G Campo, G Parrinello, P Ferraresi, B Lunghi, M Tebaldi, M Miccoli, J Marchesini, F Bernardi, R Ferrari, M Valgimigli.

1. Prospective Evaluation of On-Clopidogrel Platelet Reactivity Over Time in Patients treated with Percutaneous Coronary Intervention. Relationship with Gene Polymorphisms and Clinical Outcome G Campo, G Parrinello, P Ferraresi, B Lunghi, M Tebaldi, M Miccoli, J Marchesini, F Bernardi, R Ferrari, M Valgimigli. Cardiovascular Institute, Azienda Ospedaliero-Universitaria S.Anna, Ferrara, Italy

2. Background • Response to clopidogrel varies widely among patients, and those with a high residual on-clopidogrel platelet reactivity (PR) undergoing percutaneous coronary intervention (PCI) are at a greater risk for death, myocardial infarction (MI) and stent thrombosis (ST) • Clinical, genetic and cellular factors are involved in the clopidogrel response variability • These results have been largely provided by studies with a single phenotype assessment evaluation before or soon after PCI. • Therefore, whether clopidogrel response varies throughout follow-up and whether the role of gene polyphormism differs over time is unknown.

3. Aims • We sought to evaluate whether: • clopidogrel response in patients treated with PCI differs through follow-up • to asses the relationship of on-clopidogrel PR at different time points with gene polymorphisms and clinical outcome.

4. Methods • Individuals eligible for enrolment were patients undergoing PCI for ischemic heart disease in our centre from December 2008 to May 2009 • All patients were treated with aspirin (300 mg as loading dose at hospital admission, followed by 100 mg die, independently to previous or not chronic use). Clopidogrel 600 mg was given as loading dose (LD) at least 12 hours before PCI. • To evaluate on-clopidogrel PR we used VerifyNow P2Y12 • Clopidogrel poor response was defined as a PRU value ≥235 • CYP2C19*2, *17, CYP3A5*3 and ABCB1 polymorphisms were evaluated • Our primary analysis compared the incidence of clopidogrel poor responders at baseline vs. 1 month. • Death, reinfarction, stroke and bleeding complications are reported

5. December 2008 – May 2009 507 Pts undergoing PCI in our Cath-Lab • 202 (40%) Pts excluded for: • 130 treated for STEACS • 10 DAT controindicated • 23 clopidogrel LD <12 hours • 9 refused consent • 27 previous GPI IIa/IIIa administration • 3 recent bleedings 305 Pts with baseline PRU evaluation and genotype • 5 (1.6%) Pts without 1 month blood sample: • 1 stop DAT for major surgery (colon cancer) • 2 retired consent • 1 died • 1 readmitted for MI due to not culprit lesion 300 Pts with baseline and 1 month PRU evaluation FINAL STUDY POPULATION 6 months blood sample available in 281 (93%) Pts 1 year follow-up available in all Pts

6. Primary Analysis

7. Clinical and Genetic Determinants of On-clopidogrel PR • Age • Diabetes • Creatinine Clearance • Admission for NSTEACS • CYP2C19*2 • CYP2C19*17 • ABCB1 These three gene polymorphisms justified altogether ≈18% of PR variation (6.6%, 5.2% and 6.7%, respectively). Interestingly, the CYP2C19*2 and *17 influence appeared constant over time, whereas that of ABCB1 was higher at baseline (9%) and thereafter decreased gradually (6% at 1 month, p=0.09; 5% at 6 months, p=0.04)

8. Ischemic endpoint (death, MI, stroke)

9. Adverse events and cut-offs Ischemic Composite End Point Bleeding Composite End Point AUC (95%CI) cut-off sen spec PPV NPV AUC (95%CI) cut-off sen spec PPV NPV Baseline PRU 0.63 (0.58-0.69) ≤95 46% 85% 17% 96% Baseline PRU 0.69 (0.63-0.74) ≥214 78% 63% 14% 97% 1 month PRU 0.84 (0.79-0.88) ≤85 81% 80% 21% 98% 1 month PRU 0.87 (0.83-0.91) ≥239 81% 92% 43% 98%

10. PRU values and window effect

11. ABCB1 and CYP2C19*2 gene polymorphisms ABCB1 CC homozygotes and CYP2C19*2 GG homozygotes (53 pts.) ABCB1 T carriers and/or CYP2C19*2 A carriers (247 pts.) Baseline on-clopidogrel PR and CrCl 1 month: 0 (0%) poor R 1 month PRU: 109±77 0 (0%) adverse events PRU <258 and CrCl >52 (143 pts) PRU ≥258 or CrCl ≤52 (80 pts) PRU ≥258 and CrCl ≤52 (24 pts) Risk score 0 1 month: 6 (4%) poor R 1 month PRU: 129±69 3 (2.1%) adverse events 1 month: 17 (21%) poor R 1 month PRU: 173±84 12 (15%) adverse events OR 6.7 (95%CI 2.5-20) * 1 month: 17 (71%) poor R 1 month PRU: 248±87 6 (35%) adverse events OR 53 (95%CI 23-81) * Risk score 1 Risk score 2 Risk score 3

12. Conclusions • The main findings of this prospective investigation can be summarized as follows: • On-clopidogrel PR showed a significant reduction from index hospitalization to 1 month. The percentage of poor responders decreased from 35% (95%CI 30%-41%) at baseline to 13% (95%CI 9%-18%) at 1 month. • Gene polymorphisms justified about 18% of this trend. CYP2C19 *2 and *17 influence was apparently consistent over time, whereas ABCB1 showed a higher impact at baseline. • We found a “therapeutic window of PRU values” where both ischemic and bleeding adverse events are minimized. Then, on-clopidogrel PR may be used to predict both complications, particularly when assessed at 1 month after index procedure. • A risk-score was created by combining genotype (ABCB1 and CYP2C19*2), baseline PR and creatinine clearance, to predict 1 month poor responsiveness and 1-year poor prognosis