`
`Role of phenotypic and genetic testing in managing clopidogrel therapy
`Noel C. Chan,1 John W. Eikelboom,1,2,3 Jeffrey S. Ginsberg,2,3 Mandy N. Lauw,1,4 Thomas Vanassche,1 Jeffrey I. Weitz,2,3
`and Jack Hirsh3
`
`1Population Health Research Institute, Hamilton, Canada; 2Thrombosis and Atherosclerosis Research Institute, Hamilton, Canada; 3Department of
`Medicine, McMaster University, Hamilton, Canada; and 4Department of Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands
`
`The P2Y12 inhibitors, clopidogrel, prasu-
`grel, and ticagrelor, are administered in
`fixed doses without laboratory monitor-
`ing. Randomized trials in acute coronary
`syndrome have shown that prasugrel and
`ticagrelor are more effective than standard-
`dose clopidogrel. Nonetheless, standard-
`dose clopidogrel remains widely used
`because it causes less bleeding and is less
`expensive. Patients treated with standard-
`
`dose clopidogrel have substantial vari-
`ability in platelet inhibition, which is partly
`explained by genetic polymorphisms en-
`coding CYP2C19, the hepatic enzyme in-
`volved in biotransformation of clopidogrel
`to its active metabolite. Some advocate
`tailoring P2Y12 inhibitor therapy according
`to the results of routine laboratory testing.
`Although there is good evidence for ana-
`lytic, biological, and clinical validity of
`
`several phenotypic and genotypic bio-
`markers, the benefit of a management strat-
`egy that incorporates routine biomarker
`testing over standard of care without such
`testing remains unproven. Appropriately
`designed, adequately powered trials are
`needed but face the challenges of feasibil-
`ity, cost, and the progressive switch from
`clopidogrel to prasugrel or ticagrelor.
`(Blood. 2014;124(5):689-699)
`
`Introduction
`
`Dual antiplatelet therapy with aspirin and a P2Y12 ADP receptor an-
`tagonist is a mainstay of treatment of acute coronary syndrome (ACS).
`Clopidogrel has been the P2Y12 inhibitor of choice and is given in fixed
`doses without laboratory monitoring. Although effective, standard doses
`of clopidogrel fail to completely inhibit ADP-induced aggregation in up
`to 30% of patients, a phenomenon labeled poor response.1,2 Prasugrel
`and ticagrelor, the newer P2Y12 inhibitors, are more effective than
`clopidogrel,3,4 prompting some guidelines to recommend these
`agents over clopidogrel in ACS.5-8 Nevertheless, clopidogrel remains
`widely used because it causes less bleeding and costs less.9
`Some experts advocate individualizing P2Y12 inhibitor therapy based
`on laboratory test results,10,11 justifying their approach on 2 assumptions:
`(1) platelet function tests1,2,12 and genetic polymorphisms13-16 can
`identify poor responders to clopidogrel and (2) intensifying treatment in
`poor responders improves outcome. Treatment intensification strategies
`include doubling the clopidogrel dose or switching to prasugrel or
`ticagrelor. Although intensifying treatment increases efficacy, it also
`increases bleeding risk. Others reject routine phenotypic and genetic
`testing because its clinical utility is unknown.17-19
`This review focuses on current understanding of the value of
`phenotypic and genetic testing to identify poor responders to
`clopidogrel. We limited discussion to clopidogrel because it is the
`most widely used P2Y12 inhibitor and shows the greatest between-
`patient variability in pharmacological effect.20-22
`
`Pharmacokinetic and pharmacodynamic
`variability of clopidogrel
`
`clopidogrel is converted by esterases to a carboxylic acid metabolite
`lacking P2Y12 antagonism. Blood levels of the active metabolite vary
`widely among patients,15,27,28 and the inhibitory effect of clopidogrel on
`ADP-induced platelet aggregation is also variable.1,2,29 Increasing the
`clopidogrel dose does not eliminate variability in inhibition of ADP-
`induced platelet aggregation.30-32 Differences in drug absorption,33
`enzyme activity,15 drug-to-drug interactions (eg, statins, proton pump
`inhibitors, and calcium channel blockers),1,34 age,1 body mass index,1
`diabetes,35 high epinephrine states, hyperfibrinogenemia, and genetic
`factors contribute to the variable response to clopidogrel.14 However,
`substantial variability in response to clopidogrel remains unexplained.36
`Prasugrel is also a prodrug, but compared with clopidogrel, bio-
`activation of prasugrel involves one less step, and is less susceptible
`to genetic variation and drug interactions.25 Like clopidogrel, the
`active metabolite of prasugrel binds irreversibly to P2Y12, but
`prasugrel exhibits less between-subject variability in peak concen-
`tration and exposure in healthy subjects. The coefficients of variation
`(CVs) for maximum plasma concentrations (Cmax) of prasugrel and
`clopidogrel are 40% and 55%, respectively, whereas those for area
`under curve (AUC) are 30% and 50%, respectively.27,28 Data on
`variability of pharmacokinetic parameters in ACS and percutaneous
`coronary intervention (PCI) populations are lacking. Ticagrelor is
`a direct-acting P2Y12 inhibitor that does not require metabolic
`activation and shows similar between-subject variabilities as
`prasugrel. The CVs for Cmax and AUC are both ;40% in healthy
`subjects.37 Compared with clopidogrel, prasugrel and ticagrelor
`produce greater and more consistent platelet inhibition.20-22,27,28,37
`
`Predictive biomarkers to identify poor responders to clopidogrel
`
`Clopidogrel, a prodrug, requires bioactivation in the liver.23 About
`50% of oral clopidogrel is absorbed in the intestine,24 of which 15%
`is activated via 2 sequential oxidative steps involving the hepatic
`CYP450 system.25,26 In a competing pathway, ;85% of absorbed
`
`Predictive biomarkers, which can be phenotypic or genotypic, identify
`subgroup(s) of patients who may have a better clinical response with
`an intensified antiplatelet regimen.38-40 Phenotypic biomarkers
`measure the inhibitory effects of clopidogrel on ADP-mediated
`
`Submitted January 8, 2014; accepted June 16, 2014. Prepublished online as
`Blood First Edition paper, June 20, 2014; DOI 10.1182/blood-2014-01-
`512723.
