CYP2C19 genotyping combined with on-clopidogrel platelet reactivity in predicting major adverse cardiovascular events in Chinese patients with percutaneous coronary intervention

Thrombosis Research 147 (2016) 108–114

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CYP2C19 genotyping combined with on-clopidogrel platelet reactivity in predicting major adverse cardiovascular events in Chinese patients with percutaneous coronary intervention Xiao-Fang Tang b,1, Ya-Ling Han c,1, Jia-Hui Zhang a, Jing Wang a, Yi Yao a, Chen He a, Bo Xu b, Zhan Gao b, Shu-Bin Qiao b, Jue Chen b, Yuan Wu b, Ji-Lin Chen b, Run-Lin Gao b, Yue-Jin Yang b,⁎, Jin-Qing Yuan b,⁎ a State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Centre for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China b State Key Laboratory of Cardiovascular Disease, Department of Cardiology, Centre for Coronary Heart Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China c Department of Cardiology, Institute of Cardiovascular Research of People's Liberation Army, Shenyang Northern Hospital, Shenyang, Liaoning 110840, China

a r t i c l e

i n f o

Article history: Received 21 May 2016 Received in revised form 9 September 2016 Accepted 6 October 2016 Available online 7 October 2016 Keywords: Clopidogrel Genotyping CYP2C19 Platelet function test

a b s t r a c t Introduction: Both CYP2C19 genotyping and platelet function testing are used to predict major adverse cardiac events (MACEs) in Chinese patients treated with clopidogrel and undergoing stent implantation, but the most accurate prognostic technique is still debated. Here, we combine both techniques, to determine if a more accurate prognosis is possible. Methods: Patients undergoing stent implantation (1104) were genotyped and assessed for platelet reactivity, with a 12-month follow-up. The CYP2C19*2 (rs4244285), and *3 (rs4986893) alleles were genotyped. High on treatment platelet reactivity was defined as adenosine diphosphate (ADP)-induced platelet inhibition ≤30%. MACEs included death, nonfatal myocardial infarction, target vessel revascularization, or stent thrombosis. Results and conclusions: Hazard ratios (HRs) for cardiovascular ischemic outcomes based on the two testing methods are as follows. CYP2C19 genotyping: carriers of CYP2C19 loss-of-function alleles, HR: 2.515, 95% confidence interval (CI), 1.150–5.501, P = 0.021; ADP-induced platelet inhibition ≤30%, HR: 1.992, 95% CI, 1.040– 3.818, P = 0.038. An ischemic risk score between zero and two was calculated. Compared with the group with a score of zero, HRs for adverse cardiovascular outcomes were 4.078 for those with a score of two (95% CI: 1.525–10.905, P = 0.005). However, there was no significant difference between the group with the score of zero and the group with the score of one. CYP2C19 genotyping combined with platelet reactivity is an independent and additive predictor of 1-year MACE in Chinese patients undergoing stenting with clopidogrel treatment, which is better than either test alone. © 2016 Published by Elsevier Ltd.

1. Introduction Clopidogrel, administered with aspirin for dual antiplatelet therapy has been shown to reduce cardiovascular events in patients presenting with acute coronary syndrome (ACS), particularly in those undergoing percutaneous coronary intervention (PCI) [1,2]. However, there is a large degree of interindividual variability in the pharmacodynamic response to clopidogrel [3]. One source of the variability is the metabolism of clopidogrel, which is administered as a prodrug requiring biotransformation to generate its active metabolite. Accumulating evidence ⁎ Corresponding authors. E-mail addresses: [email protected] (Y.-J. Yang), [email protected] (J.-Q. Yuan). 1 X-F T and Y-L H contributed equally to this article.

http://dx.doi.org/10.1016/j.thromres.2016.10.008 0049-3848/© 2016 Published by Elsevier Ltd.

indicates that carriers of loss-of-function (LOF) genetic variants in the CYP2C19 gene have lower active clopidogrel metabolite levels, diminished platelet inhibition, and a higher risk of recurrent ischemic events following PCI [4–6]. Moreover, the prevalence of CYP2C19 LOF variants ranges from 55% to 70% among Asians, which is much higher than the 35%–45% and 25%–35% ranges of prevalence found among Caucasians and Africans [7,8]. After stent implantation, the rate of major adverse cardiac events (MACEs), such as stent thrombosis, myocardial infarction, or death, is significantly increased in those patients presenting with high on-treatment platelet reactivity (HTPR) [9–11]. There are two major approaches to identifying patients at high-risk for MACEs: the first assesses their metabolizing status, which involves CYP2C19 genotyping, to determine if their metabolism will have a negative impact on the bioactivation of clopidogrel; the other entails functional phenotypic testing to measure

