to treatment assignments. Definition of the outcomes has been described previously. 13 Blood Sampling Venous blood was drawn at randomization, before initiation of study treatment, with the use of a 21/22 gauge needle into Vacutainer tubes containing EDTA. The blood was centrifuged within 30 minutes at 2000g for 10 minutes. The tubes were thereafter immediately frozen at 20 C or colder. Aliquots were stored at 70 C to allow batch analysis. Laboratory Methods All plasma samples were centrally analyzed in Uppsala Clinical Research Center laboratory, Sweden. Troponin I was analyzed with the Access AccuTnI assay (Beckman Coulter, Inc, Fullerton, CA), a 2-site immunoenzymatic (sandwich) immunoassay. The lower limit of detection with this assay is g/l with g/l as the lowest concentration measurable with a coefficient of variation of 10% and 0.02 g/l as the 99th percentile upper reference limit (URL) for subjects aged 60 years 14 and 0.04 g/l as the 99th percentile URL regardless of age. 15 With present instrument calibration all troponin I concentrations g/l are reported as g/l and considered undetectable; levels are regarded as detectable and reported with 2 significant figures; levels g/l are considered elevated. NT-proBNP was analyzed by using the Sandwich Immunoassay, Elecsys, Roche Diagnostics. The analytic range extends from 20 to ng/l according to the manufacturer. The upper reference level (97.5th percentile) in men and woman aged 40 to 65 years is 184 and 268 ng/l, respectively, and age 66 to 76 years, 269 and 391 ng/l, respectively. 16 The lowest concentration measurable with a coefficient of variation 10% is 30 ng/l. 17 Statistical Analysis The sample size for the substudy was prospectively estimated at 5744 patients, based on an expected event rate of 1.6%/year (corresponding to a total event rate of 3.2%), and the assumptions that the biomarker was related to outcome in the following manner: the total event rate for the lower quartile is 1.6%, for the middle quartiles 3.2%, and for the upper quartile 6.4%. A 2-sided test of the null hypothesis of no difference in event rate between the lower quartile and a middle quartile requires 1436 patients per group when the significance level is 5% and the power is 80%. The number of samples in total was thus calculated to , to account for missing information blood samples, was planned from 6200 patients of the patients in the RE-LY study. The final number of samples in the present analysis was 6189, composed of 2561 patients participating in a comprehensive RE-LY biomarker substudy program and 3628 randomly selected patients with cardiac biomarkers (troponin I and NT-proBNP) obtained at randomization in the main RE-LY study. Patients were grouped according to quartiles of NT-proBNP levels, 387 ng/l (n 1547), 387 to 800 ng/l (n 1547), 801 to 1402 ng/l (n 1544), and 1402 ng/l (n 1551). Troponin I results were, as expected, extremely skewed, and patients were in the present study grouped according to cutoff levels based on results from previous studies, instead of 4 equally sized groups. Troponin I levels were undetectable ( g/l) in 2663 (43.0%) patients, 2006 (32.4%) patients had detectable troponin I levels to g/l, ie, up to the 99th percentile for apparently healthy individuals 60 years age. 18 One thousand twenty-three (16.5%) patients had slightly elevated troponin I levels to g/l, and 497 (8.0%) had clearly elevated troponin I levels g/l, ie, above the 99th percentile URL for the troponin I assay regardless of age. 15 Demographics and baseline characteristics were summarized for the troponin I and NT-proBNP level groups with the use of frequencies for categorical variables, and median and 25th and 75th quartiles for continuous variables. Because of the low numbers of patients in the CHADS 2 scores, 0, 4, 5, and 6 patients were grouped in CHADS 2 classes of 0 to 1, 2, and 3. For tests of differences among groups, the 2 test was used for categorical variables, and Kruskal-Wallis test was used for continuous variables. The risk of event is reported as percentage per year, which was calculated by dividing the total number of patients with events by the total number of patient-years of follow-up. Cumulative hazard plots were used to illustrate the timing of events. The relations between levels of troponin I and NT-proBNP at randomization and events were investigated by using Cox proportional hazards regression. Three different models were used. Model A was adjusted for study treatment, use of (prestudy) anticoagulant treatment at randomization and established risk factors for cardiovascular disease (age, sex, body mass index, smoking status, sitting systolic blood pressure, sitting heart rate, AF duration, AF type, creatinine clearance, diabetes, coronary artery disease, previous stroke/systemic embolism/tia, heart failure, hypertension, treatment at randomization with aspirin, angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers, and statins). Model B was only adjusted for study treatment

