1. Genotyping: Which One Should we Perform? How to Interpret the Data?
Is it Ready for Clinical Practice?
Paul A. Gurbel, M.D.
Sinai Center for Thrombosis Research Baltimore, Maryland, U.S.A. 1 1

2. Paul A. Gurbel, MD DISCLOSURES Consulting Fees
AstraZeneca, Bayer AG, Medtronic CardioVascular, Inc.
Grants/Contracted Research
AstraZeneca, Bayer AG, Boston Scientific Corporation , Haemoscope, Medtronic CardioVascular, Inc., POZEN Inc.
Honoraria
Bayer AG, Schering and Millennium

3. Clopidogrel Response Variability: A PK Problem
Intestinal Absorption
Esterases 90%
P-glycoprotein
(ABCB1 gene polymorphism) ?
Limited absorption
Two Step Conversion
Hepatic P450
Cytochromes
10%
2C19, 1A2, B6
*2,*17
Genetic polymorphisms
2C19, 2C9, 3A4, 2B6
Variable Active Metabolite Generation
Wide Pharmacodynamic Response, Nonresponsiveness
Worse Clinical Outcome
Adapted from Kauzi M et al. Drug Metab Dispos. 2009; Gurbel et al. J Inv Cardiol. 2009 3

4. 4 4 Linkage Disequilibrium and CYP2C19 Haplotypes and Diplotypes 4
Due to linkage disequilibrium between CYP2C19 *2 loss-of-function (slow metabolizer) and *17 gain-of-function (fast metabolizer) variants,
Only 3 (of 4 possible) haplotypes exist:
*1----* (fast) *1----non-* (normal) *2----non-* (slow)
These 3 haplotypes result in 6 diplotypes which define 3 main phenotypes: Diplotypes Phenotypes
fast/fast, fast/normal Extensive metabolizers (EM) normal/normal, fast/slow Normal metabolizers (NM)
normal/slow, slow/slow Poor metabolizers (PM)
Gurbel et al. ACC 2009 4 4 4

5. Polymorphisms, Platelet Reactivity, Outcomes on Clopidogrel
Study Patients (n) Gene Variants High Platelet Reactivity SNP/ Studied Outcomes
1. Taubert et al (2006) PCI (60) ABCB1 3435T  CLP absorption/active metabolite
2. Suh et al (2006) Healthy (16) CYP3A Nonexpressor more drug-drug interactions
PCI (348) Nonexpressor- more CV events
3. Giusti et al (2007) PCI (1419) P2Y12, CYP3A4, CYP2C19* CYP2C19*2
7. Geisler et al (2008) PCI (235) CYP2C19, CYP3A4, CYP3A CYP2C19*2
9. Trenk et al. (2008) PCI (797) CYP2C19* CYP2C19*2
10. Gladding et al (2008) PCI (60) CYP2C19, CYP2C9, CYP3A4, CYP2C19*2/3, CYP2C9* CYP3A5, CYP2B6, P2Y12, ABCB1 CYP2C19*2,*4,*17
11. Frere et al (2008) NSTE-ACS CYP2C19, CYP3A4, CYP3A CYP2C19*2
12. Simon et al (2008) Acute MI (2009) CYP2C19, CYP3A5, ABCB1, ABCB1, CYP2C19*2,*3, *4,*5 -HPR P2Y12
13. Mega et al (2008) Healthy (162) CYP2C19, CYP3A4, CYP2B6, CYP2C19*2
ACS (1477) CYP3A5, CYP1A CYP2C19-  1.53x- MACE, 3x- ST
14. Collet et al (2009) MI (259) CYP2C  CV events including ST
15. Shuldiner et al (2009) Amish (429) GWAS CYP2C19*2
PCI (227) C19*  1 year CV events
16. Sibbing et al (2009) PCI (2485) C19*1,*  30 d ST
17. Giusti et al (2009) PCI (772) C19*  6 month ST and ST + cardiac mortality
18. Bhatt et al (2009) Stable(4862) CYP2C19*2, *3, and * No relation between 2C19* hetero and outcomes, ? effect of homo 5 5