X.-F. Tang et al. / Thrombosis Research 147 (2016) 108–114

residual platelet reactivity in peripheral whole blood. Both prognostic tests have inherent advantages for predicting clinical outcomes [12]. However, it is not yet clear which is the more reliable method for MACE prognosis. We therefore decided to evaluate a combination of these two approaches for predicting the occurrence of 12-month MACEs following stent implantation in Chinese patients treated with clopidogrel. 2. Methods 2.1. Study population Patients presenting to the Fuwai Hospital of Chinese Academy of Medical Sciences and Peking Union Medical College between 1 January 2012 and 30 November 2012 were considered for enrollment in our prospective, randomized, clinical trial. Consecutive patients were assessed for eligibility for enrollment based on the following inclusion criteria: age of N 18 years, had undergone coronary angiography or had an uneventful PCI, and could be followed up for N1 year after PCI. The major exclusion criteria were hemodynamic instability, active bleeding and bleeding diatheses, oral anticoagulation therapy, use of intensified antiplatelet agents other than standard dual antiplatelet therapy, contraindication to antiplatelet therapy, noncardiac disease with a life expectancy of b1 year, or inability to follow the protocol. The Institutional Review Board approved the study protocol, and the patients provided written informed consent for participation and agreed to the CYP2C19 genotyping. 2.2. Study design All patients received a 300-mg loading dose of clopidogrel and aspirin at least 12 h before PCI, followed by a 300 mg/day maintenance dose of aspirin for 1 month, and then a 100 mg/day maintenance dose of aspirin for life and 75 mg/day of clopidogrel for 1 year. The decision for PCI was based on the coronary angiography results, and all interventions were conducted according to the current standard guidelines. The stent type was chosen by the operator, and tirofiban with a short halflife was administered if a glycoprotein (GP) IIb/IIIa inhibitor was required. Anticoagulation with low-molecular-weight heparin (enoxaparin) or unfractionated heparin was initiated before angiography in all patients. 2.3. Genetic analysis Blood samples were obtained from each patient from a peripheral vein and stored in 4 mL ethylenediaminetetraacetic acid-anticoagulated vacuum tubes. Genomic DNA was extracted from whole blood samples according to the salting-out protocol. The CYP2C19*2 (rs4244285, c.681G N A), and *3 (rs4986893, c.636G N A) alleles were genotyped using the improved multiple ligase detection reaction and a commercially available detection system (ABI 3730XL DNA Analyzer System, Applied Biosystems, USA). Repeat genotyping was performed on random duplicate samples (n = 60), and sequencing techniques were used to ensure quality control. 2.4. Thrombelastograph Platelet-Mapping assay Blood was collected at least 6 h after using clopidogrel, in a vacutainer tube containing 3.2% trisodium citrate and lithium heparin. The vacutainer tube was filled to capacity and inverted three to five times to ensure complete mixing of the anticoagulant. Modified thrombelastography (TEG) uses four channels to detect the effects of antiplatelet therapy acting via the arachidonic acid (AA) and adenosine diphosphate (ADP) pathways. A detailed description of this method has been outlined previously [13]. The TEG hemostasis analyzer