`
`© 2014 by The American Society of Hematology
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`Table 1. Criteria to establish clinical utility of predictive biomarker
`
`Domains
`
`Criteria
`
`Questions to be answered
`
`Specific comments
`
`Technical efficacy
`
`1. Analytical validity
`
`Does the test measure the biomarker reliably?
`
`2. Biological validity
`
`Does the test measure a variable that is uniquely
`
`Does the phenotypic test measure the inhibitory
`
`related to either the pharmacokinetics or
`
`effect of clopidogrel on ADP-induced platelet
`
`pharmacodynamics of clopidogrel?
`
`activation?
`
`Does the genetic test predict reduced (or
`
`increased) concentrations of active clopidogrel
`
`metabolite?
`
`3. Clinical validity
`
`Does the biomarker predict clinical state reliably
`
`The test predicts greater or less clinical benefit or
`
`and accurately?
`
`harm with clopidogrel in appropriately designed
`
`studies with a sufficient number of predicted
`
`outcomes to draw reliable, meaningful
`
`conclusions.
`
`Therapeutic efficacy
`
`4. Clinical utility
`
`(A) Does measurement of the biomarker and
`
`If the test is predictive of less benefit or more harm,
`
`tailoring therapy according to biomarker improve
`
`there is a strategy available that improves
`
`patient outcome?
`
`clinical outcome; either changing (increasing or
`
`decreasing) dose or using a different agent in
`
`patient identified as hypo- or hyper-responders
`
`by the test.
`
`(B) Comparative efficacy: Does a biomarker-based
`
`When the test is used to guide patient decisions
`
`strategy improve clinical outcome compared
`
`about the use of the treatment strategy that
`
`with newer therapy (prasugrel, ticagrelor, or
`
`improves patient outcomes, the benefits are
`
`higher-dose clopidogrel)?
`
`greater than if the test is not used. (Otherwise
`
`simply use the strategy without testing).
`
`platelet activation. Genotypic biomarkers identify characteristics
`that influence clopidogrel metabolism.13-15
`
`Conceptual framework for evaluating predictive biomarkers
`
`We propose 4 criteria (Table 1) to evaluate phenotypic and genotypic
`biomarkers for identifying poor responders to clopidogrel41:
`
`1. Analytical validity focuses on test precision and accuracy for
`measuring the biomarker.
`2. Biological validity informs on test ability to measure the
`inhibitory effect of clopidogrel on ADP-induced platelet activation
`(phenotypic) or the concentration of the active metabolite (genetic).
`3. Clinical validity informs on test ability to predict clinical outcome.
`Although clinical validity is important, it does not prove clinical utility.
`4. Clinical utility informs on whether modifying treatment based
`on the biomarker test result improves clinical outcome. In this
`review, we focus on the modulation of P2Y12 inhibition based
`on biomarker results rather than treatment modification involving
`alternative revascularization strategies such as avoidance of PCI or
`consideration of coronary artery bypass.
`
`Three study designs (Table 2) have been used to evaluate the
`clinical utility of phenotypic and genetic biomarker testing.42,43
`
`1. Design A. The biomarker enrichment design examines whether
`intensified treatment (high-dose clopidogrel, prasugrel, or ticagre-
`lor) is better than standard-dose clopidogrel in poor responders
`identified by biomarker testing. It is limited because any observed
`benefit of experimental treatment cannot be attributed to biomarker
`testing nor does it inform on the efficacy or safety of intensified
`treatment relative to control treatment in normal responders.
