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Background
Adverse drug reactions (ADRs) are responsible for many debilitating side effects and are a major cause of death following drug therapy. It is now clear that a significant portion of these ADRs as well as therapeutic failures are caused by genetic polymorphism and genetically based inter-individual differences in drug absorption, disposition, excretion or metabolism.
Genotyping for Cytochrome P450 Polymorphism:
Recent advances in molecular biology have improved the understanding of genetic factors underlying many ADRs. Until recently, investigations in this field have generally centered on gene coding for drug-metabolizing enzymes. Inactivating mutations have been found in gene coding for enzymes belonging to the cytochrome P450 (CYP) system, which is important in the hepatic metabolism of many drugs. Individuals with a lack of functional activity in these enzymes should be treated with very low doses of drugs metabolized by the same enzyme to avoid excessive drug levels and thus toxic effects. In recent years, research has been focused on gene coding for drug targets. As a result, most studies have been performed on single genes known to be or assumed to be functionally related to a given ADR. An alternative method is testing for complex single nucleotide polymorphisms that may be associated with ADRs, although the functional relationship between them may be completely unknown. As a consequence of influence from non-genetic factors in the development of ADRs, the association between a specific genotype and an ADR will always be less than 100 %. Thus, there is a need for well-designed clinical trials to ascertain the extent of environmental influences on the ADRs for which a genetic basis has been implicated (Guzey and Spigset, 2004). This is in agreement with the observation of Pirmohamed and Park (2003) who stated that “ADRs are common and many are suggested to have a genetic predisposition. There has been intense research in the role of CYP enzyme gene polymorphisms in the cause of ADRs. The major impact to date of polymorphic CYP expression has been on pre-clinical drug development. The direct clinical impact of CYP polymorphisms on prediction of ADRs, however, has been limited mainly because published reports have been small and retrospective and their findings conflicting. Moreover, the clinical- and cost-effectiveness of pre-prescription genotyping for CYP polymorphisms has not been established. More investigation is needed before prospective CYP genotyping can become routine clinical practice”.
Kirchheiner and Brockmoller (2005) stated that the genetic coding for the CYP enzyme 2C9 (CYP2C9) carries many inherited polymorphisms. Those coding for R144C (*2) and I359L (*3) amino acid substitutions have both significant functional effects and appreciable high population frequencies, and their in vivo consequences have been examined in humans in relation to drug metabolism -- pharmacokinetics, drug responses as well as outcomes of clinical trials in subjects with different CYP2C9 genotypes. Tentative estimates of how CYP2C9 genotyping might be applied to dose adjustments in clinical therapy were based on dose-related pharmacokinetic parameters such as clearance or trough drug concentrations. Mean clearances in homozygous carriers of the *3 allele were below 25 % of that of the wild type for S-warfarin, tolbutamide, glipizide, celecoxib, and fluvastatin. In the more frequent heterozygous carriers (genotype *1/*3), the clearances were between 40 and 75 %. In these cases in which individual dosages are derived from clinical drug effects, such as for oral anticoagulants, the pharmacogenetics-based dose adjustments showed a good correlation with the genotype-specific empirically derived doses. In addition to its role in pharmacokinetics, CYP2C9 contributes to the metabolism of fatty acids, prostanoids, and steroid hormones, and it may catalyze potentially toxic bioactivation reactions. However, the current understanding of the role of CYP2C9 in biotransformation of endogenous signaling molecules and in drug toxicity is relatively meager. These investigators concluded that the concept of therapy based on genotyping for CYP polymorphisms has not been assessed in prospective, randomized, controlled studies in which one group is dosed according to genotype while another group is dosed in a usual manner. It is unlikely that CYP2C9 genotyping will become routine practice unless its clinical value is supported by such rigorous assessments.
The following is a list* of some CYP2C9-metabolized drugs with narrow therapeutic ratios (drugs with a narrow difference between therapeutic and toxic concentrations) with serious toxic effects:
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Angiotensin II blockers (e.g., irbesartan, losartan)
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NSAIDS (e.g., diclofenac, ibuprofen, naproxen, piroxicam)
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Oral hypoglycemic agents (e.g., glipizide, tolbutamide)
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Sulfonylureas (e.g., amitriptyline, celecoxib, fluoxetine, fluvastatin, glipizide, rosiglitazone, tamoxifen, tolbutamide, S-warfarin).
Diagnostic genotyping tests for some CYP enzymes are now available commercially. The AmpliChip (Roche Diagnostics, Basel, Switzerland), cleared by the United States Food and Drug Administration (FDA) through the 510(k) process, is a microarray consisting of many DNA sequences complementary to 2 CYP genes applied in microscopic quantities at ordered locations on a solid surface (chip). The AmpliChip tests the DNA from a patient's leukocytes collected in a standard anti-coagulated blood sample for 29 polymorphisms and mutations for the CYP2D6 gene and 2 polymorphisms for the CYP2C19 gene. CYP2D6 metabolizes about 25 % of all clinically used medications (e.g., antiarrhythmics, antidepressants, beta-blockers, dextromethorphan and morphine derivatives), while CYP2C19 metabolizes several important types of drugs including anti-epileptics and proton-pump inhibitors. The AmpliChip was cleared for marketing for CYP2D6 testing in December 2004, and for CYP2C19 testing in January 2005.
The AmpliChip is marketed for use in screening of patients who are to be treated with drugs metabolized by the CYP system that have a narrow therapeutic ratio with serious toxic effects.
The following is a list* of CYP2D6-metabolized drugs with narrow therapeutic ratios (not an all inclusive list):
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Anti-depressants (e.g., amitryptiline, clomipramine, desipramine, imipramine, paroxetine)
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Anti-psychotics (e.g., chlorpromazine, haloperidol, perphenazine, risperidone, thiroidazine)
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Beta blockers (e.g., carvediol, propafenone, timolol).
The following is a list* of CYP2C19-metabolized drugs with narrow therapeutic ratios (not an all inclusive list):
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Anti-epileptics (e.g., amitryptiline, clomipramine, cyclophosphamide, diazepam, imipramine, moclobemide, phenytoin, phenobarbitone, primidone, R-warfarin)
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Proton pump inhibitors (e.g., lansoprazole, omeprazole, pantoprazole).
- Others (e.g., clopidogrel).
* These lists are based on information excerpted from the “Cytochrome P450 Drug Interaction Table”, Indiana University School of Medicine, 2009.
Randomized controlled trials are needed to ascertain if the AmpliChip will lower the incidence of ADRs by detecting patients with CYP2D6 and CYP2C19 mutations. The effectiveness of the AmpliChip in reducing toxic effects and improving health outcomes would need to be compared with standard methods of therapeutic drug monitoring (e.g., by monitoring clinical response or by measuring serum drug concentrations). Moreover, it is still unclear that the AmpliChip would eliminate the need for simultaneous use of other methods of therapeutic drug monitoring (including serum measurements) because factors other than genetic polymorphisms such as those described earlier have a significant effect on drug pharmacokinetics and pharmacodynamics.
Juran and colleagues (2006) stated that "[e]ven though the AmpliChip CYP450 has been approved by the FDA, its practical clinical utility has not yet been determined, and there is a paucity of data related to gastrointestinal and liver diseases. An understanding of the principles and opportunities provided by this new category of diagnostic test is key before planning the necessary studies to evaluate the usefulness of AmpliChip CYP450 in gastroenterologic clinical practice".
A special report (BlueCross BlueShield Technology Evaluation Center, 2004) on genotyping for CYP polymorphisms to determine drug-metabolizer status stated that diagnostic tests to identify specific polymorphisms that may be linked to increased/reduced effect or serious adverse effects are now available. Whether such testing, and for which drugs, improves patient outcomes is not yet known. While genotyping for the CYP enzymes would only need to be performed once per patient and the results could be used to consider other drugs metabolized by the same enzymes, whether genotyping is clinically useful would need to be determined for each drug. Even drugs of the same class may variably rely on specific CYP enzymes. For example, the plasma level of the selective serotonin reuptake inhibitor (SSRI) fluoxetine is significantly affected by CYPP2D6 polymorphisms, whereas the SSRI sertraline appears to be little affected and may depend more upon CYP2C19 for metabolism. The report stated that while the potential of pharmacogenetic studies is intriguing for many clinical applications, it is still unclear which are most likely to provide clinical benefit in the near future.
Ingelman-Sundberg (2005) stated that the polymorphism of CYP2D6 significantly affects the pharmacokinetics of approximately half of the drugs in clinical use, which are CYP2D6 substrates. The consequences of the polymorphism at ordinary drug doses can be either ADRs or no drug response. Predictive CYP2D6 genotyping is estimated to be beneficial for treatment of about 30 to 40 % of CYP2D6 drug substrates representing about 7 to 10 % of all clinically used drugs, although prospective clinical studies are needed to determine the exact benefit of drug selection and dosage based on the CYP2D6 genotype. Furthermore, Sanderson et al (2005) assessed the strength and quality of existing evidence about CYP2C9 gene variants and clinical outcomes in warfarin-treated patients in a meta-analysis (11 studies with a total of 2,775 patients). These investigators concluded that patients with CYP2C9*2 and CYP2C9*3 alleles have lower mean daily warfarin doses and a greater risk of bleeding. The authors also stated that while testing for gene variants could potentially alter clinical management in patients commencing warfarin, evidence for the clinical utility and cost-effectiveness of genotyping is necessary before routine testing can be recommended.
In a review article on “Drug Metabolism and Variability among Patients in Drug Response” published in the New England Journal of Medicine, Wilkinson (2005) stated that “there have not yet been prospective clinical trials showing that knowledge of a patient’s genotypic profile before prescribing drugs either increases drug efficacy, prevents or reduces adverse drug reactions, or lower the overall costs of therapy and associate sequelae …. For now, however, the individual patient is probably best served by an alert physician aware of the possibility that a genetic polymorphism in drug metabolism may be a potential factor in unexpected drug response”.