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(Haemonetics Corp, Massachusetts, USA) and automated analytical software were used to measure the physical properties. The percent of platelet inhibition by clopidogrel is computed as the contribution from ADP-stimulated platelets to maximal clot strength (ADP-inhibition): 100 − 100 × [(MAAA or ADP − MAfibrin) / (MAthrombin − MAfibrin)], where MAAA is AA-induced clot strength (measurement of aspirin effect), MAADP is ADP-induced clot strength (measurement of ADP effect), MAfibrin is activator-induced clot strength (measurement of fibrin contribution), and MAthrombin is thrombin-induced clot strength (maximum clot strength). 2.5. Outcomes and follow-up CYP2C19 genetic testing identified a genotype in 1104 patients, 449 (40.7%) were homozygous for the wild-type allele (*1/*1), 498 (45.1%) were heterozygous (*1/*2 and *1/*3), and 157 (14.2%) were homozygous for the LOF alleles (*2/*2, *3/*3 and *2/*3). Previous studies, which employed modified TEG percent platelet inhibition to measure the response to clopidogrel, used cutoff values of ≤30% to define HTPR [8,10]. In our study, 310 (28.1%) patients were assessed as having HTPR characteristics, using this definition. MACEs included all-cause death, nonfatal myocardial infarction (MI), unplanned target vessel revascularization (TVR), or stent thrombosis. MI was defined as ischemic symptoms with electrocardiogram abnormalities and upper normal limits of troponin [2,14]. Unplanned TVR was defined as any repeat percutaneous intervention or surgical bypass of any segment of the target vessel (the entire major coronary vessel proximal and distal to the target lesion, which includes upstream and downstream branches and the target lesion itself) [15]. Stent thrombosis was defined as definite stent thrombosis according to the Academic Research Consortium [15]. Two independent physicians blinded to the laboratory data adjudicated events after reviewing the source documents. 2.6. Statistical analysis Continuous variables are presented as mean ± standard deviation (SD) and compared using the Student's t-test, or one-way analysis of variance (ANOVA) test, as appropriate. Categorical variables are expressed as numbers and percentages and compared using a chisquare test (χ2) or Fisher's exact test. After demonstrating significant differences among variables using the ANOVA test, post hoc comparisons between the groups were performed using the Student–Newman–Keuls test for multiple comparisons. Clinical follow-up was censored at the day of the first cardiovascular event corresponding to the clinical endpoints. For subjects without a clinical event, clinical follow-up was censored either at the last clinic visit after 12 months of taking clopidogrel or at the day of clopidogrel discontinuation. The positive results of the genetic testing are based on the carriers of CYP2C19 LOF alleles, which includes heterozygous and homozygous for the LOF alleles. The positive platelet function results are based on the cutoff value of HTPR, as measured using the modified TEG assay. The number of positive results of the two tests was evaluated as a categorical variable. The relationship between the positive results of the two tests and adverse cardiovascular outcomes was determined using the Cox proportional hazards regression in unadjusted models and in models adjusted for established risk factors that included clinically relevant covariates (sex, body mass index, anemia, thrombocytopenia, acute MI at presentation, dyslipidemia, hypertension, diabetes mellitus, left ventricular ejection fraction, use of heparin, use of GP IIb/IIIa and proton pump inhibitors, and smoking status). Hazard ratios (HR) are presented with 95% confidence intervals (CI). To assess the cumulative event-free survival for the adverse cardiovascular events, a Kaplan– Meier analysis was performed, and the data were stratified according to the number of positive results of the tests, and were compared using a log-rank test. All statistical analyses were performed using

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SPSS version 17.0 (SPSS Inc., Chicago, IL, USA), and a two-tailed probability value b 0.05 was considered significant. To show the independent prognostic value of the number of positive results, predictors for MACE were determined using a multivariate Cox proportional hazards model using stepwise analysis, and variables with P b 0.05 were entered into the model and variables with P N 0.1 were shifted out. The variables used in the multivariate full model are as mentioned above. The additional prognostic value of the number of positive results compared with other clinical predictors was assessed by the increase in the area under the receiver-operating characteristic (ROC) curve using the test proposed by DeLong et al. [16], net reclassification improvement (NRI) and integrated discrimination improvement (IDI) [17]. Risk percentile cutoffs for NRI were at 2% and 6% according to approximately the 20th and 80th percentiles of predicted risk based on the multivariate model. All P-values given are 2-sided and P b 0.05 was considered statistically significant. All statistical analyses were performed using STATA 10.1 (Stata Corp, College Station, TX). 3. Results 3.1. Baseline demographics and clinical characteristics In total, CYP2C19 genotyping and platelet function measured using modified TEG assay were obtained in 1104 patients undergoing PCI who were treated with clopidogrel during 1 year of clinical follow-up (Fig. 1). CYP2C19 genetic testing identified genotypes in 1104 patients, and 449 (40.7%) were homozygous for the wild-type allele (*1/*1), 498 (45.1%) were heterozygous for the LOF alleles (*1/*2 and *1/*3), and 157 (14.2%) were homozygous for the LOF alleles (*2/*2, *3/*3 and *2/*3). The median level of platelet inhibition was 52.2 ± 30.3%. Platelet inhibition decreased with the number of CYP2C19 LOF alleles (55.6 ± 30.3% vs. 52.0 ± 29.7% vs. 43.1 ± 30.3%, P b 0.001), and HTPR increased with the number of CYP2C19 LOF alleles (24.7% vs. 27.5% vs. 39.7%, P = 0.001; Supplementary Table 1). The baseline characteristics of the patients, divided according to the number of positive results of the two tests, are presented in Table 1. Based on the number of positive results of the two tests, patients were classified into three groups: 0-positive results (no carriers of CYP2C19 LOF alleles and normal on-treatment platelet reactivity; n = 338); 1-

Table 1 Baseline demographics, clinical characteristics of patients undergoing PCI by aggregate positive results of CYP2C19 genotyping and on-clopidogrel platelet reactivity. Characteristics

No. of positive testing results

P value

All (n = 1104)

0 (n = 338)

1 (n = 567)

2 (n = 199)