`2. Design B. The biomarker by treatment interaction design
`randomizes patients into experimental or control arms. Biomarker
`testing is then performed to identify poor and normal responders to
`clopidogrel. Because subjects are not randomized into a biomarker
`testing or nontesting strategy, such studies are not as rigorous as
`design C. Alternatively, biomarker testing could be performed
`
`prerandomization to stratify patients into poor and normal
`responders (biomarker-stratified design).
`3. Design C. The biomarker strategy is the best design because
`it randomizes patients to use or nonuse of a biomarker strategy. If
`the biomarker strategy is used, poor responders receive intensified
`treatment and normal responders receive standard-dose clopidogrel.
`In contrast, patients randomized to nonuse of the biomarker strategy
`receive standard-dose clopidogrel. This design requires the largest
`sample size because only ;30% of patients in the biomarker
`strategy arm will be poor responders.
`
`As predictive biomarkers, several phenotypic tests (Table 3) and a
`genetic test13-15 satisfy the first and second criteria, some satisfy the
`third, but to date, none has satisfied the fourth. Consensus guideline
`committees (and clinicians) should determine whether satisfying the
`first 3 criteria, without exploring the fourth, is sufficient to recommend
`routine screening of clopidogrel-treated patients.
`
`Review of phenotypic biomarkers
`
`Table 3 lists the features of 6 commonly used phenotypic assays44,45:
`(1) light transmission aggregometry (LTA); (2) VerifyNow P2Y12;
`(3) multiplate impedance aggregometry (MEA); (4) PFA-100
`(INNOVANCE P2Y cartridge); (5) thromboelastography (TEG);
`and (6) vasodilator-stimulated phosphoprotein (VASP) assay.
`The first 5 assays measure the inhibitory effect of clopidogrel
`on ADP-induced platelet aggregation using different methods of
`detection, including light absorbance for LTA and VerifyNow,
`electrical impedance for MEA, closure time for PFA-100, and clot
`tensile strength for TEG. We consider the use of the PFA100 system
`in conjunction with the newer
`INNOVANCE P2Y cartridge
`rather than the conventional Dade PFA collagen/ADP test cartridge,
`which is insensitive to P2Y12 inhibitors.46 Using flow cytometry,
`the VASP assay measures downstream effects of clopidogrel on
`ADP-induced P2Y12 receptor activation. Of the 6 assays, only
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`Table 2. Comparison of 3 study designs to evaluate clinical utility of biomarkers
`
`Study design
`
`A. Biomarker enrichment
`
`Schematic diagram
`
`B. Biomarker stratified or by treatment
`interaction
`
`C. Biomarker strategy
`
`Primary question
`
`Is new treatment in biomarker-positive
`
`Is improvement observed with the new treatment
`
`Is a management strategy based on biomarker
`
`patients superior to standard of care?
`
`in biomarker-positive patients significantly
`
`testing with consequent treatment
`
`better than that in the biomarker-negative
`
`modification in biomarker-positive patients
`
`Inception cohort
`
`Biomarker-positive subpopulation
`
`Stratification
`
`No
`
`Randomization
`
`By treatment
`
`patients?
`
`All comers
`
`By biomarker status
`
`By treatment
`
`superior to standard of care?
`
`All comers
`
`No
`
`By biomarker testing
`
`Information
`
`obtained
`
`Informs on whether new treatment in
`
`Informs on whether biomarker status is a
`
`Informs on whether biomarker testing and
`
`biomarker-positive patient is clinically
`
`determinant of response to treatment options,
`
`treatment modification based on such
`
`useful.
`
`and whether such testing would be clinically
`
`testing is clinically useful.
`
`useful.