3 Hijazi et al Cardiac Biomarkers in Atrial Fibrillation 1607 and the CHADS 2 score (1 point for each of congestive heart failure, hypertension, age 75 years, diabetes mellitus, and 2 points for previous stroke/tia) by using 6 CHADS 2 classes (CHADS 2 score of 5 and 6 combined). Model C was only adjusted for study treatment and the CHA 2 DS 2 -VASc score (1 point for each of congestive heart failure, hypertension, age 65 but 75 years, diabetes mellitus, prior stroke or TIA, vascular disease, and sex category; and 2 points for each of age of 75 years and prior stroke or TIA) by using 7 classes (scores of 0 and 1 combined, and scores of 7, 8, and 9 combined). The 2 cardiac biomarkers were included separately and jointly, as well, in the models. The results of hazard ratios for model A are presented in Results and tables. Models B and C yielded similar hazard ratios (see online-only Data Supplement Tables I and II). The effects of randomized treatment on outcome in relation to level of biomarker was evaluated with Cox proportional hazards model with treatment group, biomarker, and their interaction as dependent variables. The proportional hazards assumption with respect to the cardiac biomarkers was assessed by visual inspection of log-cumulative hazard plots and by extending the Cox model with a time-by-biomarker interaction factor. In addition, sensitivity analyses using logistic regression were performed (results not shown). The increased discriminative values of troponin I and NT-proBNP were investigated by estimating the difference in C-statistics between models with and without respective cardiac biomarker 19 and also the integrated discrimination improvement measure (IDI) as described by Pencina et al. 20 In these analyses, the occurrence/nonoccurrence of stroke or systemic embolism and composite thromboembolic events, respectively, during the follow-up period was used as a binary response, and the C value will be the same as the area under the ROC curve. The relative IDI was calculated to facilitate interpretation of the IDI. 21 A probability value of 0.05 from 2-sided tests was considered to indicate statistical significance. The statistical software package SAS, version 9.2 for Windows (SAS institute, Cary, NC) was used for all analyses. Results Baseline Characteristics in Relation to Levels of Cardiac Biomarkers Patient characteristics are summarized in Table 1. Troponin I levels ranged from to 7.1 g/l, with a median of 0.011, 25th and 75th percentile values of and g/l. Detectable levels of troponin I ( g/l) were found in 3526 (57.0%) and elevated levels of troponin I ( g/l) in 1520 (24.6%) patients (Table 1). Several patient characteristics, eg, higher age, previous myocardial infarction, history of congestive heart failure, AF rhythm at randomization (at the time of blood sampling), and lower creatinine clearance were significantly associated with higher troponin I levels, all P (Table 1). NT-proBNP levels ranged from 5 to ng/l, with a median of 801, 25th and 75th percentile values of 387 and 1403 ng/l. Several patient characteristics, most prominently age, AF rhythm at randomization, history of congestive heart failure, and lower creatinine clearance, were significantly associated with higher NT-proBNP levels, all P (Table 1). The proportion of patients with CHADS 2 risk scores 0 to 1 was highest (37.3% and 38.6%, respectively) in groups with the lowest levels of cardiac biomarkers (troponin I g/l and NT-proBNP 387 ng/l) and lowest (25.4% and 25.6%, respectively) in groups with the highest levels of cardiac biomarkers (troponin I g/l and NT-proBNP 1402 ng/l, respectively). The opposite applied to the proportion of patients with a CHADS 2 scores 3, which was lowest (27.8% and 25.5%, respectively) in groups with undetectable troponin I ( g/l) and low NT-proBNP 387 ng/l, and highest (37.6% and 38.6%, respectively) in the groups with highest troponin I ( 0.040) g/l and highest NT-proBNP ( 1402 ng/l), respectively (Table 1). Stroke or Systemic Embolism During the median of 2.2 years follow-up, there were 183 events of stroke or systemic embolism. The annual rates of stroke or systemic embolism were lowest, 0.84%, in the group with undetectable troponin I, in comparison with 2.09% (HR, 1.99; 95% CI, ) in the highest troponin I group (P ) (Figure 1A, Table 2). In relation to NT-proBNP, the annual rates of stroke or systemic embolism were lowest, 0.92%, in the quartile group with low NT-proBNP, in comparison with 2.30% (HR, 2.40; 95% CI, ) in the highest NT-proBNP (P ) (Figure 1B, Table 2). The effects of troponin I remained significant (P ) after adjustment for NTproBNP in the multivariable model, and for NT-proBNP (P ) adjusted for troponin I, as well. Mortality During the follow up 450 patients died, 297 of which died of vascular causes. In the group with undetectable troponin I, annual vascular death rate was 1.04% in comparison with 6.56% (HR, 4.38; 95% CI, ) in the highest troponin I group (P ) (Figure 2A, Table 2). In relation to NT-proBNP, the annual rates of vascular death were 0.61% in the lowest NT-proBNP group, in comparison with 5.00% (HR, 6.73; 95% CI, ) in the highest NT-proBNP group (P ) (Figure 2B, Table 2). The effects of troponin I remained significant (P ) after adding NT-proBNP to the multivariable model, and for NT-proBNP (P ) adjusted for troponin I, as well. One hundred fifty-three patients died of nonvascular causes. The rate of nonvascular mortality was higher among patients with elevated troponin I levels, P , but without a gradual increase by troponin I levels (Table 2). There was no significant association between NT-proBNP levels and nonvascular death. Myocardial Infarction There were 103 myocardial infarctions during follow-up. The annual rates were 0.49% in the group with undetectable troponin I and 1.69% (HR 3.04; 95% CI ) in the highest troponin I group (P ) (Table 2). The association of troponin I and myocardial infarctions remained significant after adding NT-proBNP to the model. There was no significant relation between NT-proBNP levels and myocardial infarctions. Composite Thromboembolic End Point There were 482 composite thromboembolic events (ischemic stroke, systemic embolism, pulmonary embolism, myocardial infarction, and vascular death excluding hemorrhagic death). In the group with undetectable troponin, the annual rates of the composite thromboembolic end point were 2.00% in comparison with 8.85% (HR, 3.43; 95% CI, ) in the highest troponin I group (P ) (Table 2). Concerning NT-proBNP, the annual rates of the composite thromboem-

4 1608 Circulation April 3, 2012 Table 1. Baseline Characteristics According to Troponin I and NT-proBNP group Troponin I Groups All g/l g/l g/l g/l P Value* No. in substudy Follow-up time, median (25th, 75th pct), y 2.20 (1.78, 2.52) 2.22 (1.88, 2.54) 2.25 (1.74, 2.56) 2.08 (1.67, 2.40) 2.10 (1.71, 2.46) Region West Europe North America 3512 (56.7) 1553 (58.3) 1175 (58.6) 529 (51.7) 255 (51.3) Age, median (25th, 75th pct), y 72.0 (67.0, 77.0) 71.0 (66.0, 76.0) 73.0 (67.0, 78.0) 73.0 (67.0, 78.0) 72.0 (65.0, 78.0) Body mass index, median (25th, 75th pct), kg/m (25.3, 31.6) 28.2 (25.6, 31.8) 28.1 (25.2, 31.5) 28.0 (25.1, 31.9) 27.6 (24.8, 31.2) Male sex, n (%) 3944 (63.7) 1631 (61.2) 1288 (64.2) 685 (67.0) 340 (68.4) Current smoker, n (%) 483 (7.8) 202 (7.6) 157 (7.8) 79 (7.7) 45 (9.1) Systolic blood pressure, median (25th, 75th (120.0, 144.0) (120.0, 142.0) (120.0, 145.0) (120.0, 145.0) (120.0, 140.0) pct), mm Hg Heart rate median (25th, 75th pct) 72.0 (62.0, 82.0) 72.0 (63.0, 82.0) 72.0 (62.0, 82.0) 72.0 (63.0, 82.0) 72.0 (62.5, 81.0) AF rhythm at baseline (%) 4519 (73.2) 1867 (70.3) 1490 (74.5) 783 (76.8) 379 (76.4) Type of AF, n (%) Paroxysmal 1848 (29.9) 871 (32.7) 580 (28.9) 259 (25.3) 138 (27.8) Persistent 1609 (26.0) 726 (27.3) 474 (23.6) 285 (27.9) 124 (24.9) Permanent 2731 (44.1) 1066 (40.0) 951 (47.4) 479 (46.8) 235 (47.3) AF duration, n (%) 3 mo 1794 (29.0) 756 (28.4) 562 (28.0) 316 (30.9) 160 (32.2) mo 2 y 1441 (23.3) 646 (24.3) 461 (23.0) 220 (21.5) 114 (22.9) 2 y 2954 (47.7) 1261 (47.4) 983 (49.0) 487 (47.6) 223 (44.9) Heart failure, n (%) 1859 (30.0) 618 (23.2) 588 (29.3) 436 (42.6) 217 (43.7) Hypertension, n (%) 4852 (78.4) 2112 (79.3) 1575 (78.5) 791 (77.3) 374 (75.3) Age 75 y, n (%) 2356 (38.1) 883 (33.2) 838 (41.8) 426 (41.6) 209 (42.1) Diabetes mellitus, n (%) 1322 (21.4) 519 (19.5) 447 (22.3) 239 (23.4) 117 (23.5) Previous stroke/tia, n (%) 1216 (19.6) 538 (20.2) 387 (19.3) 183 (17.9) 108 (21.7) CHADS 2 score, n (%) (32.7) 992 (37.3) 636 (31.7) 271 (26.5) 126 (25.4) (35.5) 932 (35.0) 710 (35.4) 371 (36.3) 184 (37.0) (31.8) 739 (27.8) 660 (32.9) 381(37.2) 187 (37.6) Prior myocardial infarction, n (%) 1078 (17.4) 330 (12.4) 375 (18.7) 241 (23.6) 132 (26.6) Coronary artery disease, n (%) 1540 (24.9) 578 (21.7) 516 (25.7) 293 (28.6) 153 (30.8) CrCL at baseline, ml/min (25th-75th pct) 69.0 (54.2, 87.1) 73.4 (58.1, 90.3) 68.1 (53.6, 85.4) 64.3 (50.1, 82.0) 63.1 (49.3, 81.1) Medications at baseline, n (%) Aspirin 2214 (35.8) 900 (33.8) 722 (36.0) 394 (38.5) 198 (39.8) blocker 4102 (66.3) 1762 (66.2) 1352 (67.4) 656 (64.1) 332 (66.8) ACE inhibitor and/or ARB 4313 (69.7) 1761 (66.1) 1447 (72.1) 737 (72.0) 368 (74.0) Statin 2670 (43.1) 1198 (45.0) 851(42.4) 409 (40.0) 212 (42.7) Amiodarone 700 (11.3) 296 (11.1) 214 (10.7) 118 (11.5) 72 (14.5) NT-proBNP indicates N-terminal pro-b-type natriuretic peptide; ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker; CrCL, creatine clearance; AF, atrial fibrillation; pct, percentile; and TIA, transient ischemic attack. *The P value is based on Kruskal-Wallis test for continuous variables and the 2 test for categorical variables. bolic end point were 1.78% in the lowest quartile group of NT-proBNP, in comparison with 6.76% (HR, 3.55; 95% CI, ) in the highest NT-proBNP group (P ) (Table 2). The effects of troponin I remained significant (P ) after adding NT-proBNP to the multivariable model, and for NT-proBNP (P ) adjusted for effects of troponin I, as well. Major Bleeding There were 334 major bleeds. In the group with undetectable troponin I, the annual rate of major bleed was 1.72% in comparison with 4.38% (HR, 2.01; 95% CI, ) in the highest troponin I group (P ) (Table 2). There was no significant association between NT-proBNP levels and major bleeding. Cardiac Biomarkers Levels in Relation to CHADS 2 Score Figure 3A illustrates the annual rates of the composite thromboembolic end point according to troponin I levels and CHADS 2 score. The pattern of gradually higher rates of the composite thromboembolic end point concomitant with higher troponin I levels was consistent in all CHADS 2 scores, including the group with CHADS 2 scores 0 to 1. The highest annual rates of the composite end point, 11.4%, were found in the group with CHADS 2 3 and highest troponin I ( g/l) compared with the lowest annual risk of 1.48%, in the group with CHADS 2 0 to 1 and undetectable troponin I ( g/l). Annual rates of the composite thromboembolic end point according to NT-proBNP levels and CHADS 2 score are

5 Hijazi et al Cardiac Biomarkers in Atrial Fibrillation 1609 Table 1. Continued NT-proBNP 387 ng/l ng/l ng/l 1402 ng/l P Value* (1.78, 2.42) 2.23 (1.79, 2.54) 2.23 (1.84, 2.57) 2.19 (1.71, 2.50) 872 (56.4) 895 (57.9) 887 (57.4) 858 (55.3) 70.0 (65.0, 75.0) 71.0 (66.0, 76.0) 73.0 (67.0, 78.0) 74.0 (68.0, 79.0) (25.6, 32.0) 28.4 (25.7, 32.0) 27.9 (25.2, 31.6) 27.5 (24.7, 30.8) (62.4) 1056 (68.3) 977 (63.3) 946 (61.0) (7.8) 132 (8.5) 119 (7.7) 111 (7.2) (122.0, 145.0) (120.0, 144.0) (120.0, 144.0) (120.0, 140.0) (60.0, 76.0) 73.0 (64.0, 82.0) 75.0 (65.0, 84.0) 75.0 (66.0, 86.0) (32.6) 1233 (80.0) 1379 (89.4) 1405 (90.8) (61.7) 373 (24.1) 257 (16.7) 263 (17.0) (21.0) 401 (25.9) 447 (29.0) 436 (28.1) 267 (17.3) 773 (50.0) 839 (54.4) 852 (54.9) 529 (34.2) 396 (25.6) 392 (25.4) 477 (30.8) (25.4) 331 (21.4) 349 (22.6) 368 (23.7) 625 (40.4) 820 (53.0) 803 (52.0) 706 (45.5) 292 (18.9) 422 (27.3) 473 (30.6) 672 (43.3) (82.3) 1207 (78.0) 1193 (77.3) 1179 (76.0) (27.3) 538 (34.8) 660 (42.7) 736 (47.5) (23.2) 335 (21.7) 332 (21.5) 296 (19.1) (19.1) 299 (19.3) 293 (19.0) 328 (21.1) (38.6) 546 (35.3) 485 (31.4) 397 (25.6) (35.9) 548 (35.4) 537 (34.8) 556 (35.8) 394 (25.5) 453 (29.3) 522 (33.8) 598 (38.6) 190 (12.3) 214 (13.8) 296 (19.2) 378 (24.4) (20.4) 354 (22.9) 414 (26.8) 457 (29.5) (61.1, 93.9) 73.2 (57.8, 91.1) 68.3 (54.6, 85.2) 59.0 (46.1, 74.8) (37.9) 519 (33.5) 531 (34.4) 577 (37.2) (59.6) 968 (62.6) 1070 (69.3) 1142 (73.6) (68.1) 1067 (69.0) 1059 (68.6) 1134 (73.1) (45.5) 667 (43.1) 668 (43.3) 631 (40.7) (18.0) 173 (11.2) 122 (7.9) 127 (8.2) illustrated in Figure 3B. The general trend of increasing rates of the composite thromboembolic end point with increasing NT-proBNP levels was consistent in all CHADS 2 scores, including the group with CHADS 2 scores 0 to 1. The highest annual rate of the composite end point, 8.99%, was found in the group with CHADS 2 3 and highest NTproBNP ( 1402 ng/l) compared with the lowest yearly risk of 1.55%, in the group with CHADS 2 0 to 1 and lowest NT-proBNP ( 387 ng/l). Figure 3C illustrates the annual rates of the composite thromboembolic end point in relation to cardiac biomarkers only. The pattern of gradually higher rates of the composite thromboembolic end point is apparent in all biomarker groups, with the highest annual rate of the composite end point, 12.0%, found in patients with highest levels of troponin I ( g/l) in combination with highest levels of NT-proBNP ( 1402 ng/l). Study Treatment and Subgroups For all the above outcomes there were no significant interactions between troponin I or NT-proBNP groups and effects of study treatment with warfarin or dabigatran 110 mg or 150 mg. Exploratory analyses of relations between troponin or NT-proBNP levels and outcomes within subgroups defined by age ( 75; 75 years), sex, regions (North America and Western Europe versus others, Asian versus non-asian countries), CHADS 2 score (0 1; 2; 3 6), prevalent coronary artery disease versus no coronary artery disease, or prevalent