Age (years) Male, n (%) BMI, kg/m2 Hemoglobin, g/dl PLT, ×103/mm3 hs-CRP, mg/dl

58 ± 11 832 (75.4) 26.1 ± 3.3 138 ± 16 207 ± 56 4.48 ± 4.71

LDL (mg/dl)

2.54 ± 1.0

HDL (mg/dl)

1.06 ± 0.31

Glucose (mg/dl)

6.37 ± 4.49

58 ± 10 265 (78.4) 25.9 ± 3.3 140 ± 16 205 ± 52 4.54 ± 4.94 2.54 ± 1.22 1.08 ± 0.42 6.28 ± 2.65 61 ± 9

58 ± 11 442 (78.0) 26.3 ± 3.3 139 ± 16 204 ± 56 4.53 ± 4.72 2.57 ± 0.92 1.06 ± 0.25 6.42 ± 4.41 60 ± 8

59 ± 10 125 (62.8) 25.8 ± 3.2 132 ± 15 216 ± 62 4.23 ± 4.27 2.48 ± 0.80 1.06 ± 0.25 6.41 ± 6.69 61 ± 8

0.170

242 (71.6) 127 (37.6) 54 (16.0) 74 (21.9) 80 (23.7)

395 (69.7) 206 (36.3) 68 (12.0) 133 (23.5) 145 (25.6)

127 (63.8) 66 (33.2) 41 (20.6) 28 (14.1) 61 (30.7)

0.159 0.585 0.010 0.020 0.197

187 (55.3) 290 (85.8) 101 (29.9) 124 (36.7) 76 (22.5)

361 (63.7) 480 (84.7) 180 (31.7) 223 (39.3) 127 (22.4)

138 (69.3) 162 (81.4) 59 (29.6) 68 (34.2) 55 (27.6)

0.003 0.389 0.780 0.156 0.291

56 (16.6) 48 (14.2) 1 (0.3)

103 (18.2) 84 (14.8) 2 (0.4)

31 (15.6) 24 (12.1) 2 (1.0)

0.659 0.630 0.287

297 (87.9) 204 (60.4) 230 (68.0) 15 (4.4)

502 (88.5) 378 (66.7) 404 (71.3) 42 (7.4)

166 (83.4) 133 (66.8) 151 (76.9) 11 (5.5)

0.165 0.125 0.092 0.183

334 (98.8)

562 (99.1)

195 (98.0)

0.447

3 (0.9)

3 (0.5)

2 (1.0)

0.725

338 (100) 338 (100) 307 (90.8) 48 (14.2) 328 (97.0) 194 (57.4) 308 (91.1) 132 (39.1) 118 (34.9)

567 (100) 567 (100) 497 (87.7) 80 (14.1) 549 (96.8) 349 (61.6) 497 (87.7) 211 (37.2) 211 (37.2)

199 (100) 199 (100) 167 (83.9) 11 (5.5) 192 (96.5) 111 (55.8) 176 (88.4) 76 (38.2) 54 (27.1)

– – 0.057 0.004 0.938 0.257 0.270 0.856 0.037

8 (2.4) 3 (0.89) 3 (0.89) 2 (0.59) 4 (1.2)

20 (3.5) 2 (0.35) 4 (0.7) 2 (0.35) 13 (2.3)

19 (9.5) 1 (0.50) 1 (0.50) 1 (0.50) 14 (7.0)

b0.001 0.588 0.873 0.306 0.004

LVEF (%) 61 ± 8 Index clinical presentation, n (%) ACS 764 (69.2) Unstable angina 399 (36.1) NSTEMI 163 (14.8) STEMI 235 (21.3) Stable angina 286 (25.9) Risk factor, n (%) Hypertension 686 (62.1) Dyslipidemia 932 (84.4) DM 340 (30.8) Current smoking 415 (37.6) CHD family 258 (23.4) history History, n (%) Previous MI 190 (17.2) Previous PCI 156 (14.1) Previous CABG 5 (0.5) Infarct-related artery, n (%) LAD 965 (87.4) LCX 715 (64.8) RCA 787 (71.3) LM 68 (6.2) Intervention methods, n (%) Drug-eluting 1091 (98.8) stent Bare metal stent 8 (0.7) Concomitant medications, n (%) On clopidogrel 1104 (100) On aspirin 1104 (100) On heparin 971 (88.0) On GPIIb/IIIa 139 (12.6) On statin 1069 (96.8) On ARB or ACEI 654 (59.2) On beta-blocker 981 (88.9) On CCB 419 (38.0) On PPI 383 (34.7) Follow-up, n (%) MACE 47 (4.3) All-cause death 6 (0.54) Nonfatal MI 8 (0.7) Stent thrombosis 5 (0.45) Unplanned TVR 31 (2.8)

