Genetic Testing for CYP 450 Polymorphisms to Predict Response to Clopidogrel : current evidence and test availability Application : Pharmacogenomics

The anti-platelet agent clopidogrel bisulfate (sold under the trade name Plavix in the United States) is a widely prescribed medication for the prevention of blood clots in patients with acute coronary syndrome, in those who have suffered other cardiovascular disease-related events such as ischemic stroke, and in patients who are undergoing percutaneous coronary intervention. Response to clopidogrel varies substantially due to genetic and acquired factors. Patients who experience recurrent cardiovascular ischemic or thrombotic events while taking clopidogrel are typically described as non-responsive or resistant. The drug’s oxidation is mainly dependent on the cytochrome P450 enzyme 2C19 (CYP2C19). Patients with certain genetic variants in CYP2C19 have been found to have lower levels of the active metabolite, less platelet inhibition, and greater risk of major adverse cardiovascular events such as heart attack, stroke, and death. Testing for CYP2C19 polymorphisms may identify patients who will not respond adequately to the standard clopidogrel regimen and who should, consequently, be given an alternate treatment strategy. This article outlines the evidence concerning pharmacogenetic testing for clopidogrel response, including data on clinical validity and clinical utility, and summarizes the currently available tests marketed for this purpose.


Saliva No
Clopidogrel Note: These tests were found through Google searches combining terms such as "clopidogrel", "genetic test" and "CYP2C19" and by searching individual websites of known commercial genetics companies (such as Pathway Genomics).Attempts were made to make this table comprehensive.However, there are tests that genotype variants in CYP2C19 that either do not specify that the test is for pharmacogenetic purposes, or do not specifically mention that the test can be used in determining response to clopidogrel.Such tests have not been included in this table.

Public Health Importance
Clopidogrel is the second highest top-selling drug in the world [6] ; approximately 29 million prescriptions were dispensed in 2008 in the United States alone [7] .
The allele frequency of CYP2C19*17 is estimated as 18-27% among Caucasians, 17-18% among Africans/African-Americans, and 0.5 -4% among Asians [4] Recently, the U.S. Food and Drug Administration (FDA) issued a black box warning for clopidogrel due to the reduced effectiveness of the drug in poor metabolizers [17][18] .

Systematic evidence reviews
None.

Recommendations by independent group
None.

Guidelines by professional groups
In July 2010, the American College of Cardiology Foundation (ACCF) and the American Heart Association (AHA) published a Clinical Alert in response to the FDA's black box warning on clopidogrel [19] .The report stated that: The evidence base is insufficient to recommend routine genetic testing at the present time.
"Clinical judgment is required to assess clinical risk and variability in patients considered to be at increased risk.Genetic testing to determine if a patient is predisposed to poor clopidogrel metabolism ("poor metabolizers") may be considered before starting clopidogrel therapy in patients believed to be at moderate or high risk for poor outcomes.This might include, among others, patients undergoing elective high-risk PCI procedures (e.g., treatment of extensive and/or very complex disease)."

Other guidelines
The U.S. Food and Drug Administration (FDA) determined in 2009 that the available data have "provided compelling evidence that genetic variation in CYP2C19 is a significant and independent predictor of clopidogrel pharmacokinetics, pharmacodynamics and clinical response", which prompted the FDA to change clopidogrel's prescribing information [3][20] [21] .
More recently (March 12, 2010), the FDA issued a black box warning for clopidogrel [17][18] due to the reduced effectiveness of the drug in poor metabolizers.The FDA recommended that health professionals be aware that some patients may be poor metabolizers of clopidogrel because of low CYP2C19 activity and to also "be aware that tests are available to determine patients' CYP2C19 status" [17] .The FDA neither mandated nor explicitly recommended CYP2C19 genetic testing in patients prescribed clopidogrel , and the agency did not offer any specific guidance on drug dosing in CYP2C19 variant allele carriers [17] .The November 2009 label update did recommend that doctors avoid use of clopidogrel "in patients with impaired CYP2C19 function due to known genetic variation or due to drugs that inhibit CYP2C19 activity" [21] , while the more recent warning no longer specifically advises against use of clopidogrel in CYP2C19 poor metabolizers [17] .