6 1610 Circulation April 3, 2012 A µg/l µg/l µg/l <0.010 µg/l Cumulative Hazard Rate Numbers at Risk <0.010 ug/l ug/l ug/l ug/l 497 B >1402 ng/l ng/l ng/l <387 ng/l Months Figure 1. Cumulative hazard rates for stroke or systemic embolism, according to troponin I levels at randomization (A) and NT-proBNP levels at randomization (B). Cumulative Hazard Rate Numbers at Risk <387 ng/l ng/l ng/l >1402 ng/l Months cardiovascular disease (prior stroke, prevalent congestive heart failure, and/or coronary artery disease) versus no cardiovascular disease, yielded similar results as in the total material with no significant interactions (data not shown). Predictive Ability of Cardiac Biomarkers in AF for Thromboembolic Events In this cohort the C-statistic for stroke and systemic embolism was for troponin I, for NT-proBNP, and when combining the cardiac biomarkers (Table 3). A model based on the CHADS 2 score (model B) yielded a C-statistic of The separate addition of each cardiac biomarker improved the predictive model significantly, and even further when adding both cardiac biomarkers to a model simultaneously (Table 3). The IDI was significant for all models when adding cardiac biomarkers, separately or combined, with relative IDI ranging from 24% to 124% depending on the model. For the composite thromboembolic end point, the C-statistic was for troponin I, for NT-proBNP, and when combining troponin I and NT-proBNP (Table 3). A model based on the CHADS 2 score yielded a C-statistic of The separate addition of a cardiac biomarker improved the predictive model significantly, and even further when adding both cardiac biomarkers simultaneously to the model (Table 3). The IDI was significant for all models when adding cardiac biomarkers, separately or combined, with relative IDI ranging from 41% to 425% depending on the model. The improvements in C-statistic and IDI in relation to the CHA 2 DS 2 -VASc score (model C) were similar to the improvements with the CHADS 2 score (model B) (Table 3). Discussion The present RE-LY substudy demonstrated a high prevalence of detectable and elevated troponin I and considerably elevated NT-proBNP in patients with nonvalvular AF and a raised risk of stroke. The degree of troponin I and NTproBNP elevations were both independently related to a

7 Hijazi et al Cardiac Biomarkers in Atrial Fibrillation 1611 Table 2. Cox Proportional Hazards Model With Troponin I and NT-proBNP at Randomization in Relation to Outcomes Outcome Biomarkers Group Stroke and systemic embolism Vascular death Nonvascular death Myocardial infarction Composite thromboembolic outcome Major bleed Troponin I group ( g/l) NT-proBNP quartile group (ng/l) Troponin I group ( g/l) NT-proBNP quartile group (ng/l) Troponin I group ( g/l) NT-proBNP quartile group (ng/l) Troponin I group ( g/l) NT-proBNP quartile group (ng/l) Troponin I group ( g/l) NT-proBNP quartile group (ng/l) Troponin I group ( g/l) NT-proBNP quartile group (ng/l) Total Number of Events, n (%/y) HR (95% CI) P Value Effect of Biomarker Level Addition of Cardiac Biomarker* HR (95% CI) P Value Effect of Biomarker Level (0.84) (1.72) 1.79 ( ) 1.71 ( ) (1.99) 1.97 ( ) 1.76 ( ) (2.09) 1.99 ( ) 1.68 ( ) (0.92) (1.22) 1.35 ( ) 1.29 ( ) (1.22) 1.31 ( ) 1.21 ( ) (2.30) 2.40 ( ) 2.09 ( ) (1.04) (2.40) 1.94 ( ) 1.75 ( ) (3.39) 2.31 ( ) 1.88 ( ) (6.56) 4.38 ( ) 3.20 ( ) (0.61) (1.40) 2.44 ( ) 2.23 ( ) (2.19) 3.71 ( ) 3.19 ( ) (5.00) 6.73 ( ) 5.07 ( ) (0.86) (1.22) 1.19 ( ) 1.18 ( ) (2.04) 1.92 ( ) 1.87 ( ) (0.99) 0.89 ( ) 0.83 ( ) (0.83) (1.28) 1.31 ( ) 1.25 ( ) (0.82) 0.79 ( ) 0.73 ( ) (1.79) 1.42 ( ) 1.30 ( ) (0.49) (0.87) 1.59 ( ) 1.56 ( ) (1.02) 1.77 ( ) 1.72 ( ) (1.69) 3.04 ( ) 2.88 ( ) (0.64) (0.85) 1.72 ( ) 1.62 ( ) (0.73) 1.49 ( ) 1.31 ( ) (0.94) 1.93 ( ) 1.53 ( ) (2.00) (3.93) 1.73 ( ) 1.63 ( ) (5.43) 2.11 ( ) 1.83 ( ) (8.85) 3.43 ( ) 2.77 ( ) (1.78) (2.95) 1.78 ( ) 1.66 ( ) (3.40) 2.06 ( ) 1.81 ( ) (6.76) 3.55 ( ) 2.79 ( ) (1.72) (2.94) 1.48 ( ) 1.45 ( ) (3.25) 1.50 ( ) 1.45 ( ) (4.38) 2.01 ( ) 1.89 ( ) (2.03) (2.25) 1.12 ( ) 1.07 ( ) (2.43) 1.16 ( ) 1.07 ( ) (3.58) 1.47 ( ) 1.28 ( ) Six thousand ninety-two patients had values for all covariates and were included in the Cox regressions. Numbers of patients in each group were as follows: troponin I group , group , group , and group For NT-proBNP; Q1 1547, Q2 1547, Q3 1544, and Q NT-proBNP indicates N-terminal pro-b-type natriuretic peptide; and HR, hazard ratio. *Adding NT-proBNP to multivariable analysis of troponin I, and troponin I to multivariable analysis of NT-proBNP, respectively. Stroke, systemic embolism, pulmonary embolism, myocardial infarction, vascular death (excluding hemorrhagic death).