`
`VerifyNow P2Y12 is a true point-of-care assay, being easy to
`perform and having a rapid turnaround time.47
`
`Analytical validity
`
`A systematic review by the Agency for Health and Quality Research
`identified .100 studies assessing the analytical performance of
`phenotypic assays.45 All 6 tests (Table 3) were evaluated by
`
`assessing (1) reproducibility in replicate samples (intra-assay CV),
`(2) correlation between LTA and other assays, and (3) test agreement
`between LTA and other assays, summarized by k statistics. The intra-
`assay CV is reported as ,11% (;30 studies); an acceptable result
`in view of the wide between-subject variability in the pharmacody-
`namic response to clopidogrel (CV ; 70%).1 Although most studies
`reported moderate to good correlation between LTA and the other
`
`Table 3. Phenotypic biomarkers
`
`Assays
`
`LTA
`
`plasma
`
`platelet
`
`aggregation
`
`VerifyNow P2Y12
`
`Whole blood
`
`ADP-induced
`
`Sample
`
`Principle of assay
`
`Measurement
`method
`
`Analytical validity†
`(range)
`
`Biological validity Clinical validity Clinical utility
`
`Platelet-rich
`
`ADP-induced
`
`Light absorbance
`
`CV 5 3.3-11.3%
`
`†
`
`Low-quality
`
`evidence
`
`‡
`
`‡
`
`Light absorbance
`
`platelet
`
`aggregation
`
`(with PGE1
`
`modulation)
`
`Multiplate electrode
`
`Whole blood
`
`ADP-induced
`
`Electrical impedance
`
`aggregometry
`
`(MEA)
`
`PFA-100
`
`platelet
`
`aggregation
`
`Whole blood
`
`Shear-dependent
`
`Closure time: Time
`
`(INNOVANCE P2Y)
`
`ADP-induced
`
`for platelet plug
`
`platelet adhesion
`
`to stop blood
`
`and aggregation
`
`flow across
`
`aperture
`
`Thromboelastography
`
`Whole blood
`
`Kinetic changes
`
`Tensile strength
`
`(Haemoscope TEG)
`
`with ADP-
`
`induced clot
`
`formation
`
`of clot
`
`Vasodilator
`
`Whole blood
`
`ADP-induced P2Y12
`
`Flow cytometry to
`
`stimulatory protein
`
`assay (VASP)
`
`receptor activation–
`
`quantify VASP
`
`dependent
`
`phosphorylation
`
`phosphorylation
`
`CV 5 6-7.5%,
`r 5 0.35-0.86
`k 5 0.2-0.82
`
`CV 5 5-10%
`r 5 0.25-0.87
`k 5 0.1-0.7
`CV 5 7.7-9.5%
`r 5 20.7 to 20.11
`k 5 0.14-0.35
`
`CV 5 4.5-6.6%
`r 5 0.32-0.82
`k 5 20.02 to 0.81
`
`CV 5 2.3-6.6%
`r 5 0.36-0.72
`k 5 20.04-0.31
`
`†
`
`†
`
`†
`
`†
`
`†
`
`Moderate-
`
`quality
`
`evidence
`
`Low-quality
`
`evidence
`
`Low-quality
`
`evidence
`
`Insufficient
`
`evidence
`
`Low-quality
`
`evidence
`
`‡
`
`‡
`
`‡
`
`‡
`
`k, k statistics; PGE1, prostaglandin E1; r, correlation coefficient.
`*CV refers to intra-assay coefficient of variation; measures of test agreement (k) and correlation (r) refer to the comparison of given test with LTA.
`†All measure consequences of ADP-induced platelet activation.
`‡Insufficient evidence to prove or disprove clinical utility of a biomarker strategy.
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`Table 4. Meta-analyses evaluating the relation between poor response to clopidogrel and clinical outcome in PCI
`
`Meta-analyses
`
`Population
`
`Snoep et al49
`
`PCI
`
`Types of included
`studies
`
`Any observational
`
`studies/sub-analyses
`
`of RCT
`
`No of
`studies/size
`
`25, n 5 3688
`
`Poor
`responders, %
`
`Assays
`
`Outcome
`
`21
`
`LTA, VASP,
`
`flow cytometry
`
`of platelet-bound
`
`fibrinogen
`
`Composite MACE
`OR 5 8.00 (3.36-19.05)
`Stent thrombosis
`OR 5 7.03 (0.63-79.01)
`Clinical ischemic events
`OR 5 12.02 (5.91-24.42)
`Composite MACE
`OR 5 5.67 (2.97- 10.84)
`
`Composite MACE
`OR 5 4.95 (3.34-7.34)
`Stent thrombosis
`OR 5 4.14 (2.74-6.25)
`Cardiovascular death
`OR 5 3.35 (2.39-4.70)
`Non-fatal MI
`OR 5 3.00 (2.26-3.99)
`For PRU cutoff .230 U
`Composite MACE
`HR 5 2.10 (1.62-2.73)
`Stent thrombosis
`HR 5 3.11 (1.50–6.46)
`Death
`HR 5 1.66 (1.04-2.68)
`Composite MACE
`OR 5 3.05 (2.33-3.98)
`Stent thrombosis
`OR 5 3.26 (1.63-6.51)
`Death
`OR 5 2.00 (1.22-3.27)
`
`Sofi et al50
`
`PCI; 5 stable
`
`Prospective observational
`
`14, n 5 4564
`
`26.4
`
`LTA, VASP,
`
`CAD-only
`
`studies
`
`studies/sub-analyses
`
`of RCT
`
`VerifyNow P2Y12
`
`Aradi et al51
`
`PCI; 4 stable
`
`Prospective observational
`
`20, n 5 9187
`
`33.2
`
`LTA, VASP,
`
`CAD-only
`
`studies
`
`studies/sub-analyses
`
`of RCT
`
`VerifyNow P2Y12,
`
`MEA
`
`Brar et al52
`Individual patient
`
`data meta-
`
`analysis
`
`Yamaguchi
`et al53
`
`PCI; 1 stable
`
`Only prospective studies
`
`6, n 5 3059
`
`37.1
`
`VerifyNow P2Y12
`
`CAD-only
`
`study
`
`involving VerifyNow
`
`P2Y12 assay
`
`assay only
`
`PCI (98.5%)
`
`Only prospective studies
`
`8, n 5 4817
`
`46.4
`
`VerifyNow P2Y12
`
`involving VerifyNow
`
`P2Y12 assay
`
`assay only
`
`assays, test agreement was poor, in part because cutoffs were not
`rigorously evaluated (Table 3).