0.689 b0.001 0.056 b0.001 0.044 0.712 0.542 0.557 0.889

BMI, body mass index; WBC, white blood cell; PLT, platelets count; hs-CRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; LVEF, left ventricular ejection fraction; ACS, acute coronary syndrome; NSTEMI, non-ST-segment elevation myocardial infarction; STEMI, ST-segment elevation myocardial infarction; DM, diabetes mellitus; CHD, coronary heart disease; MI, myocardial infarction; PCI, percutaneous coronary intervention; CABG, coronary artery bypass grafting; LAD, left anterior descending; LCX, left circumflex; RCA, right coronary artery; LM, left main; GP, glucose protein; ARB, angiotensin receptor blocker; ACEI, angiotensin antagonist inhibitor; CCB, calcium channel blocker; PPI, proton pump inhibitor; TVR, target vessel revascularization.

Fig. 1. Flow diagram describing the study population. CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention.

positive result (carriage of CYP2C19 LOF alleles and normal on-treatment platelet reactivity, or no carriers of CYP2C19 LOF alleles and HTPR; n = 567); 2-positive results (carriage of CYP2C19 LOF alleles and HTPR; n = 199). Baseline demographics, clinical presentations,

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and treatments were mostly balanced with regard to risk score, which was calculated based on the number of positive results of the two tests (P N 0.05), with the exception of sex, hemoglobin, platelet counts, clinical presentations (NSTEMI and STEMI), diagnosis of hypertension, and concomitant medications (heparin, GP IIb/IIIa and PPI; P b 0.05). 3.2. Clinical outcomes Only six patients (0.54%) suffered all-cause death, eight patients suffered nonfatal MI (0.7%), and five patients displayed stent thrombosis (0.45%) during the 1-year follow-up period. All-cause death, nonfatal MI, and stent thrombosis were not different between the three groups. Forty seven patients (4.3%) suffered MACEs during the follow-up period, which occurred in eight patients (2.4%) with 0-positive results, 20 patients (3.5%) with 1-positive result, and 19 (9.5%) patients with 2positive results. The highest incidence of MACEs significantly increased with the number of positive results (P b 0.001; Table 1). 3.3. Relationships between individual CYP2C19 genotyping or platelet function testing and MACEs The Cox proportional hazard regression in unadjusted models, performed for individual CYP2C19 genotyping or platelet function testing demonstrates that carriage of CYP2C19 LOF alleles (HR: 2.236, 95% CI: 1.059–4.724, P = 0.035), and patients with HTPR (HR: 2.339, 95% CI: 1.237–4.421, P = 0.009), is significantly associated with the occurrence of MACEs. The Cox proportional hazard regression models, adjusted for the aforementioned covariates, demonstrate that carriers of CYP2C19 LOF alleles (HR: 2.195, 95% CI: 1.035–4.658, P = 0.04), and patients with HTPR (HR: 2.218, 95% CI: 1.131–4.351, P = 0.02), are significantly associated with the occurrence of MACEs (Table 2). 3.4. Relationships between the aggregate positive results of the two testing methods and MACEs There was a stepwise decline in survival free of MACEs with an increasing number of positive results (log rank P b 0.001; Fig. 2). The Cox proportional hazard regression models, adjusting for all the previously described covariates, demonstrate that there was no significant difference in the occurrence of MACEs between 1-positive result patients and 0-positive results patients (HR: 1.29, 95% CI: 0.525–3.173, P = 0.579). However, 2-positive results compared with 0-positive results was significantly associated with the occurrence of MACEs (HR: 3.777, 95% CI: 1.508–9.461, P = 0.005). HRs of one or two positive results, compared with zero positive results, are shown in Table 2. Table 2 Hazard ratios for 12-month major adverse cardiovascular events according to aggregate positive results of CYP2C19 genotyping and on-clopidogrel platelet reactivity. Variables

All participants; unadjusted HR (95% CI); P value

All participants; adjusted HR (95% CI); P value

Major adverse cardiovascular events Genotyping or platelet function testing in same model Carriers of 2.236 (1.059–4.724); 0.035 2.195 (1.035–4.658); 0.04 CYP2C19 LOF alleles TEG: 2.339 (1.237–4.421); 0.009 2.218 (1.131–4.351); 0.02 ADP-inhibition ≤ 30% Categorical positive testing results risk score 1 vs. 0 positive 1.282 (0.523–3.145); 0.587 1.290 (0.525–3.173); 0.579 result 2 vs. 0 positive 3.993 (1.642–9.705); 0.002 3.777 (1.508–9.461); 0.005 result HR, hazard ratios; CI, confidence intervals; MI, myocardial infarction; TVR, target vessel revascularization; LOF, loss-of-function; TEG, thrombelastograph; ADP, adenosine diphosphate.