In a multi-center 6-week study, Fux et al (2005) examined the impact of CYP2D6 polymorphism on the tolerability of metoprolol in a primary care setting. The adverse effects studied comprised effects related to the central nervous system, cardiovascular effects, and sexual dysfunction. The dosage of metoprolol was determined on an individual basis and could be adjusted on clinical grounds. The indication for treatment was hypertension in about 90 % of cases. CYP2D6 genotyping covered alleles *3 to *10 and *41 and the duplications. Possible ADRs of metoprolol were systematically assessed over the study period using standardized rating scales and questionnaires. The final study population comprised 121 evaluable patients: 5 ultrarapid metabolizers (UMs) (4.1 %), 91 extensive metabolizers (EMs) (75 %), 21 intermediate metabolizers (IMs) (17 %), and 4 poor metabolizers (PMs) (3.3 %). Plasma metoprolol concentrations normalized for the daily dose and metoprolol/alpha-hydroxymetoprolol ratios at steady state were markedly influenced by CYP2D6 genotype and displayed a gene-dose effect. The median of the dose-normalized metoprolol concentration was 0.0088 ng/ml, 0.047 ng/ml, 0.34 ng/ml, and 1.34 ng/ml among UMs, EMs, IMs, and PMs, respectively (p < 0.0001). There was no significant association between CYP2D6 genotype-derived phenotype (EMs and UMs combined versus PMs and IMs combined) and ADRs during treatment with metoprolol. There was a tendency toward a more frequent occurrence of cold extremities in the PM plus IM group as compared with the EM plus UM group (16.0 % versus 4.2 %, p = 0.056; relative risk, 3.8 [95 % confidence interval [CI]: 1.03 to 14.3]). These investigators concluded that CYP2D6 genotype-derived phenotype was not significantly associated with a propensity for ADRs to develop during treatment with metoprolol. However, the results concerning tolerability of metoprolol in PMs were inconclusive because of the small number of PMs enrolled.
The mechanisms of variable response to tamoxifen have been the subject of much scrutiny in the published literature. Early studies attempting to link a clinical response to tamoxifen therapy with plasma tamoxifen concentrations reported no statistically significant differences in outcomes between women who received 20 mg of tamoxifen daily and those who received 40 mg of tamoxifen daily, even though women in the 40 mg tamoxifen group had higher plasma tamoxifen concentrations than those in the 20 mg tamoxifen group. These results have been reported as evidence that plasma tamoxifen concentration is not a predictor of clinical outcome. Because there is evidence that tamoxifen is converted to anti-estrogenic metabolites, one hypothesis is that altered patterns of metabolism of tamoxifen might contribute to inter-individual variability in effects (Jin et al, 2005). Plasma concentrations of the active tamoxifen metabolite endoxifen are associated with the cytochrome P450 (CYP) 2D6 genotype.
Goetz et al (2005) stated that polymorphisms in tamoxifen metabolizing genes affect the plasma concentration of tamoxifen metabolites, but their effect on clinical outcome is unknown. These investigators determined cytochrome P450 (CYP)2D6 (*4 and *6) and CYP3A5 (*3) genotype from paraffin-embedded tumor samples and buccal cells (living patients) in tamoxifen-treated women enrolled onto a North Central Cancer Treatment Group adjuvant breast cancer trial. The relationship between genotype and disease outcome was determined using the log-rank test and Cox proportional hazards modeling. Paraffin blocks were obtained from 223 of 256 eligible patients, and buccal cells were obtained from 17 living women. CYP2D6 (*4 and *6) and CYP3A5 (*3) genotypes were determined from 190, 194, and 205 patient samples and in 17 living women. The concordance rate between buccal and tumor genotype was 100 %. Women with the CYP2D6 *4/*4 genotype had worse relapse-free time (RF-time; p = 0.023) and disease-free survival (DFS; p = 0.012), but not overall survival (p = 0.169) and did not experience moderate to severe hot flashes relative to women heterozygous or homozygous for the wild-type allele. In the multi-variate analysis, women with the CYP2D6 *4/*4 genotype still tended to have worse RFS (hazard ratio [HR], 1.85; p = 0.176) and DFS (HR, 1.86; p = 0.089). The CYP3A5*3 variant was not associated with any of these clinical outcomes. The authors concluded that in tamoxifen-treated patients, women with the CYP2D6 *4/*4 genotype tend to have a higher risk of disease relapse and a lower incidence of hot flashes, which is consistent with their previous observation that CYP2D6 is responsible for the metabolic activation of tamoxifen to endoxifen. They noted that "[t]hese findings have the potential to improve the ability of physicians to select the optimal hormonal therapy for the treatment of ER-positive breast cancer. Further studies are needed in women receiving tamoxifen to fully define the effect of CYP2D6 genetic polymorphisms and medications that inhibit CYP2D6 on tamoxifen response."
An assessment of CYP2D6 pharmacogenomics of tamoxifen treatment conducted by the BlueCross BlueShield Association Technology Evaluation Center (2008) evaluated the evidence for CYP2D6 genotyping and tamoxifen treatment efficacy. The hypothesis examined by the assessment is that CYP2D6 poor metabolizers, whether by genotype or by co-administration of CYP2D6 inhibitory medication, have reduced tamoxifen metabolism and lower endoxifen levels compared to better metabolizers, and as a result have poorer clinical outcomes. The reviewers stated that this hypothesis is based on the assumption, not yet supported by evidence, that some level of endoxifen is necessary for tamoxifen efficacy and that this level is not achieved in genotypic and functional CYP2D6 poor metabolizers and possibly not in some intermediate metabolizers. However, the reviewers found no scientific evidence for a significant association between endoxifen and clinical outcomes. In addition, they reported several limitations on the association of genotype with clinical outcomes. The reviewers stated, "Because tamoxifen metabolism is complex and CYP2D6 does not appear to account for all variability in endoxifen levels, it is conceivable that polymorphisms in other tamoxifen metabolic pathway enzymes may affect active metabolite levels, and direct measurement of the metabolite(s) itself may be the better predictor of benefit from tamoxifen treatment. However, since it takes 8 weeks for tamoxifen metabolites to reach steady-state concentrations, measuring metabolite levels is not practical for clinical applications outside of a retrospective study." Furthermore, the reviewers noted that multiple enzyme genotypes may be needed to confidently predict tamoxifen versus aromatase inhibitors treatment benefit; however, there are little data at present to recommend any genotype combinations. The assessment concluded, "There is insufficient evidence to permit conclusions regarding the use of CYP2D6 genotyping for directing endocrine therapy regimen selection for women at high risk for or with breast cancer."
Visvanathan et al (2009) updated the 2002 American Society of Clinical Oncology guideline on pharmacologic interventions for breast cancer risk reduction. The cytochrome P450 2D6 gene (CYP2D6) encodes the enzyme responsible for catalyzing the conversion of tamoxifen to endoxifen, an active metabolite of tamoxifen. Functional allelic variants (*4 most common in whites and *10 most common in Asians), have been identified in approximately 7 % of the population. Lower levels of endoxifen have been observed in women taking tamoxifen who are heterozygous and homozygotes for variant alleles in CYP2D6 in a dose-dependent manner, or in women treated with concomitant medications that block CYP2D6, including certain selective serotonin reuptake inhibitors such as paroxetine. Further, in a small nested case-control study of women who took part in the Italian prevention trial, a higher prevalence of the CYP2D6 *4/*4 phenotype was observed among women with breast cancer who took tamoxifen compared with controls. The authors concluded that confirmation of these results in larger studies is needed. Given the limited evidence, CYP2D6 testing is currently not recommended in the preventive setting.
The BlueCross BlueShield Association's technology assessment on CYP2D6 pharmacogenomics of tamoxifen treatment (2011) stated that studies showing no evidence of association between CYP2D6 genotype and either tamoxifen- or aromatase inhibitor-treated patient outcomes has suggested that using the results of CYP2D6 genetic testing to influence decisions about tamoxifen treatment is not currently warranted.
A BlueCross BlueShield Association Technology Evaluation Center (TEC, 2013) assessment of CYP2D6 pharmacogenetics of tamoxifen treatment concluded that CYP2D6 genotyping does not meet the TEC criteria for directing endocrine therapy regimen selection for women at high risk for primary breast cancer or breast cancer recurrence. The assessment stated that there are several limitations to the overall body of evidence, but the largest, most well-designed studies do not support clinical validity of the test. In the absence of evidence for clinical validity, evidence to support clinical utility is lacking.
In a review on pharmacogenomics and individualized drug therapy, Eichelbaum et al (2006) stated that "[t]here is also a growing list of genetic polymorphisms in drug targets that have been shown to influence drug response. A major limitation that has heretofore moderated the use of pharmacogenetic testing in the clinical setting is the lack of prospective clinical trials demonstrating that such testing can improve the benefit/risk ratio of drug therapy". Furthermore, Humphries and Hingorani (2006) noted that the full potential of the field of pharmacogenetics will only be realized with much further work.
An assessment of CYP450 genetic testing by the Canadian Coordinating Office for Health Technology Assessment (Palylyk-Colwell, 2006) concluded: "No published studies show that patient outcomes can be predicted or altered by knowledge of DME status in the absence of other confounding variables. Prospective studies are needed to assess the benefits and potential risks of this technology in guiding drug selection and dose adjustment."
Since their introduction in the late 1980s, SSRIs such as citalopram, fluoxetine, paroxetine, and sertraline have become the most commonly prescribed class of drugs for treating depression. However, the likelihood that a patient will experience relief from all symptoms of depression after 1 year of treatment is approximately 40 %, and adverse events cause 12 to 15 % of patients who start treatment to stop taking the drug. Following the recent FDA approval of a test to predict differences in the CYP450 gene, physicians and patients must decide if using such tests to choose a type or dose of an SSRI might improve the patient's response to treatment.