8 1612 Circulation April 3, 2012 A µg/l µg/l µg/l <0.010 µg/l 0.15 Cumulative Hazard Rate Numbers at Risk <0.010 ug/l ug/l ug/l ug/l 497 B >1402 ng/l ng/l ng/l <387 ng/l Months Figure 2. Cumulative hazard rates for vascular death, according to troponin I levels at randomization (A) and NT-proBNP levels at randomization (B) Cumulative Hazard Rate Numbers at Risk <387 ng/l ng/l ng/l >1402 ng/l Months raised risk of stroke or systemic embolism, mortality, and other cardiovascular events. The levels of these biomarkers added prognostic information beyond currently used clinical risk scores, eg, the CHADS 2 or CHA 2 DS 2 -VASc risk scores. Elevation of troponin was initially identified as a sensitive indicator of myocardial damage and myocardial infarction and also as an indicator of raised risk of reinfarction and mortality in patients with acute coronary syndromes. 5,6,22 At a later stage, slight elevations of troponin were observed in a proportion of patients with stable coronary artery disease and also associated with a worse outcome. 8 In advanced heart failure, elevated troponin is associated with progressive left ventricular dysfunction and increased mortality. 9 Recently, more sensitive assays have identified detectable troponin levels also in elderly healthy men, predicting coronary heart disease events and mortality independent of conventional major coronary risk factors. 12 The prognostic value of natriuretic peptides has previously been established for a variety of cardiovascular diseases Even in community-based populations without heart failure, natriuretic peptide levels predict risk of death and cardiovascular events. 11,23 BNP and NT-proBNP are also prognostic of short- and long-term mortality in patients with acute coronary syndromes 24,25 and stable coronary artery disease. 7 Elevated levels of BNP also provide prognostic information in acute decompensated heart failure 26 and in chronic heart failure. 10 The simultaneous use of both cardiac biomarkers has displayed even further improvements in risk stratification in patients with acute coronary syndromes. 27 Our findings in the present RE-LY substudy extend these observations to a novel population by demonstrating the prognostic importance of troponin I and NT-proBNP in patients with AF. At present, risk stratification in AF is based on clinical variables, with the CHADS 2 risk score being the most widely used. Although easy to apply, all clinical risk scores including the novel CHA 2 DS 2 -VASc score have, at best, only a modest discriminating ability for the individual patients, in this respect not very different from CHADS 2 risk score, with C-statistics in

9 Hijazi et al Cardiac Biomarkers in Atrial Fibrillation 1613 Figure 3. Stroke, systemic embolism, pulmonary embolism, myocardial infarction, vascular death (excluding hemorrhagic death) in relation to troponin I levels and CHADS 2 scores (A), NT-proBNP levels and CHADS 2 -scores (B), and troponin I and NT-proBNP levels (C). Total number of patients given in each bar. the range from 0.54 to We demonstrated the additive value of troponin I and NT-proBNP to CHADS 2 score by stratifying the RE-LY biomarker cohort based on cardiac biomarkers and CHADS 2 score (Figure 3A). Within every CHADS 2 score stratum, there was also a further gradation of thromboembolic risk in relation to the troponin I and NTproBNP levels. Even in low-risk patients with a CHADS 2 score of 0 to 1, any increase in troponin I ( g/l) doubled the risk, and highly elevated troponin I levels ( g/l) raised the risk 5-fold, surpassing the annual risk of patients with a CHADS 2 score of 2 and undetectable troponin I levels. The incremental information from NT-proBNP levels was similar, with a 2.5-fold increase in risk when comparing patients with the highest and lowest quartiles of NT-proBNP within the group of patients with CHADS 2 score of 0 to 1 (Figure 3B). The significantly improved C-statistics and IDI support the improvement of risk prediction of thromboembolic events when adding cardiac biomarkers to the CHADS 2 and the CHA 2 DS 2 -VASc risk scores. A CHADS 2 score 2 is currently the generally accepted indication for treatment with oral anticoagulants in AF patients. 