Public Health Importance
Clopidogrel is the second highest top-selling drug in the world [6] ; approximately 29 million prescriptions were dispensed in 2008 in the United States alone [7] .
The allele frequency of CYP2C19*17 is estimated as 18-27% among Caucasians, 17-18% among Africans/African-Americans, and 0.5 -4% among Asians [4] Recently, the U.S. Food and Drug Administration (FDA) issued a black box warning for clopidogrel due to the reduced effectiveness of the drug in poor metabolizers [17][18] .

Systematic evidence reviews
None.

Recommendations by independent group
None.

Guidelines by professional groups
In July 2010, the American College of Cardiology Foundation (ACCF) and the American Heart Association (AHA) published a Clinical Alert in response to the FDA's black box warning on clopidogrel [19] .The report stated that: The evidence base is insufficient to recommend routine genetic testing at the present time.
"Clinical judgment is required to assess clinical risk and variability in patients considered to be at increased risk.Genetic testing to determine if a patient is predisposed to poor clopidogrel metabolism ("poor metabolizers") may be considered before starting clopidogrel therapy in patients believed to be at moderate or high risk for poor outcomes.This might include, among others, patients undergoing elective high-risk PCI procedures (e.g., treatment of extensive and/or very complex disease)."

Other guidelines
The U.S. Food and Drug Administration (FDA) determined in 2009 that the available data have "provided compelling evidence that genetic variation in CYP2C19 is a significant and independent predictor of clopidogrel pharmacokinetics, pharmacodynamics and clinical response", which prompted the FDA to change clopidogrel's prescribing information [3][20] [21] .
More recently (March 12, 2010), the FDA issued a black box warning for clopidogrel [17][18] due to the reduced effectiveness of the drug in poor metabolizers.The FDA recommended that health professionals be aware that some patients may be poor metabolizers of clopidogrel because of low CYP2C19 activity and to also "be aware that tests are available to determine patients' CYP2C19 status" [17] .The FDA neither mandated nor explicitly recommended CYP2C19 genetic testing in patients prescribed clopidogrel , and the agency did not offer any specific guidance on drug dosing in CYP2C19 variant allele carriers [17] .The November 2009 label update did recommend that doctors avoid use of clopidogrel "in patients with impaired CYP2C19 function due to known genetic variation or due to drugs that inhibit CYP2C19 activity" [21] , while the more recent warning no longer specifically advises against use of clopidogrel in CYP2C19 poor metabolizers [17] .

Public Health Importance
Clopidogrel is the second highest top-selling drug in the world [6] ; approximately 29 million prescriptions were dispensed in 2008 in the United States alone [7] .
The allele frequency of CYP2C19*17 is estimated as 18-27% among Caucasians, 17-18% among Africans/African-Americans, and 0.5 -4% among Asians [4] Recently, the U.S. Food and Drug Administration (FDA) issued a black box warning for clopidogrel due to the reduced effectiveness of the drug in poor metabolizers [17][18] .

Systematic evidence reviews
None.

Recommendations by independent group
None.

Guidelines by professional groups
In July 2010, the American College of Cardiology Foundation (ACCF) and the American Heart Association (AHA) published a Clinical Alert in response to the FDA's black box warning on clopidogrel [19] .The report stated that: The evidence base is insufficient to recommend routine genetic testing at the present time.
"Clinical judgment is required to assess clinical risk and variability in patients considered to be at increased risk.Genetic testing to determine if a patient is predisposed to poor clopidogrel metabolism ("poor metabolizers") may be considered before starting clopidogrel therapy in patients believed to be at moderate or high risk for poor outcomes.This might include, among others, patients undergoing elective high-risk PCI procedures (e.g., treatment of extensive and/or very complex disease)."

Other guidelines
The U.S. Food and Drug Administration (FDA) determined in 2009 that the available data have "provided compelling evidence that genetic variation in CYP2C19 is a significant and independent predictor of clopidogrel pharmacokinetics, pharmacodynamics and clinical response", which prompted the FDA to change clopidogrel's prescribing information [3][20] [21] .
More recently (March 12, 2010), the FDA issued a black box warning for clopidogrel [17][18] due to the reduced effectiveness of the drug in poor metabolizers.The FDA recommended that health professionals be aware that some patients may be poor metabolizers of clopidogrel because of low CYP2C19 activity and to also "be aware that tests are available to determine patients' CYP2C19 status" [17] .The FDA neither mandated nor explicitly recommended CYP2C19 genetic testing in patients prescribed clopidogrel , and the agency did not offer any specific guidance on drug dosing in CYP2C19 variant allele carriers [17] .The November 2009 label update did recommend that doctors avoid use of clopidogrel "in patients with impaired CYP2C19 function due to known genetic variation or due to drugs that inhibit CYP2C19 activity" [21] , while the more recent warning no longer specifically advises against use of clopidogrel in CYP2C19 poor metabolizers [17] .