Kirchheiner and colleagues (2003) noted that antidepressants are characterized by a high rate of drug failure. There is evidence that genetic factors are contributing to the inter-individual variability in response to these medications. Genetic polymorphisms in drug metabolizing enzymes are well established and have significant effects on oral clearances or elimination half-lives of antidepressants. These differences can be compensated by adapting the individual dose to genotype in addition to other factors such as age, gender, weight, drug interactions, diseases (cardiovascular, hepatic, renal and respiratory) as well as environmental influences on drug metabolism (e.g., diet and smoking). Genetic variability is found in molecular structures of antidepressant effects. Furthermore, Kirchheiner et al (2003) noted that studies on response of antidepressants have revealed influences of polymorphisms in neurotransmitter receptors and transporters altering sensitivity of patients to treatment with antidepressants; however, results were often contradictory.
The Agency for Healthcare Research and Quality (AHRQ, 2006) released an evidence report that found there is insufficient evidence to determine if current gene-based tests intended to personalize the dose of SSRIs improve patient outcomes or aid physicians or patients in making treatment decisions. The available studies indicated that the tests are largely accurate at evaluating differences in genes belonging to the CYP450 family that affect the rate at which a person metabolizes SSRIs. However, additional well-designed studies are needed to determine the usefulness of test results in the clinical setting. This report is the first step in the 2-step process of Centers for Disease Control and Prevention's Evaluation of Genomic Applications in Practice and Prevention (EGAPP) pilot project to evaluate and make recommendations regarding the use of gene-based tests.
The Evaluation of Genomic Applications in Practice and Prevention Working Group (EGAPP, 2007) found insufficient evidence to support a recommendation for or against use of cytochrome P450 (CYP450) testing in adults beginning SSRI treatment for non-psychotic depression. In the absence of supporting evidence, and with consideration of other contextual issues, EGAPP discourages use of CYP450 testing for patients beginning SSRI treatment until further clinical trials are completed. EGAPP found that use of genetic testing for CYP450 polymorphisms and impact on physician decision-making with regard to use of SSRIs is not known. EGAPP noted that, in the absence of evidence supporting clinical utility, widespread use of CYP450 genetic testing is potentially costly and may not lead to changes in treatment that improve patient outcomes.
The eradication rates of Helicobacter pylori by triple therapy consisting of 1 proton pump inhibitor (PPI) and 2 antimicrobial agents are mainly influenced by bacterial susceptibility to anti-microbial agents and the magnitude of acid inhibition during treatment with a PPI (e.g., omeprazole, lansoprazole, rabeprazole). Tailored therapy using CYP2C19 pharmacogenomics with a PPI has been proposed as a method to help improve the efficacy of H. pylori eradication rates.
A review on the use of pharmacogenomics-based treatment of H. pylori infection conducted by the BlueCross BlueShield Association Technology Evaluation Center (2008) examined the scientific evidence of a pharmacogenomics-based treatment regimen for the eradication of H. pylori. The reviewers found 1 randomized, controlled study that met their inclusion criteria. The reviewers reported that this study found higher eradication rates after first-line treatment for the pharmacogenomics group compared with the standard treatment group, however, because of numerous variations in treatment protocol within the pharmacogenomics group, it was not possible to determine whether the improvement resulted from the tailored PPI dosages according to CYP2C19 genetic status, or if it was due to other variations in the treatment protocol unrelated to CYP2C19 status. Furthermore, the review noted that it was possible other clinical factors, such as clarithromycin resistance, or other treatment factors, such as length of antibiotic treatment, influenced eradication rates. In addition, the study was performed in a Japanese population and did not employ a diagnostic approach or a treatment regimen that is standard care in the United States. The review concluded, "The scientific evidence does not permit conclusions on whether the use of a pharmacogenomics-based treatment regimen for H. pylori improves eradication rates."
Clopidogrel bisulfate (Plavix) (Bristol-Myers Squibb/Sanofi Pharmaceuticals, Bridgewater, NJ) is an orally administered antiplatelet drug that is used to prevent blood clots that could lead to heart attacks or strokes in individuals with established cardiovascular atherosclerotic disease. The degree of platelet inhibition seen following use of the second-generation thienopyridine clopidogrel varies from patient to patient in a normal or bell-shaped distribution. The variability in non-response is such that, when laboratory measurements of platelet aggregation are performed, 4 to 30 % of patients treated with clopidogrel do not have an adequate anti-platelet response.
Studies have linked reduced response to clopidogrel to variants in the gene CYP2C19. Mega et al (2009) reported that in healthy subjects who were treated with clopidogrel, carriers of at least one CYP2C19 reduced-function allele had a relative reduction of 32.4 % in plasma exposure to the active metabolite of clopidogrel, as compared with non-carriers. The CYP2C19*1 allele corresponds to fully functional metabolism while the CYP2C19*2 and *3 alleles are non-functional. CYP2C19*2 and *3 account for the majority of reduced function alleles in white (85 %) and Asian (99 %) poor metabolizers. Other alleles associated with absent or reduced metabolism are less frequent, and include, but are not limited to, CYP2C19*4, *5, *6, *7, and *8. A patient with poor metabolizer status will possess 2 loss-of-function (LOF) alleles as defined above. Published frequencies for poor CYP2C19 metabolizer genotypes are approximately 2 % for whites, 4 % for blacks and 14 % for Chinese. Tests are available to determine a patient's CYP2C19 genotype.
The results of this research are now included in the prescribing label for clopidogrel. The new label includes that reduced CYP2C19 metabolism in intermediate and poor metabolizers is associated with diminished response to clopidogrel and that pharmacogenetic testing can identify genotypes associated with variability in CYP2C19 activity. However, the information in the new labeling does not include a recommendation for genetic testing prior to administration of the drug and does not provide information on how to determine dosing once a patient’s individual genotype has been established.
On March 12, 2010, the labeling of Plavix was updated to include a boxed warning about the diminished effectiveness of clopidrogel in poor metabolizers. The boxed warning states that the effectiveness of Plavix is dependent on its activation to an active metabolite by the cytochrome P450 (CYP) system, principally CYP2C19. The labeling states that Plavix at recommended doses forms less of that metabolite and has a smaller effect on platelet function in patients who are CYP2C19 poor metabolizers. Citing cohort studies and retrospective analyses of clinical trials, the labeling states that poor metabolizers with acute coronary syndrome or undergoing percutaneous coronary intervention (PCI) treated with Plavix at recommended doses exhibit higher cardiovascular event rates than do patients with normal CYP2C19 function. The labeling states that tests are available to identify a patient's CYP2C19 genotype, and that these tests can be used as an aid in determining therapeutic strategy. The labeling states that clinicians should consider alternative treatment or treatment strategies in patients identified as CYP2C19 poor metabolizers.
The new label notes that reduced CYP2C19 metabolism in intermediate and poor metabolizers is associated with diminished response to clopidogrel and that pharmacogenetic testing can identify genotypes associated with variability in CYP2C19 activity. However, the information in the new labeling does not include a recommendation for genetic testing prior to administration of the drug. The label states, "although a higher dose regimen (600 mg loading dose followed by 150 mg once-daily) in poor metabolizers increases antiplatelet response, an appropriate dose regimen for this patient population has not been established in clinical outcome trials." In addition, the label states that there may be genetic variants of other CYP450 enzymes with effects on the ability to form clopidogrel's active metabolite.
A Clinical Alert from the American College of Cardiology and the American Heart Association (Holmes et al, 2010) stated that the clopidogrel boxed warning leaves the issue of whether to perform CYP2C19 testing up to the individual physician. It does not specifically require genetic testing or other changes in evaluation or treatment and does not imply that there are solid evidence-based reasons for such actions. Rather, it serves to make clinicians aware of the imperfect, but significant, knowledge that we have about genetic variations in response to clopidogrel and to emphasize that clinicians should use this knowledge to make decisions about how to treat individual patients. The Clinical Alert concluded that the evidence base is insufficient to recommend either routine genetic or platelet function testing at the present time. There is no information that routine testing improves outcome in large subgroups of patients. In addition, the clinical course of the majority of patients treated with clopidogrel without either genetic testing or functional testing is excellent. The Clinical Alert stated that clinical judgment is required to assess clinical risk and variability in patients considered to be at increased risk. The Clinical Alert stated that 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). The Clinical Alert stated that, if such testing identifies a potential poor metabolizer, other therapies, particularly prasugrel for coronary patients, should be considered. With these other therapies, the balance of potential ischemic benefit with the known increased risk of bleeding should be considered either with alternative clopidogrel dosing or newer agents such as prasugrel.
Guidelines issued in December 2009 from the American College of Cardiology, the American Heart Association, and the Society for Cardiac Angiography and Interventions stated that, although clopidogrel in combination with ASA has been shown to reduce recurrent coronary events in the post-hospitalized acute coronary syndrome (ACS) population, the response to clopidogrel varies among patients, and clopidogrel resistance has been observed (Kushner et al, 2009). The guidelines noted that information is accumulating about the variations in the anti-platelet effect of clopidogrel in patients with LOF alleles in the gene encoding CYP450 2C19. These patients form a subgroup in which failure of clopidogrel effectiveness has been linked to adverse clinical outcomes. The guidelines stated, accordingly, that the effective clopidogrel dose for an individual undergoing PCI for STEMI may not be known. The guidelines stated that a large randomized trial is attempting to determine whether adjustment of clopidogrel therapy on the basis of platelet function testing with a point-of-care assay safely improves outcomes after PCI with drug-eluting stent (DES). The guidelines stated that the current recommended loading dose for clopidogrel is uncertain. In addition, a period of several hours is required to metabolize clopidogrel to its active metabolite, which leaves a window of time during which there is a reduced level of effectiveness even in responders.