Fig. 2. Kaplan-Meier analysis. Kaplan–Meier analysis for the cumulative risk of major adverse cardiac events in patients based on the number of positive results obtained by CYP2C19 genotyping combined with on-treatment platelet reactivity measured by TEG assay. mTEG, modified thrombelastograph.

3.5. Aggregate positive results of the two testing methods as an independent and additive predictor for MACEs Using Cox proportional hazard models that included all the aforementioned covariates, independent predictors of MACEs during the follow-up period other than the aggregate positive results of the two testing methods were dyslipidemia (HR: 2.71, 95% CI: 0.84–8.74, P = 0.094), acute MI (HR: 1.88, 95% CI: 1.05–3.34, P = 0.033), anemia (HR: 2.67, 95% CI: 1.25–5.74, P = 0.012), and thrombocytopenia (HR: 1.51, 95% CI: 1.05–2.18, P = 0.027). We evaluated the additive prognostic value of the aggregate positive results of the two testing methods for 12-month MACEs. The area under the curve (AUC) was 0.6699 (95% CI: 0.592–0.748) for the multivariate full model without the aggregate positive results. When the aggregate positive results were added (Table 3), it improved the prognostic performance of the model significantly, based on the increase of the AUC (AUC 0.67 vs. 0.73, P = 0.074; Fig. 3). In the NRI analysis, using risk categories of b 2%, 2%–6%, and N6%, net reclassification after adding aggregate positive results to the conventional predictors in the multivariate model placed five (10.6%) patients in the deceased group at higher risk (i.e., 12 were pushed to higher risk and 7 were pushed to lower risk) while 181 (17.1%) of the survivors were reclassified into lower risk categories (i.e., 285 were pushed to lower risk and 104 were pushed to higher risk). The overall NRI was 27.7% (P = 0.003) and the IDI was 0.016 (P = 0.013, Table 4).

4. Discussion Several major findings of the present study applying to Chinese patients undergoing stent implantation are: 1) each testing method (CYP2C19 genotyping or on-clopidogrel platelet reactivity) was an independent predictor of risk of MACEs in Chinese patients undergoing Table 3 Multivariate Cox proportional hazard analyses for major adverse cardiovascular events in patients undergoing PCI using aggregate positive results of CYP2C19 genotyping and onclopidogrel platelet reactivity. Variables

HR (95% CI)

P-value

Dyslipidemia Acute myocardial infarction Anemia Thromboeytopenia 1 positive result 2 positive results

2.98 (0.93–9.64) 1.90 (1.07–3. 40) 2.25 (1.04–4.87) 1.44 (1.003–2.069) 1.57 (0.69–3.56) 3.99 (1.74–9.17)

0.067 0.03 0.041 0.048 0.282 0.001

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Fig. 3. Receiver operating characteristic curve assessing the CYP2C19 genotyping combined with platelet function to detect the additional risk predictive power of severe adverse cardiovascular events during 1 year follow-up of patients with coronary artery disease. AUC, area under the curve.

PCI. 2) The aggregate positive results based on the two testing methods were a more powerful predictor of higher risk. In comparison to patients with 0-aggregate positive results, those who had 2-aggregate positive results (almost 18% of the population) experienced almost a four-fold increased risk of MACEs. 3) The aggregate positive results based on the two testing methods showed independent and additive prognostic values for 1-year MACEs in patients undergoing PCI after adjustment for other clinical predictors, as evidenced by improvement in the area under the ROC curve and NRI. Carriers of loss-of-function CYP2C19 alleles (1 or 2-LOF) have an attenuated pharmacologic response and worse clinical outcomes on treatment with the standard dose of clopidogrel [5,6,8,18]. The present study strengthens the concept that CYP2C19 genetic status can be used to predict the occurrence of MACEs after stenting in Chinese patients. Hence, a point-of-care genetic test was designed for the CYP2C19 LOF alleles, to guide the double dosing of clopidogrel or new antiplatelet agents (prasugrel or ticagrelor) to reduce the rate of inadequate platelet inhibition in the context of loss-of-function CYP2C19 genotypes after PCI [19, 20]. However, the prevalence of the CYP2C19 LOF variant is 35% to 45% and 25% to 35% among Caucasians and Africans, respectively, whereas it is 55% to 70% among Asians. In our population, 59.5% of patients presented with CYP2C19 LOF alleles. These patients suffered an increased risk of cardiac ischemic events compared with those without CYP2C19 LOF alleles, which was almost 2.2-fold, after adjusting for clinical variables, risk factors, and medication use (sex, anemia, thrombocytopenia, NSTEMI or STEMI at presentation, diagnosis of hypertension, use of heparin, use of GP IIb/IIIa and proton pump inhibitors). Therefore, single