Evidence Overview
Analytic Validity : Test accuracy and reliability in identifying CYP2C19 genotypes (analytic sensitivity and specificity).
Cross validation of the techniques used inCYP2C19genotyping assays, along with their reliability, specificity, and reproducibility, is extremely limited [19] .Only one clinical trial explicitly reported measures of clinical validity.In the EXCELSIOR (Impact of Extent of Clopidogrel-Induced Platelet Inhibition During Elective Stent Implantation on Clinical Event Rate) trial, the sensitivity and specificity ofCYP2C19*2carrier status for detecting high on-clopidogrel residual platelet aggregation (RPA) was 45.1% and 75.0%, respectively [35] .Measures of sensitivity and specificity can be calculated from data reported in some of the other published studies.

Clinical Validity
No investigators explicitly included estimates of positive predictive value (PPV) or negative predictive value (NPV) for clopidogrel response in their original published reports.Using data from the EXCELSIOR report [35] , estimates of PPV and NPV are 41.6% and 77.6%, respectively.
Clinical Utility : Net benefit of test in improving health outcomes.
A recent meta-analysis of seven prospective cohort studies was published that examined the CYP2C19*2 variant and cardiovascular recurrences in 8,043 coronary artery disease patients taking clopidogrel.The authors found a statistically significant increased risk of major adverse cardiovascular events in CYP2C19*2 carriers (RR = 1.96) and an even greater risk when stent thrombosis was analyzed separately using four studies that included 4,975 patients (RR = 3.82) [56] .
A more recent meta-analysis that was not restricted to prospective cohort studies examined the CYP2C19*2 variant and the incidence of major adverse cardiovascular events (MACE) and mortality in patients taking clopidogrel [57] .The pooled data showed that CYP2C19*2 carriers had a statistically significant increased risk of MACE (OR = 1.29) using data from ten studies (11,959 patients).Carriers of *2 also had statistically significant increases in risk for stent thrombosis (OR = 3.45 using data from 4 studies including 4,905 patients) and death (OR = 1.79 using data from 5 studies including 6,225 patients).Four studies including 5,694 patients allowed the separate assessment of heterozygotes and homozygotes of the *2 allele.Increased risks for MACE and stent thrombosis were noted for both groups, though the odds ratio for MACE was significant only for homozygotes [57] .
Hulot and Fuster [58] calculated the PPV for clinical outcomes ofCYP2C19loss-of-function variants using data from two studies.They estimate the PPV for a cardiovascular event is between 12% [36] and 20% [59] .
Most studies have examined CYP2C19 genotypes only among patients taking clopidogrel.In the only published study to date that included a placebo-controlled group, neither CYP2C19* 2or *3 allele carriage nor metabolizer phenotype influenced the effect of clopidogrel on cardiovascular disease-related outcomes compared to the placebo group [60] .
Tests that assay CYP2C19*17 may also identify individuals at increased risk for bleeding events [13][14] or those who may derive a larger benefit (reduction in cardiovascular events) with clopidogrel treatment compared to placebo [60] .
There are no published clinical trial data evaluating the net benefit of CYP2C19 testing prior to administration of clopidogrel in improving health outcomes.
It is unknown if the risk from a given individual's genomic profile changes over time, depending on the specific clinical