6. 1) First genome-wide association study (~400,000 SNP’s):
Event-Free Survival Over 1 Year of Follow-up in Sinai Hospital of Baltimore Patients Treated With Clopidogrel Following Percutaneous Coronary Intervention, Stratified by CYP2C19*2 Genotype
1) First genome-wide association study (~400,000 SNP’s):
- Pharmacogenomics of Antiplatelet Intervention (PAPI) study Identify genes associated with clopidogrel response variability Homogeneous founder population - Healthy Older Order Amish (n=429)
- Minimized confounding factors-medications, lifestyle, etc.
- Clopidogrel 300 mg load then 75 mg qd x 6 days Association analyses between SNP’s and ADP-induced aggregation by variance component model (additive effect of genotype)
Genotyping was performed with the Affymetrix GeneChip Human Mapping 500K or 1 M (version 6.0) arrays (Affymetrix Inc, Santa Clara, CA)
2) Replication and clinical outcome study in PCI patients (n=227)
TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA)

7. 1) Genome-Wide Association Study of Adenosine Diphosphate-Stimulated Platelet Aggregation in Response to Clopidogrel
13 SNP’s
1) 13 SNP’s cluster (within and flanking CYP2C18-2C19-2C9-2C8 cluster) (1.5 megabases on 10q24) strongly associated with clopidogrel response (p <10-7).
2) SNP’s in strong linkage disequilibrium.
3) None of the SNP’s associated with pre-clopidogrel platelet function or response to ASA.
4) No other region with signals meeting genome-wide significance (p <10-7).
5) No relation of ABCB1 variant or CYP2C19*17 with clopidogrel response.
6) CYP2C19*2 accounted for most or all of the 10q24 association signal (~12% of the variable response)
7) Majority of clopidogrel response variability remains unexplained. .

8. Patients With Ischemic Events (%) 20 µM ADP-Induced Aggregation (%)
CYP 2C19*2 Frequency, Platelet Function and Ischemic Events
Patients (n)
Patients With Ischemic Events (%)
160 64 3 40 80
120
-/ /* *2/*2
(Frequency = 30%)
CYP2C19*2 Frequency in Total Population
Relation of Ischemic Events Frequency to Genotype 5 10 15 20 25
21%
10%
+ CYP2C19* (n=67)
- CYP2C19* (n=160)
p=0.02
(n = 14)
(n = 16) 15 30 45 60 75
-/ /* *2/*2
p=0.02
Without Events
With Events
p=0.004
20 µM ADP-Induced Aggregation (%)
Platelet Function Measurement 8 8 8

9. High Platelet Reactivity (HPR) and CYP 2C19*2 Frequency
and Their Relation to Post-PCI Ischemic Event Occurrence
Total Patients
No Events
With Events
Neither 56%
HPR 13%
Both 20%
CYP* %
Neither
61%
HPR
11%
Both
10%
CYP*2
18%
25%
29%
17%
Specificity CYP*2 = 72%
HPR = 79%
Sensitivity CYP*2 = 46%
HPR = 46%
Gurbel PA et al Presented at i2 Summit Symposium, Orlando 2009 9

10. Cox Model HRs by Treatment Arm
CHARISMA Genomics Study
Cox Model HRs by Treatment Arm
Effect
p value HR
95% CI for HR
Effect of Genotype in Placebo Group
*2/WT vs WT/WT
0.54
0.852
(0.51, 1.42)
*2/*2 vs WT/WT
0.19
1.852
(0.74, 4.65)
*2/*17 vs WT/WT
0.47
1.285
(0.65, 2.54)
*17/WT vs WT/WT
0.052
1.486
(1.00, 2.21)
*17/*17 vs WT/WT
0.71
0.841
(0.33, 2.11)
Effect of Genotype in Clopidogrel Group
*2/WT vs WT/WT
0.63
1.112
(0.72, 1.71)
*2/*2 vs WT/WT
0.022
2.383
(1.14, 5.00)
*2/*17 vs WT/WT
0.36
1.322
(0.72, 2.42)
*17/WT vs WT/WT
0.72
0.927
(0.61, 1.40)
*17/*17 vs WT/WT
0.44
0.696
(0.28, 1.74)
*2/*2 has increased risk in clopidogrel arm, but much of this risk is also present in placebo arm
Presented at TCT by Bhatt et al.