`
`Biological validity
`
`All 6 assays are biologically valid because each measures $1
`consequence of P2Y12 receptor stimulation by ADP: platelet
`activation, platelet aggregation, or clot formation. The VASP assay
`quantifies phosphorylated VASP levels downstream to the P2Y12
`receptor, which is a measure of platelet activation.48 The TEG
`measures clot tensile strength. The other assays capture clopidog-
`rel’s inhibition of P2Y12 by measuring platelet aggregation and
`are susceptible to variables that influence the optical (LTA and
`VerifyNow) and impedance (MEA) end points. Test selection
`depends on feasibility in clinical trials. The most convenient test is
`the VerifyNow P2Y12 assay. Clinical outcome studies are required
`to determine a test’s cutoff values.12 An optimal cutoff value is
`identified by performing an exploratory study to identify the cutoff,
`which is then prospectively tested in a confirmatory clinical outcome
`study.
`
`Clinical validity
`
`Adverse cardiovascular outcomes. Most studies were performed
`in the setting of PCI and used major adverse cardiovascular events
`(MACE) and stent thrombosis as efficacy outcomes.12,45 Five meta-
`analyses of prospective observational studies and subanalyses of
`randomized controlled trials (RCTs) involving .10 000 PCI
`patients have been published (Table 4).49-53 All reported strong
`
`associations between poor response to clopidogrel and adverse
`cardiovascular outcomes with the 4 commonly evaluated assays
`(LTA, VerifyNow P2Y12, VASP, and MEA). The odds ratios
`(ORs) were significant for MACE (range, 2.1-8.0) and stent
`thrombosis (range, 3.1-7.0).
`Limited information is available in medically managed patients
`with coronary artery disease (CAD). The largest study in medically
`managed ACS patients, a nested substudy (n 5 2,564) of the
`targeted platelet inhibition to clarify the optimal strategy to
`medically manage acute coronary syndromes (TRILOGY ACS)
`trial, failed to show an independent association between poor
`response and MACE (adjusted hazard ratio [HR], 1.03; 95%
`confidence interval [CI], 0.96-1.11).54
`Bleeding. Results of studies examining the relationship between
`enhanced response to clopidogrel and bleeding have been incon-
`sistent. Two observational studies support a relationship between
`clopidogrel response and bleeding. In the first, enhanced respon-
`siveness to clopidogrel by MEA showed a 3.5-fold increase in major
`bleeding in a PCI population (n 5 2533).55 The second,
`the
`assessment of dual antiplatelet therapy with drug eluting stents
`(ADEPT-DES) prospective registry (n 5 8665), reported that poor
`responders had less clinically relevant bleeding (adjusted HR, 0.65;
`95% CI, 0.43-0.99).56 In contrast, 2 large RCTs57,58 failed to show
`an association between clopidogrel response and bleeding but
`were probably underpowered.
`Parallel comparisons of phenotypic assays in the PCI
`population. The meta-analyses do not provide information about
`relative capacities of the various assays to predict clinical out-
`comes. The “Do platelet function assays predict clinical outcomes
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`in clopidogrel pretreated patients undergoing elective PCI”
`(POPULAR) study performed parallel comparison of 8 pheno-
`typic assays to predict 1-year MACE outcome and bleeding in
`1069 consecutive patients.59 The assays differed in their asso-
`ciations with clinical outcomes. Only LTA, VerifyNow P2Y12,
`and Plateletworks (an uncommonly used assay because it needs to
`be performed within 10 minutes) showed significant associations
`with MACE, but the ability to differentiate between responders
`and poor responders was modest (AUC range, 0.61-0.63). None
`of the assays predicted bleeding.
`
`Clinical utility
`
`The clinical utility of phenotypic testing was evaluated in several
`older RCTs in .1500 patients using enrichment designs (design A)
`(Table 5).60-66 Although poor responders to clopidogrel who were
`treated with an alternative P2Y12 inhibitor had improvement in
`clinical outcome,67 these studies do not inform on whether routine
`biomarker testing and treatment intensification in poor responders
`were responsible for the improved outcome.
`Three more recent randomized studies (double randomization
`of a monitoring adjusted antiplatelet treatment vs a common
`antiplatelet treatment for DES implantation, and Interruption vs
`continuation of double antiplatelet therapy [ARCTIC], gauging
`responsiveness with a VerifyNow assay-impact on thrombosis and
`safety [GRAVITAS], and testing platelet reactivity in patients
`undergoing elective stent placement on clopidogrel to guide alternative
`therapy with prasugrel [TRIGGER-PCI]) used VerifyNow to identify
`poor responders.57,58,68 Of these, only ARCTIC used a biomarker
`strategy design (design C) to compare a tailored approach with
`standard-dose clopidogrel in all-comers. The other 2 used an
`enrichment design (design A).