Evidence Overview
Analytic Validity : Test accuracy and reliability in identifying CYP2C19 genotypes (analytic sensitivity and specificity).
Cross validation of the techniques used inCYP2C19genotyping assays, along with their reliability, specificity, and reproducibility, is extremely limited [19] .
Clinical Validity : Test accuracy and reliability in predicting response to clopidogrel (predictive value).
Numerous published studies have examined the relationship of CYP2C19alleles to clopidogrel response [33].
HuGE Navigator : query "clopidogrel and CYP2C19" At least 22 studies have examined pharmacokinetic and/or pharmacodynamic responses byCYP2C19genotype; these studies differed in the populations tested and the specific pharmacokinetic or pharmacodynamic measure(s) that were employed (most studies reviewed in [1][34] ).[55] .Only one of these studies measured plasma levels of the clopidogrel active metabolite.However, *17 allele carriers were grouped with *1/*1 extensive metabolizers; and so individual effects of the *17 allele on clopidogrel metabolism cannot be determined [44] .
Only one clinical trial explicitly reported measures of clinical validity.In the EXCELSIOR (Impact of Extent of Clopidogrel-Induced Platelet Inhibition During Elective Stent Implantation on Clinical Event Rate) trial, the sensitivity and specificity ofCYP2C19*2carrier status for detecting high on-clopidogrel residual platelet aggregation (RPA) was 45.1% and 75.0%, respectively [35] .Measures of sensitivity and specificity can be calculated from data reported in some of the other published studies.
No investigators explicitly included estimates of positive predictive value (PPV) or negative predictive value (NPV) for clopidogrel response in their original published reports.Using data from the EXCELSIOR report [35] , estimates of PPV and NPV are 41.6% and 77.6%, respectively.
Clinical Utility : Net benefit of test in improving health outcomes.
A recent meta-analysis of seven prospective cohort studies was published that examined the CYP2C19*2 variant and cardiovascular recurrences in 8,043 coronary artery disease patients taking clopidogrel.The authors found a statistically significant increased risk of major adverse cardiovascular events in CYP2C19*2 carriers (RR = 1.96) and an even greater risk when stent thrombosis was analyzed separately using four studies that included 4,975 patients (RR = 3.82) [56] .
A more recent meta-analysis that was not restricted to prospective cohort studies examined the CYP2C19*2 variant and the incidence of major adverse cardiovascular events (MACE) and mortality in patients taking clopidogrel [57] .The pooled data showed that CYP2C19*2 carriers had a statistically significant increased risk of MACE (OR = 1.29) using data from ten studies (11,959 patients).Carriers of *2 also had statistically significant increases in risk for stent thrombosis (OR = 3.45 using data from 4 studies including 4,905 patients) and death (OR = 1.79 using data from 5 studies including 6,225 patients).Four studies including 5,694 patients allowed the separate assessment of heterozygotes and homozygotes of the *2 allele.Increased risks for MACE and stent thrombosis were noted for both groups, though the odds ratio for MACE was significant only for homozygotes [57] .
Hulot and Fuster [58] calculated the PPV for clinical outcomes ofCYP2C19loss-of-function variants using data from two studies.They estimate the PPV for a cardiovascular event is between 12% [36] and 20% [59] .
Most studies have examined CYP2C19 genotypes only among patients taking clopidogrel.In the only published study to date that included a placebo-controlled group, neither CYP2C19* 2or *3 allele carriage nor metabolizer phenotype influenced the effect of clopidogrel on cardiovascular disease-related outcomes compared to the placebo group [60] .
Tests that assay CYP2C19*17 may also identify individuals at increased risk for bleeding events [13][14] or those who may derive a larger benefit (reduction in cardiovascular events) with clopidogrel treatment compared to placebo [60] .
There are no published clinical trial data evaluating the net benefit of CYP2C19 testing prior to administration of clopidogrel in improving health outcomes.
It is unknown if the risk from a given individual's genomic profile changes over time, depending on the specific clinical