The guidelines observed that thienopyridine prasugrel has a higher level of inhibition of platelet aggregation than clopidogrel and a more rapid onset of action (Kushner et al, 2009). The guidelines stated that its metabolism is not affected by the 2C19 allele variant. However, the guidelines do not endorse explicitly one of the thienopyridines over the other. Furthermore, there are some emerging studies that suggest there may be some patients who are resistant to clopidogrel, but there is little information about the use of strategies to select patients who might do better with prasugrel. There is not yet experience with the use of prasugrel in routine community practice. As a result, the guidelines state that there is some uncertainty regarding the net benefit and risks of one drug over another for a given patient.
More recently published evidence casts doubt over the role of genetic variation in CYP2C19 and clopidrogel treatment effects. A recent study published in the New England Journal of Medicine (Pare et al, 2010) found that, among patients with ACS or atrial fibrillation, the effect of clopidogrel as compared with placebo is consistent, irrespective of CYP2C19 genotype. Investigators genotyped patients from 2 large, randomized, placebo-controlled trials of the effect of clopidogrel on the rate of cardiovascular events among patients with ACS and among patients with atrial fibrillation. Investigators found that, in both studies, clopidogrel's superiority to placebo was similar, regardless of patients' genotype. In addition, CYP2C19 genotype was not associated with adverse events during treatment.
A randomized controlled clinical study published in the Lancet (Wallentin et al, 2010), comparing clopidrogel to ticagrelor in subjects with ACS, found no significant effect difference of clopidrogel by CYP2C19 genotype. In this study, ticagrelor was found to be more effective than clopidrogel in reducing the risk of cardiovascular events, but the effect difference did not vary significantly by CYP2C19 genotype. In addition, CYP2C19 genotype was not associated with adverse events during treatment.
Tetrabenazine (Xenazine) is a monoamine depletor for oral administration, and is indicated indicated for the treatment of chorea associated with Huntington’s disease. The precise mechanism by which tetrabenazine exerts its anti-chorea effects is unknown, but is believed to be related to its effect as a reversible depletor of monoamines (such as dopamine, serotonin, norepinephrine, and histamine) from nerve terminals. Tetrabenazine is extensively metabolized in the liver to metabolites alpha-HTBZ and beta-HTBZ. Alpha-HTBZ and beta-HTBZ are metabolized by CYP450 enzymes, principally CYP2D6, to another major circulating metabolite, O-dealkylated-HTBZ. These metabolites are primarily renally excreted. The labeling of Xenazine states that, although the pharmacokinetics of tetrabenazine and its metabolites in subjects who do not express the drug metabolizing enzyme CYP2D6 (poor metabolizers) have not been systematically evaluated, it is likely that the exposure to beta-HTBZ would be increased compared to subjects who express the enzyme (extensive metabolizers), with an increase similar to that observed in patients taking strong CYP2D6 inhibitors. The labeling of Xenazine states that "[p]atients should be genotyped for CYP2D6 prior to treatment with daily doses of tetrabenazine over 50 mg", and patients who are poor metabolizers should not be given daily doses greater than 50 mg.
Knowing how an individual will respond to warfarin would help in tailoring the dose needed to maintain appropriate anticoagulation. Toward that end, researchers studied variability in the recently discovered gene for the warfarin target, vitamin K epoxide reductase complex 1 (VKORC1). VKORC1 is the key enzyme of the vitamin K cycle and the molecular target of coumarins, which represent the most commonly prescribed drugs for therapy and prevention of thromboembolic conditions. Recent studies have identified variants of the VKORC1 gene as responsible for about 25 % of the inter-individual response to warfarin, and for significant inter-ethnic response variability. Whether or not this is sufficient to successfully direct initial dosing, achieve a shorter time to stable dose, and reduce bleeding events has yet to be shown in a prospective trial. There are several such prospective clinical studies that are currently ongoing both in the United States and Europe.
Wadelius and Pirmohamed (2007) explained that the most important genes affecting the pharmacokinetic and pharmacodynamic parameters of warfarin are CYP2C9 and VKORC1. These 2 genes, together with environmental factors, partly explain the inter-individual variation in warfarin dose requirements.
Although studies have shown that genetic polymorphisms in CYP2C9 and VKORC1 affect warfarin dosing, no randomized controlled trials have linked the use of pharmacogenomic testing to improvements in clinical outcomes. An assessment by the Canadian Agency for Drugs and Technologies in Health (Ndegwa, 2007) found that most of the studies performed to date have been of retrospective or cross-sectional design. Consequently, individuals who stop warfarin early because of adverse effects or those who have difficulty attaining a therapeutic maintenance dose may have been excluded. Furthermore, many studies were underpowered to investigate the risk of bleeding.
Rieder et al (2005) analyzed genetic data from 186 American patients of European descent who were recruited from anticoagulation clinics and were receiving long-term warfarin therapy. They identified 10 single-nucleotide polymorphisms of VKORC1 that showed significant associations with warfarin maintenance doses and that had an overall frequency of more than 5 % in this cohort. From these 10 single-nucleotide polymorphisms, the researchers inferred five common VKORC1 haplotypes (a haplotype is a set of closely linked genetic markers present on one chromosome that tend to be inherited together). Four of the haplotypes had independent associations with warfarin maintenance doses, 2 with a low-dose requirement and 2 with a high-dose requirement. Ultimately, subjects were linked with 1 of 3 haplotype groupings; the mean daily warfarin maintenance dose differed significantly across these 3 subgroups (2.7 mg, 4.9 mg, and 6.2 mg, respectively). The authors report that VKORC1 haplotype explained 25 % of the variance in warfarin dose, and they replicated these findings in a larger European American population. The researchers also examined VKORC1 haplotype frequencies in Asian American and African American populations and found significant variability by race.
The FDA has cleared for marketing the Verigene warfarin metabolism test, manufactured by Nanosphere, which detects variants of two genes, CYP2C9 and VKORC1 that can contribute to changes in warfarin metabolism (FDA, 2007).
Large ongoing studies of genes involved in the actions of warfarin, together with prospective assessment of environmental factors, will increase the capacity to accurately predict warfarin dose. Kamali (2006) stated that prospective studies that incorporate both CYP2C9 and VKORC1 genes and environmental factors in warfarin dose calculation will be needed to demonstrate the safety, cost-effectiveness, and feasibility of individualized dosing regimens.
ECRI Institute's Health Technology Trends (2007) reported that "[a]lthough genetic testing can currently identify who has these variants [CYP2C9 and VKORC1], more studies are needed to explore the precise starting doses for these patients".
Anderson et al (2007) stated that pharmacogenetic-guided dosing of warfarin is a promising application of "personalized medicine" but has not been adequately examined in randomized studies. In a randomized trial, these investigators assessed genotype-guided versus standard warfarin dosing in patients initiating oral anticoagulation (n = 206). Buccal swab DNA was genotyped for CYP2C9 *2 and CYP2C9 *3 and VKORC1C1173T with a rapid assay. Standard dosing followed an empirical protocol, whereas pharmacogenetic-guided dosing followed a regression equation including the 3 genetic variants as well as age, sex, and weight. Prothrombin time INR was measured routinely on days 0, 3, 5, 8, 21, 60, and 90. A research pharmacist un-blinded to treatment strategy managed dose adjustments. Patients were followed-up for up to 3 months. Pharmacogenetic-guided predicted doses more accurately approximated stable doses (p < 0.001), resulting in smaller (p = 0.002) and fewer (p = 0.03) dosing changes and INRs (p = 0.06). However, percent out-of-range INRs (pharmacogenetic = 30.7 %, standard = 33.1 %), the primary end point, did not differ significantly between arms. Despite this, when restricted to wild-type patients (who required larger doses; p = 0.001) and multiple variant carriers (who required smaller doses; p < 0.001) in exploratory analyses, results (pharmacogenetic = 29 %, standard = 39 %) achieved nominal significance (p = 0.03). Multiple variant allele carriers were at increased risk of an INR of greater than or equal to 4 (p = 0.03). The authors concluded that an algorithm guided by pharmacogenetic and clinical factors improved the accuracy and efficiency of warfarin dose initiation. Despite this, the primary end point of a reduction in out-of-range INRs was not achieved. In subset analyses, pharmacogenetic guidance showed promise for wild-type and multiple variant genotypes.
A review by Hynicka et al (2007) concluded that the use of pharmacogenomic testing in the initiation of warfarin therapy does not show improved outcomes in either safety or efficacy with warfarin therapy. Studies of pharmacogenomic testing to improve outcomes with initiation of warfarin therapy were eligible for inclusion. All patients were adults; however, the included studies varied in their population and treatment regimens. The genotypes included CYP2C9 and VKORC1 wild-types and variants. Four studies met the inclusion criteria: 2 randomized controlled trials (n = 238) and 2 prospective cohort studies (n = 345). None of the studies reported a significant difference in efficacy between dosing strategies, although the prospective cohort studies reported a tendency towards fewer adverse events in patients with pharmacogenomic-guided dosing.
An assessment published by the Canadian Agency for Drugs and Technology in Health (Ndegwa, 2007) on "Pharmacogenomics and Warfarin Therapy" concluded that "prospective studies are needed to determine whether pharmacogenomic testing improves patient outcomes, identify which subgroups of patients may benefit, and clarify the risks and costs associated with the use of these tests. Several randomized controlled trials are currently evaluating the impact of pharmacogenomics on dosing accuracy, time to achieve and maintain target international normalized ratio (INR), incidence of bleeding or thromboembolic events, and monitoring requirements".
In August 2007, the FDA updated the product label for warfarin to include information on the impact of genetic variations in CYP2C9 and VKORC1 on warfarin metabolism; however the FDA does not require the use of these genetic tests in dosing individual patients initiating warfarin therapy. On January 28, 2008, the FDA cleared the Infiniti 2C9-VKORC1 Multiplex Assay (AutoGenomic, Inc., Carlsbad, CA) for detection of Warfarin sensitivity. Guidelines for pharmacogenomics-based warfarin dosing are under development.