29 Therefore, it is noteworthy that, in the present population, a group with low CHADS 2 score of 0 to 1 and elevated levels of any or both of these cardiac biomarkers had a higher annual rate of a composite of thromboembolic events than patients with higher CHADS 2 scores and undetectable troponin I and/or low NT-proBNP levels. Conversely, patients without elevated cardiac biomarkers might have substantially lower risk than perceived by the CHADS 2 score. Similar findings were shown in relation to the novel CHA 2 DS 2 -VASc risk score. Patients with high CHADS 2 or CHA 2 DS 2 -VASc risk score and elevated cardiac biomarkers remain at high risk for thromboembolic events despite preventive treatment with effective oral anticoagulants. Such patients might be considered for intensified pharmacological treatment with angiotensin-converting enzyme inhibitors, angiotensin receptor blockers or statins, ablation therapy, left atrial appendage closure devices, left atrial volume reduction, and, perhaps, also myocardial perfusion stress test or coronary angiogram for further risk stratification and potential percutaneous coronary interventions. The results of this study also document an association between elevated troponin I levels and risk of major bleeding. In AF, several of the variables in scores for estimating stroke risk are the same components used for assessing bleeding risk. 30 There are some previous studies in acute coronary syndrome populations linking peak troponin I levels to subsequent increase of bleeding rate. 31 The causality is

10 1614 Circulation April 3, 2012 Table 3. Receiver Operating Characteristics for Stroke or Systemic Embolism, and the Composite of Stroke, Systemic Embolism, Pulmonary Embolism, Myocardial Infarction, Vascular Death (Excluding Hemorrhagic Death) Stroke and Systemic Embolism Composite Thromboembolic Outcome* Model C-statistic P Value IDI Relative IDI, % C-statistic P Value IDI Relative IDI, % Troponin I Referent Referent NT-proBNP Troponin I NT-proBNP CHADS Referent Referent Troponin I NT-proBNP Troponin I NT-proBNP CHA 2 DS 2 -VASc Referent Referent Troponin I NT-proBNP Troponin I NT-proBNP CV risk factors Referent Referent Troponin I NT-proBNP Troponin I NT-proBNP NT-proBNP indicates N-terminal pro-b-type natriuretic peptide; IDI, integrated discrimination improvement; CV, cardiovascular; AF, atrial fibrillation; TIA, transient ischemic attack. *Stroke, systemic embolism, pulmonary embolism, myocardial infarction, vascular death (excl hemorrhagic death). All P CVs risk factors: Adjusted for study treatment, use of (prestudy) anticoagulant treatment at randomization and established risk factors for cardiovascular disease, ie, age, sex, body mass index, smoking status, sitting systolic blood pressure, sitting heart rate, AF duration, AF type, creatinine clearance, diabetes, coronary artery disease, previous stroke/systemic embolism/tia, heart failure, hypertension, treatment at randomization with aspirin, angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers, and statins. unknown, but elevated troponin I levels might contribute to the identification of a more fragile AF subpopulation more likely to bleed during anticoagulation. The NT-proBNP levels were not related to major bleeding risk in the present analysis. The mechanism for the prognostic value of elevated troponin I levels in patients with AF can currently only be an area for speculation. In acute coronary syndrome, troponin levels reflect necrosis of myocytes as a consequence of myocardial ischemia. In patients with chronic heart failure, stable coronary disease, apparently healthy elderly individuals, or as in this cohort of stable AF patients, there might be alternative explanations such as the increased ventricular rate that might lead to oxygen demand/mismatch and myocardial ischemia, volume and pressure overload, changes in microvascular blood flow, atrial calcium overload, oxidative stress, or alterations in tissue structure. 29,32,33 The pathogenesis of thrombi in AF involves poorly contractile atrium, 34 hypercoagulable state, 35 and endothelial dysfunction. 