Evidence Overview
Analytic Validity : Test accuracy and reliability in identifying CYP2C19 genotypes (analytic sensitivity and specificity).
Cross validation of the techniques used inCYP2C19genotyping assays, along with their reliability, specificity, and reproducibility, is extremely limited [19] .
Clinical Validity : Test accuracy and reliability in predicting response to clopidogrel (predictive value).
Numerous published studies have examined the relationship of CYP2C19alleles to clopidogrel response [33].
HuGE Navigator : query "clopidogrel and CYP2C19" At least 22 studies have examined pharmacokinetic and/or pharmacodynamic responses byCYP2C19genotype; these studies differed in the populations tested and the specific pharmacokinetic or pharmacodynamic measure(s) that were employed (most studies reviewed in [1][34] ).[35] .Measures of sensitivity and specificity can be calculated from data reported in some of the other published studies.
No investigators explicitly included estimates of positive predictive value (PPV) or negative predictive value (NPV) for clopidogrel response in their original published reports.Using data from the EXCELSIOR report [35] , estimates of PPV and NPV are 41.6% and 77.6%, respectively.
Clinical Utility : Net benefit of test in improving health outcomes.
A recent meta-analysis of seven prospective cohort studies was published that examined the CYP2C19*2 variant and cardiovascular recurrences in 8,043 coronary artery disease patients taking clopidogrel.The authors found a statistically significant increased risk of major adverse cardiovascular events in CYP2C19*2 carriers (RR = 1.96) and an even greater risk when stent thrombosis was analyzed separately using four studies that included 4,975 patients (RR = 3.82) [56] .
A more recent meta-analysis that was not restricted to prospective cohort studies examined the CYP2C19*2 variant and the incidence of major adverse cardiovascular events (MACE) and mortality in patients taking clopidogrel [57] .The pooled data showed that CYP2C19*2 carriers had a statistically significant increased risk of MACE (OR = 1.29) using data from ten studies (11,959 patients).Carriers of *2 also had statistically significant increases in risk for stent thrombosis (OR = 3.45 using data from 4 studies including 4,905 patients) and death (OR = 1.79 using data from 5 studies including 6,225 patients).Four studies including 5,694 patients allowed the separate assessment of heterozygotes and homozygotes of the *2 allele.Increased risks for MACE and stent thrombosis were noted for both groups, though the odds ratio for MACE was significant only for homozygotes [57] .
Hulot and Fuster [58] calculated the PPV for clinical outcomes ofCYP2C19loss-of-function variants using data from two studies.They estimate the PPV for a cardiovascular event is between 12% [36] and 20% [59] .
Most studies have examined CYP2C19 genotypes only among patients taking clopidogrel.In the only published study to date that included a placebo-controlled group, neither CYP2C19* 2or *3 allele carriage nor metabolizer phenotype influenced the effect of clopidogrel on cardiovascular disease-related outcomes compared to the placebo group [60] .
Tests that assay CYP2C19*17 may also identify individuals at increased risk for bleeding events [13][14] or those who may derive a larger benefit (reduction in cardiovascular events) with clopidogrel treatment compared to placebo [60] .
There are no published clinical trial data evaluating the net benefit of CYP2C19 testing prior to administration of clopidogrel in improving health outcomes.
It is unknown if the risk from a given individual's genomic profile changes over time, depending on the specific clinical scenario (for example, acute coronary syndrome versus stable angina pectoris) [19] .However, a meta-analysis demonstrated that the increased occurrence of major adverse cardiovascular events and of mortality in CYP2C19*2 carriers was independent of patients' baseline cardiovascular risk [57] .
Any benefits of DNA testing may be hampered by the following considerations: The response to clopidogrel is unclear for some CYP2C19 genotypes, such as reduced-function alleles in the presence of *17.The only study to directly assess the combined effect of *17 and reduced-function alleles (in this case, *2) found a gradient of effect on pharmacodynamic response to clopidogrel [53] .
Genotyping only for CYP2C19 alleles does not capture all of the genetic variability in pharmacodynamic, pharmacokinetic, or clinical responses to clopidogrel.Variants in the ABCB1 gene also seem to be potentially important to the interindividual variability in these phenotypes [3][14][61] [62] .
Currently, there are no standardized clinical guidelines to manage patients with an inadequate response to clopidogrel.Typically, one or more alternatives are tried: higher loading or maintenance doses of clopidogrel, dual therapy with aspirin if this has not already been initiated, or treatment with another antiplatelet medication [19] .

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Table 1 . Pharmacogenetic Tests for Clopidogrel Response
made to make this table comprehensive.However, there are tests that genotype variants in CYP2C19 that either do not specify that the test is for pharmacogenetic purposes, or do not specifically mention that the test can be used in determining response to clopidogrel.Such tests have not been included in this table.Disclaimer:whether *1/*17 carriers have a different phenotype than *1/*1 carriers].

Table 1 . Pharmacogenetic Tests for Clopidogrel Response
Note: These tests were found through Google searches combining terms such as "clopidogrel", "genetic test" and "CYP2C19" and by searching individual websites of known commercial genetics companies (such as Pathway Genomics).Attempts were made to make this table comprehensive.However, there are tests that genotype variants in CYP2C19 that either do not specify that the test is for pharmacogenetic purposes, or do not specifically mention that the test can be used in determining response to clopidogrel.Such tests have not been included in this table.

:
Test accuracy and reliability in predicting response to clopidogrel (predictive value).
Seven studies have examined the association of the *17 variant with pharmacokinetic or pharmacodynamic responses specifically to clopidogrel, with inconsistent results[2][39][44][48][53][54][55].Only one of these studies measured plasma levels of the clopidogrel active metabolite.However, *17 allele carriers were grouped with *1/*1 extensive metabolizers; and so individual effects of the *17 allele on clopidogrel metabolism cannot be determined[44].