Table 4 Risk reclassification by adding aggregate positive testing results to the multivariate Cox model without positive testing results. Multivariate model without positive testing results

Patients with 1-year MACE Low risk (b2%) Intermediate risk (2–6%) High risk (≥6%) Total Patients without 1-year MACE Low risk (b2%) Intermediate risk (2–6%) High risk (≥6%) Total

Multivariate model with positive testing results

Total

Low risk

Intermediate risk

High risk

b2%

2%–6%

≥6%

1 5 0 6

0 15 2 17

0 12 12 24

1 32 14 47

108 238 0 346

12 460 47 519

0 92 100 192

120 790 147 1057

CYP2C19 genotyping may be not suitable for the optimization of clopidogrel therapy in the Chinese population. Recent evidence has clearly demonstrated that inadequate platelet inhibition, the occurrence of stent thrombosis, and recurrent ischemic events are significantly related [10,21,22]. Standard light transmittance aggregometry (LTA) has been the most widely used technique in this regard and has clearly demonstrated the relationship between HTPR and subsequent atherothrombotic events [22,23]. There have also been several studies, which have reported an increased ADP-induced aggregation measured by TEG, in patients suffering a higher risk of cardiac ischemic events [10,24,25]. Our findings are concordant with the relationship between HTPR and subsequent atherothrombotic events. The modified TEG technique was implemented in this study to assess its reliability as a monitoring tool for analyzing the response to anti-platelet therapy. The TEG technique, combined with the Platelet-Mapping assay, was sufficient to identify a sub-therapeutic response, and was highly correlated with the more labor intensive LTA method [10,24, 26]. Therefore, the TEG assay might affect the delivery of individualized anti-platelet therapy. In this study, 28% of patients presented with HTPR, which entailed an increased risk of MACEs compared with those without HTPR, which is almost 2.2-fold after adjusting for clinical variables, risk factors, and medication use, as described previously. Our study established the value of CYP2C19 genotyping combined with on-treatment platelet reactivity measured using a TEG assay, in the Chinese population treated with clopidogrel and undergoing PCI. This is different from many studies that used a single assay to predict MACEs in patients treated with clopidogrel. In our study, aggregate positive results were established based on the number of positive results of the two testing methods, and almost 18% of patients presented with 2positive results (CYP2C19 LOF alleles and HTPR). These patients demonstrated a significant 3.8-fold increase in the risk of MACEs, compared with patients with a 0-positive result; however, the manifestation of MACEs was not different between patients with a 1-positive result and those with a 0-positive result. Therefore, aggregate positive results based on the two testing methods predicted MACEs better than a single assay. In our study, aggregate positive results based on the two testing methods were a potent independent predictor for MACEs in patients treated with stent implantation. Adding the aggregate positive results to conventional clinical predictors improved the prognostic performance of the prediction model significantly, as evidenced by improvement in the area under the ROC curve (AUC 0.67 vs. 0.73) and NRI (27.7%, P = 0.003). Several mechanisms have been proposed to explain the association between MACEs and the aggregate positive results based on CYP2C19 genotyping and platelet function testing, including acute MI, dyslipidemia, anemia and thrombocytopenia [27–31]. The high risk strategy is aimed at identification of patients with multiple cardiovascular risk factors, identification of those with genetic lipoprotein disorders and initiation of treatment with medications if necessary [27]. Clinical data show that a 1% decrease in serum concentrations of highdensity lipoprotein cholesterol can increase cardiovascular risk by 2%– 3% [28,29]. The presence of anemia is associated with a higher 30-day level of MACEs, and anemia is an independent predictor of mortality after PCI [30]. Thrombocytopenia, a common complication of ACS, is associated with increased mortality and adverse outcomes [31]. Application of a combined definition for thrombocytopenia using both absolute and relative thresholds permits for increased sensitivity for patients at high risk of adverse outcomes. 5. Study limitations The limitations of this study include a one-time measurement of platelet function (measured using a TEG assay) that may not reflect levels at future time points. The best time for the detection of platelet function (potentials include time of admission, before or after PCI, and in the weeks after discharge) has not been evaluated in our study.