`ARCTIC study: is a phenotypic biomarker based strategy better
`than conventional use of antiplatelet in a PCI population? The
`ARCTIC study (n 5 2440), an open-labeled RCT, enrolled patients
`with stable angina (73%) or ACS (27%) who underwent PCI.57
`Patients were randomized to either standard antiplatelet therapy
`(aspirin and clopidogrel) or the experimental arm of VerifyNow-
`directed antiplatelet therapy. Poor responders to clopidogrel in the
`experimental arm were identified using a cutoff of .235 platelet
`reactivity units (PRUs) or platelet inhibition of ,15% from baseline.
`Prior to PCI, 34.5% of patients were identified as poor responders at
`initial testing and were treated with a glycoprotein IIb/IIIa inhibitor
`and/or an increased loading dose of clopidogrel (600 mg) or prasugrel
`(60 mg), in addition to either maintenance clopidogrel (150 mg daily)
`or prasugrel (10 mg daily). On days 14 to 30 after stent implantation,
`a second VerifyNow test was performed in patients allocated to
`the experimental arm; 15.6% were found to be poor responders.
`The clopidogrel dose was increased further in these patients, or
`they were switched to prasugrel. At 1 year, the MACE rates in the
`experimental and control arms were similar (34.6% and 31.1%,
`respectively; HR, 1.13; 95% CI, 0.98-1.29) as were the rates of
`stent thrombosis (1.0% vs 0.7%, respectively; HR, 1.34; 95% CI,
`0.56-3.18). In addition, there was no significant difference in overall
`rates of bleeding between the groups (4.5% vs 3.1%, respectively;
`HR, 0.90; 95% CI, 0.46-1.05).
`GRAVITAS study: is high-dose clopidogrel better than standard-
`dose clopidogrel in PCI patients identified as poor responders by
`VerifyNow P2Y12? The GRAVITAS study, a blinded RCT, enrolled
`2214 patients with stable angina (60.2%) or ACS (39.8%) who had
`undergone PCI. Poor responders identified with the VerifyNow
`assay (using the consensus cutoff of PRUs $230 at 12-24 hours after
`
`PCI) were randomized to either increased-dose clopidogrel (150 mg
`daily) or standard clopidogrel (75 mg daily). At 6 months, the rates of
`MACE, the primary outcome, in the experimental and control arms
`were similar (2.3% and 2.3%, respectively; HR, 1.01; 95% CI,
`0.58-1.76), as were the rates of stent thrombosis (0.5% and 0.7%,
`respectively; HR, 0.63; 95% CI, 0.21-1.93) and bleeding (1.4% and
`2.3%, respectively; HR, 0.59; 95% CI, 0.31-1.11).
`GRAVITAS is limited because the MACE rate of 2.3% in the
`control group was lower than the projected rate of 5.0%.
`Furthermore, the cutoff PRU value $230 used to classify poor
`responders to clopidogrel may have been too high because a post
`hoc analysis identified a PRU value .208 as being a more
`appropriate cutoff value.69 In addition increasing the clopidogrel
`dose to 150 mg was not sufficient to overcome a poor response to
`clopidogrel because .35% of patients in the experimental arm
`remained poor responders when VerifyNow testing was repeated
`at 1 and 6 months.58
`TRIGGER PCI: is prasugrel better than standard clopidogrel
`in PCI patients identified to be poor responders by VerifyNow
`P2Y12? The TRIGGER PCI study, a blinded RCT, enrolled
`patients with stable angina who had received drug-eluting stents.68
`Poor responders to clopidogrel, identified with the VerifyNow
`assay using a cutoff PRU value of .208 (the cutoff tested post hoc
`in GRAVITAS) were randomized to either standard clopidogrel
`(75 mg) or prasugrel (10 mg) starting in the morning after PCI. The
`trial was stopped for futility after enrollment of only 413 patients
`because of 6-month MACE rates of 0.5% in the control arm and 0%
`in the experimental arm. Therefore, TRIGGER PCI contributes
`little useful information.
`In summary, the 3 largest studies conducted to date have failed
`to show clinical utility of phenotypic assays in ACS patients to
`identify poor responders so that they can be targeted for intensified
`therapy. Two ongoing RCTs are exploring the clinical utility of
`VerifyNow in the PCI population (dual antiplatelet therapy tailored
`on the extent of platelet inhibition [DANTE] and tailored antiplatelet
`therapy vs recommended dose of prasugrel [ANTARCTIC]),70,71
`with the latter focusing on elderly patients.