A technology assessment by the California Technology Assessment Forum (CTAF, 2008) reviewed the scientific evidence for the use of genetic testing to guide the initial dosing of warfarin when initiating therapy for anticoagulation. The assessment stated that genotyping studies of patients at a stable, therapeutic dose of warfarin demonstrated that patients who have CYP2C9*2 and CYP2C9*3 require lower doses of warfarin on average than patients with the more common CYP2C9*1 allele; in addition, patients with the A haplotype of VKORC1 require lower doses of warfarin on average than patients with the B haploytpe. The assessment reported that these genetic variations have been shown to predict an increased risk of excessive anticoagulation and major bleeding among patients prescribed warfarin and that statistical models have been developed in an attempt to predict the dose of warfarin needed to achieve stable anticoagulation. The assessment examined the results of a small case series and 3 small, randomized clinical trials and reported that the model used by the case series did reasonably well at predicting the warfarin dose, but that patients with the CYP2C9*2 and CYP2C9*3 alleles were still at more than a 4-fold risk of excessive anticoagulation; only 1 of the randomized trials used a model incorporating genotyping information from both CYP2C9 and VKORC1 and that study found almost no difference in outcomes between patients receiving warfarin using a pharmacogentic model and those treated according to a standard approach. The assessment stated, "Significant uncertainty remains in the field. There is no widely accepted, standard pharmacogenetic model to determine the starting dose of warfarin and new models are being developed in 2008. Current models only explain 50 % to 60 % of the variability in warfarin dosing and the remaining variability is unexplained. Only 1 of the randomized trials used a model incorporating information from both genes with variants known to influence warfarin dosing. Furthermore, all of the trials to date have been under-powered to meaningfully evaluate the effect of genotyping on major bleeding, the most important clinical outcome." The assessment concluded that the use of genetic testing to guide initial warfarin dosing does not meet Technology Assessment Criteria 3 through 5 for safety, effectiveness and improvement in health outcomes. Several large clinical trials are ongoing in both the United Stated and Europe to clarify the role of genetic testing in warfarin management.
The Centers for Medicare & Medicaid Services (CMS, 2013) concluded that the available evidence does not demonstrate that pharmacogenomic testing of CYP2C9 or VKORC1alleles to predict warfarin responsiveness improves health outcomes in Medicare beneficiaries. Therefore, CMS determined that pharmacogenomic testing of CYP2C9 or VKORC1 alleles to predict warfarin responsiveness is not reasonable and necessary.
A randomized controlled clinical trial found that genotype-guided dosing of warfarin did not improve anticoagulation control during the first four weeks of therapy. Kimmel, et al. (2013) randomly assigned 1015 patients to receive doses of warfarin during the first 5 days of therapy that were determined according to a dosing algorithm that included both clinical variables and genotype data or to one that included clinical variables only. All patients and clinicians were unaware of the dose of warfarin during the first 4 weeks of therapy. The primary outcome was the percentage of time that the international normalized ratio (INR) was in the therapeutic range from day 4 or 5 through day 28 of therapy. At 4 weeks, the mean percentage of time in the therapeutic range was 45.2% in the genotype-guided group and 45.4% in the clinically guided group (adjusted mean difference, [genotype-guided group minus clinically guided group], -0.2; 95% confidence interval, -3.4 to 3.1; P=0.91). There also was no significant between-group difference among patients with a predicted dose difference between the two algorithms of 1 mg per day or more. There was, however, a significant interaction between dosing strategy and race (P=0.003). Among black patients, the mean percentage of time in the therapeutic range was less in the genotype-guided group than in the clinically guided group. The rates of the combined outcome of any INR of 4 or more, major bleeding, or thromboembolism did not differ significantly according to dosing strategy.
The Invader UGT1A1 Molecular Assay:
Colorectal cancer (CRC) is one of the most common malignancies in western countries showing an increasing incidence, and has been associated with genetic as well as lifestyle factors. About 150,000 new cases of CRC are diagnosed each year in the United States, 40 to 50 % of which are metastatic. Irinotecan (Camptosar) is a chemotherapeutic agent approved as a combination therapy with 5-FU/leucovorin for the treatment of advanced CRC. The response to irinotecan is variable, possibly because of individuals' variation in the expression of the enzymes that metabolize irinotecan. Although multiple genes may play a role in irinotecan activity, the uridine diphosphate glycuronosyltransferase 1 family, polypeptide A1 (UGT1A1) enzyme has been strongly associated with irinotecan-related toxicity. The UGT1A1 gene is responsible for glucuronidation of the active metabolite of irinotecan. A common di-nucleotide repeat polymorphism in the UGT1A1 promoter region (UGT1A1*28) has been correlated with toxicity in cancer patients receiving irinotecan-containing therapy.
Lentz et al (2005) stated that hepatic metastases occur in about 50 % of patients with CRC. Since hepatic metastases are often inaccessible for surgery, chemotherapy of metastases is important. The most commonly used chemotherapeutic agents for hepatic metastases are fluorouracil, irinotecan, and oxaliplatin. Several enzymes are known to be involved in the metabolism of these drugs, and the activity of these enzymes varies greatly between individuals. The causes of this variation include genetic polymorphisms, different regulation between normal and cancer tissue, and the influence of chemotherapy on enzyme expression. The varying enzyme activity may have an important effect on the outcome of chemotherapy. Lentz et al (2005) reported that several studies confirm the influence of the activity of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase on the outcome of fluorouracil therapy for CRC, with higher enzyme activities predicting lower treatment efficacy. Although fewer studies are available regarding therapy of hepatic metastases, the same relationship between thymidylate synthase activity and outcome of fluorouracil therapy observed for primary CRC was found. For the other 2 enzymes, only a few studies are available, but the results indicate similarly that higher enzyme activity seems to be disadvantageous. Enzymes that are responsible for the activation, metabolism, and mechanism of action of irinotecan (e.g., CYP 3A4 and UGT1A1) also exhibit variable inter-individual activity. The authors concluded therefore, that there may be an association between enzyme activity and response to therapy. For example, in patients with CRC, higher enzyme activity of topoisomerase-I seems to be predictive of a better response to irinotecan. CYP3A4 and UGT1A1 activity levels might be predictive of irinotecan toxicity rather than efficacy. These authors stated that available data indicate the importance of the different enzyme activities on the outcome of chemotherapy of hepatic metastases in CRC. The authors noted that more information is needed, especially for the newer drugs irinotecan and oxaliplatin. However, the existing data are very promising in respect to the potential to guide dose and drug selection for more efficient and less toxic chemotherapy of hepatic metastases from CRC.
The Invader UGT1A1 molecular assay (Genzyme Corporation, Cambridge, MA) was cleared by the FDA on August 22, 2005. The test can be performed before starting irinotecan therapy and is designed to identify patients who may be at risk for ADRs to the chemotherapeutic agent by detecting a genetic variation in the UGT1A1 gene.
A clinical study (Innocenti et al, 2004) indicated that patients with one of these variations in the UGT1A1 gene have a significantly greater risk of experiencing drug-related toxicity from irinotecan than those without it. The product labeling for irinotecan (Camptosar) was updated to recommend that a reduced initial dose should be considered for patients homozygous for UGT1A1*28 allele, although the precise dose reduction in this group of patients is unknown (Waknine, 2005). The product labeling, however, does not include a recommendation for assessment of UGT1A1 status prior to initiation of irinotecan therapy.
In a prospective study, Innocenti and colleagues (2004) assessed the association between the prevalence of severe toxicity and UGT1A1 genetic variation. A total of 66 cancer patients with advanced disease refractory to other treatments received irinotecan 350 mg/m2 every 3 weeks. Toxicity and pharmacokinetic data were measured during cycle 1. UGT1A1 variants (-3279G>T, -3156G>A, promoter TA indel, 211G>A, 686C>A) were genotyped. The prevalence of grade 4 neutropenia was 9.5 %. Grade 4 neutropenia was much more common in patients with the TA indel 7/7 genotype (UGT1A1(*)28 homozygous) (3 of 6 patients; 50 %) compared with 6/7 (3 of 24 patients; 12.5 %) and 6/6 (0 of 29 patients; 0 %) (p = 0.001). The TA indel genotype was significantly associated with the absolute neutrophil count nadir (7/7 less than 6/7 less than 6/6, p = 0.02). The relative risk of grade 4 neutropenia was 9.3 (95 %) for the 7/7 patients versus the rest of the patients. Pre-treatment total bilirubin levels were significantly higher in patients with grade 4 neutropenia (0.83 +/- 0.08 mg/dL) compared to those without grade 4 neutropenia (0.47 +/- 0.03 mg/dL; p < 0.001). The -3156G>A variant seemed to distinguish different phenotypes of total bilirubin within the TA indel genotypes. The -3156 genotype and the SN-38 area under the concentration versus time curve were significant predictors of absolute neutrophil count nadir (r2 = 0.51). These investigators concluded that UGT1A1 genotype and total bilirubin levels are strongly associated with severe neutropenia, and could be used to identify cancer patients predisposed to the severe toxicity of irinotecan. Furthermore, the hypothesis that the -3156G>A variant is a better predictor of UGT1A1 status than the previously reported TA indel requires further testing.