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Some studies have demonstrated that the detection of platelet function in the 24 h after interventional therapy can predict clinical prognosis [21,32]. Future research is needed to clarify the best time for routine assessment of platelet function in clinical practice. In addition, the mean of the left ventricular ejection fraction and renal function (creatinine clearance rate) was 61% and 97.4 mL/min, respectively, in our study subjects, and this phenomenon may be related to the screening of patients with interventional therapy in our hospital. Therefore, our study could not confirm the prognostic performance of the other traditional clinical predictors such as the left ventricular ejection fraction and creatinine clearance rate. 6. Conclusions The incidence of severe adverse cardiovascular events was highest in Chinese patients who presented with CYP2C19 LOF alleles and HTPR during a 1-year follow-up. An ischemic risk score based on CYP2C19 LOF allele genotyping and platelet function as assessed by TEG assay, is a predictor of future risk of adverse cardiovascular outcomes in Chinese patients treated with clopidogrel and undergoing coronary stent implantation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.thromres.2016.10.008. Conflicts of interest The authors state that they have no conflicts of interest. Acknowledgments This work was supported by grants from the National Research Key Project of The Twelfth Five-Year Plan of Republic of China (No. 2011BAI11B07) and National Natural Science Foundation of China (81170194). We are grateful to the Department of Cardiology, Cardiovascular Institute of Fuwai Hospital for its help in recruiting patients. We thank all members who contributed to the study. References [1] C.W. Hamm, J.P. Bassand, S. Agewall, J. Bax, E. Boersma, H. Bueno, P. Caso, D. Dudek, S. Gielen, K. Huber, M. Ohman, M.C. Petrie, F. Sonntag, M.S. Uva, R.F. Storey, W. Wijns, D. Zahger, J.J. Bax, A. Auricchio, H. Baumgartner, C. Ceconi, V. Dean, C. Deaton, R. Fagard, C. Funck-Brentano, D. Hasdai, A. Hoes, J. Knuuti, P. Kolh, T. McDonagh, C. Moulin, D. Poldermans, B.A. Popescu, Z. Reiner, U. Sechtem, P.A. Sirnes, A. Torbicki, A. Vahanian, S. Windecker, S. Achenbach, L. Badimon, M. Bertrand, H.E. Botker, J.P. Collet, F. Crea, N. Danchin, E. Falk, J. Goudevenos, D. Gulba, R. Hambrecht, J. Herrmann, A. Kastrati, K. Kjeldsen, S.D. Kristensen, P. Lancellotti, J. Mehilli, B. Merkely, G. Montalescot, F.J. Neumann, L. Neyses, J. Perk, M. Roffi, F. Romeo, M. Ruda, E. Swahn, M. Valgimigli, C.J. Vrints, P. Widimsky, Esc guidelines for the management of acute coronary syndromes in patients presenting without persistent st-segment elevation: the task force for the management of acute coronary syndromes (acs) in patients presenting without persistent st-segment elevation of the European society of cardiology (esc), Eur. Heart J. 32 (2011) 2999–3054. [2] G.N. Levine, E.R. Bates, J.C. Blankenship, S.R. Bailey, J.A. Bittl, B. Cercek, C.E. Chambers, S.G. Ellis, R.A. Guyton, S.M. Hollenberg, U.N. Khot, R.A. Lange, L. Mauri, R. Mehran, I.D. Moussa, D. Mukherjee, B.K. Nallamothu, H.H. Ting, 2011 accf/aha/scai guideline for percutaneous coronary intervention: a report of the American college of cardiology foundation/American heart association task force on practice guidelines and the society for cardiovascular angiography and interventions, Catheter. Cardiovasc. Interv. 82 (2013) E266–E355. [3] D.J. Angiolillo, A. Fernandez-Ortiz, E. Bernardo, F. Alfonso, C. Macaya, T.A. Bass, M.A. Costa, Variability in individual responsiveness to clopidogrel: clinical implications, management, and future perspectives, J. Am. Coll. Cardiol. 49 (2007) 1505–1516. [4] B. Giusti, A.M. Gori, R. Marcucci, C. Saracini, A. Vestrini, R. Abbate, Determinants to optimize response to clopidogrel in acute coronary syndrome, Pharmgenomics Pers. Med. 3 (2010) 33–50. [5] J.L. Mega, T. Simon, J.P. Collet, J.L. Anderson, E.M. Antman, K. Bliden, C.P. Cannon, N. Danchin, B. Giusti, P. Gurbel, B.D. Horne, J.S. Hulot, A. Kastrati, G. Montalescot, F.J. Neumann, L. Shen, D. Sibbing, P.G. Steg, D. Trenk, S.D. Wiviott, M.S. Sabatine, Reduced-function cyp2c19 genotype and risk of adverse clinical outcomes among patients treated with clopidogrel predominantly for pci: a meta-analysis, JAMA 304 (2010) 1821–1830.

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