`
`Genotypic biomarkers
`
`Most genetic biomarker testing has focused on the CYP2C19 gene
`because it is the only one independently associated with variability
`in the platelet inhibitory response to clopidogrel in genome-wide
`or whole-exome association studies.14,72 The CYP2C19 gene
`encodes an enzyme involved in both steps of conversion of
`clopidogrel to its active metabolite.26 This gene is highly poly-
`morphic, with $34 identified polymorphisms, some of which result
`in loss of function (LOF) and others in gain of function (GOF).73
`CYP2C19*2 and CYP2C19*3 are the most common LOF alleles
`(with an estimated carrier prevalence of 30% in whites, 40% in
`blacks, and 55% in East Asians).74 The other LOF alleles
`(CYP2C19*4, *5, *6, *7, and *8) are much less common (,1%
`allelic frequency each)75 and have not been adequately evaluated in
`clinical studies. Individuals who are heterozygous for LOF alleles are
`intermediate metabolizers, whereas those who are homozygous are
`poor metabolizers of clopidogrel.
`Although LOF CYP2C19 genotypes are associated with reduced
`ADP-induced platelet aggregation in response to clopidogrel, it is
`estimated that the common CYP2C19*2 allele explains only 12% of
`the variation in platelet response.14,72 With other factors collectively
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1030, p. 5 of 11
`
`
`
`694
`
`CHAN et al
`
`BLOOD, 31 JULY 2014 x VOLUME 124, NUMBER 5
`
`Table 5. RCTs evaluating clinical utility of phenotypic testing in the PCI setting
`
`Studies
`(author/acronym)
`
`RCT
`design/size
`
`Collet et al57
`ARCTIC
`
`Design C
`n 5 2440
`
`Population
`
`Assay/cutoff
`
`PCI with DES
`
`ACS 27%
`
`(no STEMI)
`
`VerifyNow P2Y12
`$235 U
`(at 2 time points)
`
`Poor
`responders (%)
`
`Intervention in poor responders
`
`Outcome intervention vs control
`
`34.5
`
`Clopidogrel (600 mg reloading,
`
`MACE:
`
`75 or 150 maintenance), or
`
`34.6% vs 31.1%
`
`prasugrel, or GpIIb/IIIa
`
`(HR: 1.13; 95% CI: 0.98-1.29)
`
`Stent thrombosis:
`
`1.0% vs 0.7%
`
`(HR: 1.34; 95% CI: 0.56-3.18)
`
`Major bleeding:
`
`2.3% vs 3.3%
`
`(HR:0.70; 95% CI:0.43-1.14)
`
`Price et al58
`GRAVITAS
`
`Design A
`n 5 2214
`
`Trenk et al68
`TRIGGER-PCI
`
`Design A
`n 5 423
`
`PCI with DES
`
`VerifyNow
`
`41
`
`600/150 mg clopidogrel
`
`MACE:
`
`ACS 10.5%
`
`P2Y12
`$230 U
`
`(VerifyNow)
`
`2.3% vs 2.3%,
`
`(HR: 1.01; 95% CI: 0.58-1.76)
`
`Severe or moderate bleeding:
`
`1.4% vs 2.3%
`
`(HR: 0.59; 95% CI: 0.31-1.11)
`
`Elective PCI
`
`VerifyNow
`
`19
`
`Prasugrel 10 mg maintenance
`
`(Stopped early because of futility)
`
`with DES
`
`ACS 0%
`
`P2Y12
`.208 U
`
`CV death or MI:
`
`0 vs 1 event
`
`Stent thrombosis:
`
`0 vs 0 event
`
`Major bleeding:
`
`3(1.4%) vs 1(0.5%) events
`
`Hazarbasanov
`et al99
`
`Design C
`n 5 192
`
`PCI
`
`ACS 56.8%
`
`MEA
`$46 U
`
`18.5
`
`Second loading dose
`
`clopidogrel 600 mg and
`
`150 mg maintenance for
`
`1 month
`
`Ari et al60
`EFFICIENT
`
`Design A
`n 5 94
`
`Elective PCI
`
`VerifyNow
`
`48.9
`
`Clopidogrel 150 mg maintenance
`
`ACS 0%
`
`P2Y12
`,40% inhibition
`
`Aradi et al61
`DOSER
`
`Design A
`n 5 74
`
`PCI
`
`ACS 0%
`
`LTA $34% max agg
`
`38
`
`150 mg maintenance clopidogrel
`
`Wang et al62
`
`Design A
`n 5 306
`
`PCI
`
`ACS 20%
`
`VASP-PRI
`.50%
`
`57
`
`Dynamic adjustment of