In an accompanying editorial, McLeod and Watters (2004) raised questions regarding the findings of Innocenti et al (2004): (i) is the relative risk of grade 4 neutropenia in a patient with the UGT1A1(*)28 homozygous genotype the same after 300 to 350 mg/m2 every 3 weeks (9.3-fold risk) as after the 100 to 125 mg/m2 weekly regimen? and (ii) does this marker retain its predictive power when irinotecan is used as part of a combined treatment? These researchers also noted that it would be difficult to perform randomized controlled trials to ascertain if there is a predictable, safe, and effective dose of irinotecan that can be given to patients with the UGT1A1(*)28 homozygous genotype, or if physicians should choose a non-irinotecan-containing regimen, since only 10 % of all patients have this genotype. Moreover, studies are planned to determine the impact of dosage on the safety of irinotecan in patients with either the UGT1A1 6/6/ or 6/7 genotype, which are the 2 most common genotypes in the general patient population.
While several investigators (And et al, 2000; Iyer et al, 2002, and Innocenti et al, 2004) reported that testing of patients carrying the UGT1A1*28 polymorphism may detect their susceptibility to irinotecan-related toxicity, others (Marcuello et al, 2004, Carlini et al, 2005, Dervieux et al, 2005, and Eichelbaum et al, 2005) have questioned its clinical value. In a clinical trial, Marcuello et al (2004) examined the influence of the UGT1A1 gene promoter polymorphism in the toxicity profile, in the response rate and in the overall survival (OS) in 95 patients with metastatic CRC treated with an irinotecan-containing chemotherapy. Genotypes were determined by analyzing the sequence of TATA box of UGT1A1 of genomic DNA from the patients. Clinical parameters and genotypes were compared by uni-variate and multi-variate statistical methods. The more frequent ADRs were asthenia (n = 34), diarrhea (n = 29) and neutropenia (n = 20). Severe diarrhea was observed in 7/10 (70 %) homozygous and 15/45 (33 %) heterozygous in comparison to 7/40 (17 %) wild-type patients (p = 0.005). These results maintained the statistical significance in logistic regression analysis (p = 0.01) after adjustment for other clinical relevant variables. The presence of severe hematological toxicity increased from wild-type patients to UGT1A1(*)28 homozygotes, but without achieving statistical significance. No relationship was found between the UGT1A1(*)28 genotypes and infection, nausea or mucositis. In uni-variate studies, patients with the UGT1A1(*)28 polymorphism showed a trend to a poorer OS (p = 0.09). In the multi-variate analysis, the genotype was not related to clinical response or to OS. These investigators stated that the role of the UGT1A1 genotype as a predictor of toxicity in patients with CRC receiving irinotecan demands the performance of randomized controlled studies to determine if genotype-adjusted dosages of the drug can help to establish safe and effective doses not only for patients with the UGT1A1(*)28 homozygous genotype, but also for those with the most common UGT1A1 6/6 or 6/7 genotype.
In a phase II clinical study, Carlini and co-workers (2005) examined whether germ-line polymorphisms within genes related to drug target (thymidylate synthase) or metabolizing enzymes (UGT isozymes) would alter response and toxicity to the combination of capecitabine plus irinotecan. A total of 67 patients with measurable CRC were treated with intravenous irinotecan (100 or 125 mg/m2) on days 1 and 8 and capecitabine orally (900 or 1,000 mg/m2, twice daily) on days 2 through 15 of each 3-week cycle. Genomic DNA was obtained from peripheral blood and genotyped using Pyrosequencing, GeneScan, and direct sequencing technologies. The overall objective response rate was 45 % with 21 patients (31 %) exhibiting grade 3 or 4 diarrhea and 3 patients (4.5 %) demonstrating grade 3 or 4 neutropenia in the first 2 cycles. Low enzyme activity UGT1A7 genotypes, UGT1A7*2/*2 (6 patients) and UGT1A7*3/*3 (7 patients), were significantly associated with anti-tumor response (p = 0.013) and lack of severe gastrointestinal toxicity (p = 0.003). In addition, the UGT1A9 -118 (dT)(9/9) genotype was significantly associated with reduced toxicity (p = 0.002) and increased response (p = 0.047). There were no statistically significant associations between UGT1A1, UGT1A6, or thymidylate synthase genotypes and toxicity or tumor response. The authors concluded that these data strongly suggest that UGT1A7 and/or UGT1A9 genotypes may be predictors of response and toxicity in CRC patients treated with capecitabine plus irinotecan. Specifically, patients with genotypes conferring low UGT1A7 activity and/or the UGT1A9 (dT)(9/9) genotype may be particularly likely to exhibit greater anti-tumor response with little toxicity. However, it is interesting to note that the allele frequencies of UGT1A7 gene in Taiwan Chinese are different from those in Caucasians and Japanese (Huang et al, 2005).
In a review, Dervieux et al (2005) stated that several proofs of principle have established that pharmacogenetic testing for mutations altering expression and functions of genes associated with drug disposition and response can reduce the "trial-and-error" dosing and decrease the risk of ADRs. These proofs of principle include UGT1A1 and irinotecan therapy, as well as CYP450 2C9 and S-warfarin therapy. These evidences advocate for the prospective identification of mutations associated with drug response, serious ADRs and treatment failure. The authors stated that with the convergence of rising drug costs and evidence supporting the clinical benefits of pharmacogenetic testing, it will be important to demonstrate the improved net health outcomes attributed to the additional costs for this testing.
This in agreement with the observation of Eichelbaum et al (2005) who noted that there is also a growing list of genetic polymorphisms in drug targets that have been demonstrated to influence drug response. A major limitation that has moderated the use of pharmacogenetic testing in the clinical setting is the lack of prospective clinical trials showing that such testing can improve the benefit/risk ratio of drug therapy. Moreover, Gardiner and Begg (2005) stated that currently pharmacogenetic tests for drug metabolizing enzymes are rarely carried out in clinical practice, despite repeated claims that they may benefit patient care. They noted that "the only tests performed with any regularity in Australasia are for thiopurine methyltransferase and pseudocholinesterase, and CYP2D6 phenotyping in 1 center for patients on perhexilene. The low clinical utilization reflects a poor evidence base, un-established clinical relevance and, in the few cases with the strongest rationale, a slow translation to the clinical setting".
Han et al (2006) determined if uridine diphosphate-glucuronosyltransferase 1A1, UGT1A7, and UGT1A9 polymorphisms affect the pharmacokinetics (PK) of irinotecan and treatment outcome of Korean patients with advanced non-small-cell lung cancer (NSCLC). A total of 81 patients with advanced NSCLC were treated with irinotecan (80 mg/m2) on day 1 and 8 and cisplatin (60 mg/m2) on day 1 intravenously of each 3-week cycle. Genomic DNA was extracted from peripheral blood and genotyped using direct sequencing. These researchers analyzed the association of UGT1A genotypes with irinotecan PK and clinical outcomes. All statistical tests were two-sided. In genotype-PK association analysis, UGT1A1*6/*6 (n = 6), UGT1A7*3/*3 (n = 6), and UGT1A9-118(dT)9/9 (n = 11) were associated with significantly lower area under the time-concentration curve (AUC) SN-38G to SN-38 (AUC(SN-38G)/AUC(SN-38)) ratio (p = 0.002, p =0 .009, and p = 0.001, respectively). In linkage disequilibrium analysis, the UGT1A7 variants were highly linked with the UGT1A1*6 (D' = 0.85, r2 = 0.63) and UGT1A9*22 (D'= 0.95, r2 = 0.88), which was substantiated in haplotype analysis. Patients with UGT1A1*6/*6 had lower tumor response and higher incidence of severe neutropenia. UGT1A9-118(dT)9/9 also showed a trend for high incidence of severe diarrhea, but not tumor response. In survival analysis, patients with UGT1A1*6/*6 had significantly shorter progression-free survival (p = 0.001) and overall survival (p = 0.017). The authors concluded that UGT1A1*6 and UGT1A9*22 genotypes may be important for SN-38 glucuronidation and associate with irinotecan-related severe toxicity. Specifically, UGT1A1*6 might be useful for predicting tumor response and survival outcome of Korean patients with NSCLC treated with irinotecan-based chemotherapy. These investigators also stated that "[a]lthough it is still hypothetical, we suggest that UGT1A1*6 and/or UGT1A9*22 genotypes might be important for predicting severe toxicity and treatment outcome after irinotecan-based chemotherapy. To confirm the data observed in this study, further larger studies are needed in an independent data set, preferably in a group of patients of similar ethnicity".
In an editorial that accompanied the study by Han et al (2006), Innocenti and associates (2006) stated that "[t]he study by Han et al provides evidence that the UGT1A1*6 polymorphism can be considered a biomarker of severe toxicity of irinotecan in Asians. The impact of this variant on efficacy in irinotecan-containing regimens should be prospectively investigated in patients of Asian descent ….".
In a review on genetic polymorphisms of drug-metabolizing enzymes and drug transporters in the chemotherapeutic treatment of cancer, Bosch et al (2006) focused on the clinical significance of polymorphisms in drug-metabolizing enzymes (CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, dihydropyrimidine dehydrogenase, UGT1A1, glutathione S-transferase, sulfotransferase [SULT] 1A1, N-acetyltransferase [NAT], thiopurine methyltransferase [TPMT]) and drug transporters (P-glycoprotein [multi-drug resistance 1], multi-drug resistance protein 2 [MRP2], breast cancer resistance protein [BCRP]) in influencing toxicity and effectiveness of chemotherapy. The authors stated that the clinical application of pharmacogenetics in cancer treatment will require more detailed information of the different polymorphisms in drug-metabolizing enzymes and drug transporters; and that larger studies, in different ethnic populations, and extended with haplotype and linkage disequilibrium analysis, are needed for each anti-cancer drug separately.