11. All GUSTO Bleeding Events: Logistic Regression ORs by Treatment Arm
Effect
p value OR
95% CI for OR
Treatment vs Placebo
<0.0001
2.4110
(1.978, 2.943)
Effect of Genotype in Placebo Group
*2/WT vs WT/WT
0.6392
1.0640
(0.821, 1.374)
*2/*2 vs WT/WT
0.3528
0.7250
(0.340, 1.400)
*2/*17 vs WT/WT
0.5803
1.1180
(0.748, 1.641)
*17/WT vs WT/WT
0.8618
0.9790
(0.769, 1.243)
*17/*17 vs WT/WT
0.3477
0.7910
(0.469, 1.278)
Effect of Genotype in Clopidogrel Group
0.1712
0.8550
(0.683, 1.070)
4 x 10-4
0.3290
(0.160, 0.619)
0.0652
0.7270
(0.513, 1.020)
0.9222
1.0100
(0.824, 1.238)
0.2236
1.2770
(0.860, 1.891)
Significantly reduced risk of bleeding events for the *2/*2 genotype vs wt/wt on clopidogrel
Significantly more bleeding events in wt/wt subjects on clopidogrel compared with placebo
Bleeding risk in *2/*2 is not significantly different between treatments
Presented at TCT by Bhatt et al.

12. 2C19*17, Platelet Aggregation and Bleeding
1524 Post PCI Patients
6.4 M ADP-Induced Aggregation
30- Day Incidence of TIMI Bleedings
Caveats: - *2 not analyzed relation of platelet function to bleeding not reported *2 and *17 variants are 19,959 base pairs apart and in linkage disequilibrium Therefore, pts. with one or two *17 alleles are less likely to carry the *2 allele.
- Pts with zero *17 alleles (“wild type” homozygotes at the *17 locus) are more likely to carry the *2 allele.
- Greater clopidogrel response in *17 group can be partly or completely attributed to a decreased frequency of the *2 allele.
Sibbing D et al. Circulation 2010;121: 12

13. Clinical Outcomes in High Risk Patients (PCI/ACS)
Conclusions
Pharmacogenetic Measurement (CYP2C19*2)
Pharmacokinetic Measurement (reduced clopidogrel active metabolite)
Pharmacodynamic Measurement
(high on-treatment platelet reactivity)
? Independent Effect CHARISMA Placebo Group
Clinical Outcomes in High Risk Patients (PCI/ACS)
Definitive association between: pharmacogenetic, pharmacokinetic and pharmacodynamic measurements and clinical outcomes has not yet been confirmed in a single adequately powered prospective study

14. Future of Personalized Antiplatelet Therapy
Larger prospective studies are needed to confirm the Association of :
- CYP2C19*2 - High on-treatment platelet reactivity - Increased ischemic risk
- CYP2C19*17- Low on-treatment platelet reactivity - Increased bleeding risk
- Establish the role of genotyping vs. platelet function measurements for personalized antiplatelet therapy (alternative or complementary?).
VerifyNow
Multiplate Analyzer
Blood Volume ~ 3 mL
Time ~ 30 minutes
POC Candidates for Measurement of Phenotype 14

15. POC Candidates for Measurement of Genotype
Blood Volume = 1 mL
Single Cell Cartidge
Time ~ 3 hours
Blood Volume = 1 mL
24-Well Plate Format
Time ~ 3 hours 15

16. Genotyping: Which One Should we Perform?
None at this time Most of data are on 2C19*2
How to Interpret the Data?
Genotype is not a surrogate for phenotype
*2 – ischemic risk
*17- Independent bleeding risk?
Diplotype analysis may enhance risk assessment
Is it Ready for Clinical Practice?
Not yet- No prospective studies available 16 16