`
`maintenance clopidogrel up
`
`Valgimigli et al63
`
`Design A
`n 5 147
`
`PCI
`
`ACS 32.6%
`
`VerifyNow P2Y12
`,40% inhibition
`
`Bonello et al64
`
`Design A
`n 5 429
`
`PCI
`
`ACS 52.3%
`
`VASP-PRI .50%
`
`Bonello et al65
`
`Design A
`n 5 162
`
`PCI
`
`ACS 48%
`
`VASP-PRI .50%
`
`Cuisset et al66
`
`Design A
`n 5 149
`
`PCI
`
`ACS 0%
`
`LTA .70% max agg
`
`to 375 mg daily
`(VASP # 50%)
`Tirofiban
`
`Clopidogrel 600 mg reloading,
`aim VASP # 50%
`
`Clopidogrel 600 mg reloading
`
`Abciximab
`
`27
`
`45
`
`52
`
`23
`
`MACE:
`0 (0.0%) vs 5(2.6%) P 5 .03
`Stent thrombosis:
`9 (0.0%) vs 4(2.1%) P 5 .06
`Major bleeding:
`
`1 vs 0 event
`
`MACE:
`2(4.3%) vs 8(17%) P 5 .02
`Major bleeding:
`
`1(2.1%) vs 0 (0%) ns
`
`MACE:
`1(3.1%) vs 8(24.6%), P 5 .01
`Major bleeding:
`
`1(2.8%) vs 0, ns
`
`MACE:
`9.3% vs 20.4%, P 5 .008
`Major bleeding
`
`0 vs 0
`
`MACE:
`3.8% vs 10.7%, P , .05
`Major bleeding:
`
`0% vs 0%
`
`MACE:
`0.5% vs 8.9%, P , .001
`Major bleeding:
`0.9% vs 0.9%, P 5 .1
`MACE:
`0% vs 8(10%), P 5 .007
`Major bleeding: 1.3% vs 1.3%
`
`MACE:
`19% vs 40%,OR 5 2.8, P 5 .006
`Major bleeding:
`
`0% vs 0%
`
`DES, drug eluting stent; Max agg, maximum aggregation; MEA, multiplate electrode; ns, not significant; STEMI, ST elevation myocardial infarction.
`
`explaining .70% of the variation,14 treatment modification based on
`CYP2C19 testing alone is unlikely to have a major impact on outcome.
`CYP2C19*17, a GOF allele, occurs in 2% to 5% of Asians and
`20% to 25% of whites and blacks.76 Although initially reported
`to be associated with an exaggerated response to clopidogrel, subjects
`with this GOF haplotype lack the CYP2C19*2 LOF allele, raising the
`
`possibility that the gain of effect attributed to CYP2C19*17 allele is
`caused, at least in part, by the absence of CYP2C19*2 allele.77
`
`Analytical validity
`
`A systematic review of 11 studies reported good reproducibility
`of CYP2C19 genotyping methods and high levels of interassay
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1030, p. 6 of 11
`
`
`
`BLOOD, 31 JULY 2014 x VOLUME 124, NUMBER 5
`
`TAILORED ANTIPLATELET THERAPY
`
`695
`
`Table 6. Meta-analyses evaluating association between CYP2C19 LOF and clinical outcome
`
`Authors
`
`Hulot et al82
`Mega et al83
`
`No of studies
`
`No of patients
`
`LOF vs non-LOF MACE 95% CI
`
`LOF vs non-LOF stent thrombosis 95% CI
`
`10
`
`9
`
`15
`
`32
`
`8
`
`18
`
`7
`
`16
`
`13
`
`11 959
`
`9 685
`
`19 328
`
`42 016
`
`8 280
`
`21 441
`
`8 043
`
`20 785
`
`16 360
`
`OR 1.29 (1.12-1.49)
`
`OR 1.55 (1 LOF)
`
`(1.11-2.17)
`
`OR 1.76 (2 LOF)
`
`(1.24-2.50)
`
`OR 1.11 (0.89-1.39)
`
`RR 1.18 (1.09-1.28)
`
`N/R
`
`OR 1.26 (1.06-1.50)
`
`RR 1.96 (1.14-3.37)
`
`OR 1.42 (1.13-1.78)
`
`HR 1.23 (0.97-1.55)
`
`OR 3.45 (2.14-5.57)
`
`OR 2.67 (1 LOF)
`
`(1.69-4.22)
`
`OR 3.97 (2 LOF)
`
`(1.75-9.02)
`
`OR 1.77 (1.31-2.40)
`
`RR 1.75 (1.50-2.03)
`
`OR 3.81 (2.27-6.40)
`
`OR 2.58 (1.77-3.77)
`
`RR 3.82 (2.22-6.54)
`
`OR 2.41 (1.76-3.30)
`
`HR 2.24 (1.5203.30)
`
`Bauer et al84
`Holmes et al29
`Jin et al85
`Liu et al86
`Sofi et al87
`Jang et al88
`Zabalza et al89
`Mao et al90
`Yamaguchi et al53
`AHRQ45
`
`21
`
`7
`
`N/R
`
`23 035
`
`5 307
`
`N/R
`
`OR 1.56 (1.21-1.87)
`
`N/R
`
`RR 1.
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