While initial reports described above suggested that UGT1A1*28 homozygotes were at high risk for worse irinotecan-related hematologic and gastrointestinal toxicity, more recent reports suggest that the magnitude of the problem (particularly the association with worse diarrhea) is not as great as was initially suspected. In a prospective study of 250 patients with metastatic colorectal cancer starting irinotecan, fluorouracil and leukovorin, the relative risk for grade 3 or 4 hematologic toxicity was significantly higher among UGT1A1*28 homozygotes (odds ratio 8.63, 95 % CI: 1.31 to 56.55) (Toffoli et al, 2006). However, the absolute magnitude of risk was relatively low (13.6 % versus 1.7 % for those with the wild-type alleles), and relevant for the first cycle only. Furthermore, there was no significant association between the presence of a UGT1A1*28 polymorphism and severity of diarrhea, or the need for irinotecan dose reduction.
One study found higher rates of neutropenia in persons homozygous for the UGT1A1*28 allele, regardless of whether the combination chemotherapy regimen included irinotecan (McLeod et al, 2006). In a preliminary analysis of data from 520 patients with colorectal cancer enrolled in the United States Intergroup (INT) 9741 trial, which compared a variety of first-line oxaliplatin and irinotecan-containing chemotherapy regimens, the risk of grade 3 or 4 neutropenia was significantly higher for homozygotes (but not heterozygotes) regardless of whether they received irinotecan or oxaliplatin-based chemotherapy (36.2 % versus 18.2 % and 14.8 % for homozygotes, heterozygotes, and wild-type alleles, respectively). Similar to the study by Toffoli et al (2006) described above, the investigators found no association between inheritance of UGT1A1*28 alleles and treatment-related diarrhea. UGT1A1*28 was also not a predictor of tumor response, time to progression, or overall survival.
The Evaluation of Genomic Applications in Practice and Prevention Working Group (EGAPP, 2009) found that the evidence is currently insufficient to recommend for or against the routine use of UGT1A1 genotyping in patients with metastatic colorectal cancer who are to be treated with irinotecan, with the intent of modifying the dose as a way to avoid adverse drug reactions (severe neutropenia). The EGAPP Working Group found no evidence to support clinical utility. Preliminary modeling suggests that, even if targeted dosing were to be highly effective, it is not clear that benefits (reduced adverse drug events) outweigh harms (unresponsive tumors).
In summary, although it has been advocated that pharmacogenetic testing of patients with CRC before chemotherapy with irinotecan may reduce the frequency of severe toxicities by allowing alternate therapy selections for patients carrying the UGT1A1*28 polymorphism, the clinical value of this testing (i.e., whether testing will lead to better health outcomes) has yet to be established by prospective, randomized, controlled trials. Only about 1 in 10 patients will be identified as being homozygous, and the excess risk of severe neutropenia that is attributable to the inheritance of this polymorphism appears to be small. There is a lack of consensus on whether initial dose reduction is needed for UGT1A1*28 homozygotes, and the precise dose reduction that is warranted in this patient population has not been determined.
Genotyping for HLA-B*1502
Carbamazepine (brand names Carbatrol, Equetro and Tegretol) is used for the treatment of patients with epilepsy, bipolar disorder, and neuropathic pain. The use of carbamazepine is associated with rare but severe and sometimes life-threatening skin reactions, which includes toxic epidermal necrolysis and Stevens-Johnson syndrome, characterized by multiple skin lesions, blisters, fever, itching and other symptoms. The risk of these reactions is estimated to be about 1 to 6 per 10,000 new users of the drug in countries with mainly white populations. However, the risk is estimated to be about 10 times higher in some Asian countries. Studies have demonstrated a strong association between certain serious skin reactions and an inherited variant of an immune system gene, HLA-B*1502, found almost exclusively among individuals with Asian ancestry (Hung et al, 2006; Chung et al, 2007).
In December 2007, the FDA announced that manufacturers of drugs containing carbamazepine have agreed to add to the drugs' labeling a recommendation that, before starting therapy with the drugs patients with Asian ancestry should receive genotyping of HLA-B*1502.
HLA-B*5701 Screening
Abacavir (Ziagen) is a nucleoside analogue reverse transcriptase inhibitor indicated for use in combination with other antiretroviral drugs for the treatment of HIV-1 infection. Review of reports of hypersensitivity in patients receiving abacavir (Glaxo Wellcome Inc., Research Triangle Park, NC) indicated that respiratory symptoms (including cough, dyspnea, and pharyngitis) have occurred in approximately 20 % of patients who have had hypersensitivity reactions. The frequency of the HLA-B*5701 allele varies in different populations, occurring in whites 5 to 8 %, Hispanics 4 to 7 %, Asians less than 1 %, Spaniards 1 to 4 %, and rarely in Sub-Saharan Africans. A delay in diagnosis of hypersensitivity can result in abacavir being continued or re-introduced, leading to more severe hypersensitivity reactions, including life-threatening hypotension and death.
In a double-blind, prospective, randomized study, Mallal et al (2008) examined whether HLA-B*5701 screening could prevent hypersensitivity reaction to abacavir. Patients who were infected with HIV-1 infection (n = 1956) who had not previously received abacavir were randomly assigned to undergo prospective HLA-B*5701 screening, with exclusion of HLA-B*5701 positive patients from abacavir treatment (prospective screening group), or to undergo a standard of care approach of abacavir use without prospective HLA-B*5701 screening (control group). All patients who started abacavir were observed for 6 weeks. Epicutaneous patch testing with abacavir was performed to immunologically confirm and enhance the specificity of the clinical diagnosis of hypersensitivity reaction to abacavir. The prevalence of HLA-B*5701 in this predominantly white study population was 5.6 %. Hypersensitivity reaction was diagnosed in 93 patients with a significantly lower incidence in the prospective-screening group (3.4 %) than in the control group (7.8 %). Of the patients receiving abacavir, 72 % were men, 84 % were white, and 18 % had not previously received anti-retroviral therapy. Screening eliminated immunologically confirmed hypersensitivity reaction (0 % in the prospective-screening group versus. 2.7 % in the control group), with a negative predictive value of 100 % and a positive predictive value of 47.9 %. The authors concluded that HLA-B*5701 screening reduced the risk of hypersensitivity reaction to abacavir. Although the population in Mallal's study was predominantly white, other investigators have reported comparable sensitivity results of HLA-B*5701 for abacavir hypersensitivity in different ethnic groups, including blacks (Saag M et al, 2007) and Spaniards (Rodríguez-Nóvoa et al, 2007). In addition, Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents (2008) developed by the Department of Health and Human Services recommends:
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Screening for HLA-B*5701 before starting patients on an abacavir-containing regimen to reduce the risk of hypersensitivity reaction (strength of recommendation: Strong evidence with at least 1 randomized trial with clinical results);
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HLA-B*5701-positive patients should not be prescribed abacavir (strength of recommendation: Strong evidence with at least 1 randomized trial with clinical results);
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The positive status should be recorded as an abacavir allergy in the patient’s medical record (strength of recommendation: Strong evidence with clinical trials with laboratory results);
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When HLA-B*5701 screening is not readily available, it remains reasonable to initiate abacavir with appropriate clinical counseling and monitoring for any signs of hypersensitivity reaction (strength of recommendation: Optional with expert opinion).
Genotyping for Apolipoprotein E (Apo E)
Apolipoprotein E (Apo E), a member of the apolipoprotein gene family, is essential in the formation of very low density lipoprotein (VLDL) and chylomicrons. Among the variants of this gene, alleles e2, e3, and e4 are the common polymorphism found in most populations. Of these variants, Apo e3 is the most frequent (greater than 60 %) in all populations studied. The polymorphism has functional effects on lipoprotein metabolism mediated through the hepatic binding, uptake, and catabolism of chylomicrons, chylomicron remnants, VLDL, and high density lipoprotein subspecies. Apolipoprotein E is the primary ligand for 2 receptors, the low density lipoprotein (LDL) receptor (also known as the B/E receptor) found on the liver and other tissues and an Apo E-specific receptor found in the liver. The interactions of these lipoprotein complexes with their receptors form the basis for the metabolic regulation of cholesterol. Allelic variation in apolipoprotein is consistently associated with plasma concentrations of total cholesterol, LDL cholesterol, and Apo B (the major protein of LDL, VLDL, and chylomicrons). Apolipoprotein has been studied in disorders associated with elevated cholesterol levels or lipid derangements (i.e., hyperlipoproteinemia type III, coronary heart disease, strokes, peripheral artery disease, and diabetes mellitus). The apolipoprotein genotype yields poor predictive values when screening for clinically defined atherosclerosis despite positive, but modest associations with plaque and coronary heart disease outcomes. In addition to genotype-phenotype associations with vascular disease, the alleles and isoforms of apolipoprotein have been related to dementias, most commonly Alzheimer's disease.
Several studies have been published assessing the interactions between Apo E and cholesterol in response to lipid-lowering drugs.
Gerdes et al (2000) examined whether the beneficial effects of simvastatin treatment differed by apolipoprotein genotype. After providing dietary advice, they randomized men and women aged 35 to 70 years with a history of myocardial infarction or angina, serum total cholesterol concentrations in the range of 5.5 to 8.0 mmol/L, and serum triglyceride levels of less than 2.5 mmol/L to placebo or simvastatin groups. Simvastatin treatment reduced the mortality risk more in e4 carriers than in other patients, although the difference was not statistically significant for the treatment by genotype interaction.
At least 2 other studies have examined the influence of the Apo E polymorphism in response to lipid-lowering drug treatments in patients with combined hyper-lipoproteinemia and familial hypercholesterolemia.
Knijff et al (1990) examined the influence of the Apo E polymorphism on pre-treatment plasma lipid levels and on the response to simvastatin treatment in a sample of 120 Dutch patients with heterozygous familial hypercholesterolemia. They found that differences in pre-treatment lipid levels were not related to the Apo E polymorphism in these patients. With respect to the effect of 12 weeks of simvastatin treatment, a reduction of 33 %, 38 %, and 19 % (on average) was found in the plasma levels of total cholesterol, LDL cholesterol, and triglycerides, respectively. Inter-individual variation in response to simvastatin treatment was not related to the Apo E polymorphism.