36 Troponin release in AF patients may be connected to several of these mechanisms associated with myocardial dysfunction, apoptosis, inflammation, and fibrosis in the atrial and ventricular musculature, as well. The higher proportion of patients with troponin elevation in permanent AF as an indicator of increased AF burden and a more advanced cardiac disease supports this hypothesis and also the relation to major bleeding events. The mechanism for the prognostic value of elevated NT-proBNP levels in AF seems easier to understand. Elevated natriuretic peptides reflect the myocyte response to increased wall tension. This is usually seen in settings of left ventricular systolic or diastolic dysfunction, ventricular hypertrophy, 37 increasing age, and female sex. 38 An acute coronary syndrome, renal dysfunction, and high-output states may cause elevation of NT-proBNP as well. 39 Concerning natriuretic peptides in AF, NT-proBNP level is a predictor of future development of AF, independent of other risk factors, including echocardiographic parameters in older adults. 40 In accordance with the present results, the levels of NT-proBNP have previously been shown to be elevated in patients with AF, either with or without structural heart disease in comparison with matched controls in sinus rhythm. 41 After restoration of sinus rhythm, either by cardioversion or ablation therapy, the level of natriuretic peptides falls rapidly. 42,43 Therefore, there are arguments for NT-proBNP being of atrial origin in AF, 44 in contrast to the pathophysiology of heart failure, where it is derived mainly from the ventricles. 45 Some studies support this by indicating that atrial stretch is a source of BNP in patients with AF. 46 The level of natriuretic peptides in AF may therefore, to some extent reflect, atrial dysfunction, which is an established marker of atrial thrombus formation, 34 and thereby provide a plausible mechanism for the prognostic importance of elevated NT-proBNP levels and thromboembolic events as shown in the present study. There are several limitations of this study. The findings concern a population with nonvalvular AF with at least 1 risk factor for stroke. To extend and apply the results to AF patients without any clinical stroke risk factors, further studies in other AF populations are warranted. Furthermore, the study design, with all study participants receiving oral

11 Hijazi et al Cardiac Biomarkers in Atrial Fibrillation 1615 anticoagulants, does not allow final conclusions concerning the optimal cutoff value of cardiac biomarkers as a decisionsupport tool for improved selections of AF patients for oral anticoagulation. The mechanisms behind the release and origin of NT-proBNP in AF patients without congestive heart failure need further clarification, and the exact mechanisms behind troponin elevations and their relations to events such as stroke and bleeding in AF, as well. Although the current troponin I assay has shown robust analytic performance in patients who have coronary artery disease and are older, 12,14 newly developed assays with even higher sensitivity and precision might provide even better identification of patients at increased risk and with a potential benefit of oral anticoagulation. Conclusion Elevated levels of troponin I and NT-proBNP are common in patients with AF and at least 1 risk factor for stroke and associated with an increase in the risk for stroke or systemic embolism and vascular events. The prognostic information from the troponin I and NT-proBNP levels are independent of and additive to established clinical risk factors such as the CHADS 2 and CHA 2 DS 2 -VASc risk scores. Source of Funding The RE-LY trial was funded by Boehringer Ingelheim Pharmaceuticals. Disclosures All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Hijazi reports receiving lecture fees and an institutional research grant from Boehringer- Ingelheim. Dr Oldgren reports receiving consulting and lecture fees, and grant support from Boehringer Ingelheim, and consultant and lecture fees from Bayer and Bristol-Myers Squibb. Ms Andersson reports no conflicts of interest. Dr Connolly reports receiving consulting fees, lecture fees, and grant support from Boehringer Ingelheim. Dr Ezekowitz reports receiving consulting fees, lecture fees, and grant support from Boehringer Ingelheim and Aryx Therapeutics; consulting fees from Sanofi-aventis; and lecture fees and grant support from Portola Pharmaceuticals. Dr Hohnloser reports receiving consulting fees and lecture fees from Boehringer Ingelheim, St. Jude Medical, and Sanofi-aventis, and lecture fees from Cardiome. Dr Reilly is an employee of Boehringer Ingelheim. Dr Vinereanu reports receiving lecture fees and grant support from Boehringer Ingelheim. Dr Siegbahn reports consulting fees, lecture fees, and grant support from Boehringer Ingelheim; lecture fees and grants from Eli Lilly; and grant support from AstraZeneca. Dr Yusuf reports receiving consulting fees, lecture fees, and grant support from Boehringer Ingelheim; and consulting fees from AstraZeneca, Bristol- Myers Squibb, and Sanofi-aventis. 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