Nestel et al (1997) conducted a cross-over, randomized trial to examine the efficacy of simvastatin and gemfibrozil in patients with combined hyperlipoproteinemia. Efficacy was noted after 6 and 12 weeks on each treatment for the 66 subjects enrolled. The lipid-lowering responsiveness was greatest in those with the Apo E2 isoform with both medications.
An Agency for Healthcare Research and Quality (AHRQ, 2008) technology assessment on pharmacogenetic testing reviewed the available evidence of Apo E genotype (e2, e3, and e4) and statin treatment and found that genotyping for Apo E has not been shown to help select patients for treatment. "No studies addressed the effects of therapeutic choice: there were no data on the benefits, harms, or adverse effects on patients from subsequent therapeutic management after pharmacogenetic testing for the three Apo E genotypes."
The AHRQ assessment found that the pooled reduction in total and LDL cholesterol from baseline values was lower for all 3 genotypes but did not differ significantly among them.
The AHRQ also found significant between-study heterogeneity. "Although few studies included certain subgroups, factors that may affect the associations between all three Apo E genotypes and response to statin therapy were ethnicity, sex, familial hyperlipidemia, the type of statin used, and possibly the presence of diabetes."
In addition, there are no prospective data showing improved clinical management of hypercholesterolemia patients as a result of genotyping for Apo E.
Genotyping for Methylenetetrahydrofolate Reductase (MTHFR)
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating intracellular folate levels, which in turn affects DNA synthesis and methylation. Two MTHFR gene polymorphisms, C677T and A1298C, influence the metabolism of folates and could modify the pharmacodynamics of antifolates and many other drugs whose metabolism, biochemical effects, or target structures require methylation reactions. Several studies have shown these two polymorphisms may reduce cancer susceptibility and increase drug-related toxicity when folate antagonists (e.g., methotrexate, fluorouracil) are utilized, but data are inconsistent and contradictory. According to the National Cancer Institute, 5 of 6 patients who experienced grade-4 toxicity in their first cycle of adjuvant chemotherapy with cyclophosphamide, methotrexate and 5-FU for early breast cancer had the variant C677T MTHFR genotype.
An Agency for Healthcare Research and Quality (AHRQ, 2008) technology assessment on pharmacogenetic testing reviewed the available evidence on MTHFR gene polymorphisms for their associations with patient's response to therapy with antifolate chemotherapy and found that the evidence indicates that MTHFR gene polymorphisms do not predict response to chemotherapy.
AHRQ found limited data on MTHFR gene testing and therapeutic choice which preclude making meaningful inferences about the relationship between common variants in MTHFR and chemotherapy of the folate metabolic pathway.
AHRQ also found considerable variation in study designs, study populations, medication dosages, and the type of medications.
Studies have shown that MTHFR polymorphisms may affect the sensitivity to antifolate chemotherapy, however, there is insufficient evidence of its clinical effectiveness.
Measurement of Thromboxane Metabolites in Urine
The U.S. Preventive Services Task Force (USPSTF, 2009) has developed guidelines for chronic aspirin therapy based upon a person's age and Framingham risk score. Long-term aspirin administration has clear benefits for the secondary prevention of cardiovascular diseases with a significant 21 % reduction in the risk of cardiovascular events over 2 years (Berger et al, 2008). However, this indicates that not all individuals respond equally to aspirin therapy and cardiovascular events may occur during aspirin therapy. This is often described as "clinical aspirin resistance". A systematic review and meta-analysis on aspirin resistance indicated that patients who are resistant to aspirin are at a greater risk (odds ratio [OR]: 3.85) of clinically important cardiovascular morbidity than patients who are sensitive to aspirin (Krasopoulos et al, 2008). The effect of aspirin administration varies considerably among patients at high risk for cardiovascular events. Gum and co-workers (2001) found insufficient inhibition of platelet aggregation by aspirin in 6 to 24 % of patients with stable coronary artery disease, while other estimates range from 5 to 60 % (Martin and Talbert, 2005).
Many authors believe that aspirin resistance can be detected by biochemical tests and several commercially available products are being marketed for this purpose. Tests used in research laboratories are aggregometry (turbidometric and impedance), tests based on activation-dependent changes in platelet surface, and tests based on activation-dependent release from platelets. Point-of-care tests include PFA-100, IMPACT, and VerifyNow, which can detect platelet dysfunction that may be due to aspirin effect.
It has been proposed that aspirin resistance can also be detected by thromboxane metabolites in urine. Aspirin inhibits platelet activation through the permanent inactivation of the cyclooxygenase (COX) activity of prostaglandin H synthase-1 (referred to as COX-1), and consequently inhibits the biosynthesis of thromboxane A2(TXA2), a platelet agonist. The urinary concentrations of the metabolite 11-dehydrothromboxane B2(11 dhTxB2) indicate the level of TXA2 generation.
Eikelboom et al (2002) studied whether aspirin resistance, defined as failure of suppression of thromboxane generation, increases the risk of cardiovascular events in a high-risk population. Baseline urine samples were obtained from 5,529 Canadian patients enrolled in the Heart Outcomes Prevention Evaluation (HOPE) Study. Using a nested case-control design, the investigators measured urinary 11 dhTxB2 levels, a marker of in vivo thromboxane generation, in 488 cases treated with aspirin who had myocardial infarction, stroke, or cardiovascular death during 5 years of follow-up and in 488 sex- and age-matched control subjects also receiving aspirin who did not have an event. After adjustment for baseline differences, the odds for the composite outcome of myocardial infarction, stroke, or cardiovascular death increased with each increasing quartile of 11 dhTxB2, with patients in the upper quartile having a 1.8-times-higher risk than those in the lower quartile (OR, 1.8; 95 % CI: 1.2 to 2.7; p = 0.009). Those in the upper quartile had a 2-times-higher risk of myocardial infarction (OR, 2.0; 95 % CI: 1.2 to 3.4; p = 0.006) and a 3.5-times-higher risk of cardiovascular death (OR, 3.5; 95 % CI: 1.7 to 7.4; p < 0.001) than those in the lower quartile. The authors concluded that in aspirin-treated patients, urinary concentrations of 11 dhTxB2 predict the future risk of myocardial infarction or cardiovascular death and that these findings raise the possibility that elevated urinary 11 dhTxB2 levels identify patients who are relatively resistant to aspirin and who may benefit from additional anti-platelet therapies or treatments that more effectively block in vivo thromboxane production or activity. However, Altman et al (2004) reviewed this study and stated that the authors support the view that failure to suppress thromboxane generation defines aspirin resistance and that this hypothesis assumes a direct association between the rise of urinary 11 dhTxB2 levels and increment of vascular events (e.g., myocardial infarction, stroke and cardiovascular death). Altman and colleagues explained that failure of aspirin to produce the expected inhibition of platelet function might be attributed to several mechanisms and that it can not be defined by the level of serum thromboxane or its urinary metabolites because these measurements do not correlate with the reduction of inhibition of platelet aggregation in response to multiple stimuli, and also because (i) although most of the thromboxane is believed to come from the platelets, there are additional cellular origins (e.g., monocytes/macrophages are also a rich source of TXA2), (ii) unlike the platelet, the macrophage is capable of synthesizing new COX-2 after aspirin has inhibited it; COX-2 is the enzyme responsible for most of the metabolism of arachidonic acid in the macrophage, and low dose aspirin is not sufficient to inhibit COX-2 maximally, (iii) macrophages in atheromata may contribute significantly to the pool of TXA2, and (iv) aspirin only inhibits monocyte PGHS-2, which is inducible by inflammatory stimuli, transiently at very high concentrations.
The AspirinWorks Test Kit (Corgenix Medical Corp; Broomfield, CO) is an enzyme-linked immunoassay test that can be used to determine levels of 11 dhTxB2 in human urine. AspirinWorks received 510(k) marketing clearance from the FDA in May, 2007 and is intended to aid in the qualitative detection of aspirin in apparently healthy individuals post ingestion.
The AspirinWorks Test Kit was compared to the Accurnetrics VerifyNow Aspirin Assay as the predicate device. The manual AspirinWorks Test Kit measures urinary 11 dhTxB2, while the automated Accumetrics VerifyNow Aspirin Assay is a turbidimetric-based optical detection system, which measures platelet-induced aggregation in whole blood. The two devices have similar intended uses in that they both measure aspirin effect. The AspirinWorks kit detects a metabolite of TxA2, a direct inducer of platelet aggregation, while the Accumetrics kit measures ex vivo platelet aggregation caused by TxA2 by artificially inducing aggregation and measuring an optical signal. Ultimately, both are analyzing aspirin's effect through the reduction of TxA2 production or the resulting inhibition of platelet aggregation.
According to the FDA, 2 different clinical studies were employed for the evaluation of the AspirinWorks Test Kit. Results from these studies established a cutoff for aspirin effect at less than 1500 pg 11d hTxB2/mg creatinine. Further analysis revealed that 180/204 (88.2 %) of samples from individuals not taking aspirin were above the cut-off value. Analysis of samples from individuals taking various doses of aspirin revealed that 7/163 (4.3 %) of 81 mg/day aspirin users indicated a lack of aspirin effect (greater than 1500 pg 1 ldhTxB2/mg creatinine) and 4/38 (10.5 %) of the 325 mg/day aspirin users indicated a lack of aspirin effect. In total, 11/201 (5.5 %) of all aspirin users tested indicated a lack of aspirin effect. These percentages are consistent with those in published literature for aspirin non-responsiveness or lack of aspirin effect.
Lordkipanidze et al (2007) compared the results obtained from 6 major platelet function tests in the assessment of the prevalence of aspirin resistance in patients with stable coronary artery disease. Patients with stable coronary artery disease (n = 201) receiving daily aspirin therapy (80 mg or more) were recruited. Platelet aggregation was measured by: (i) light transmission aggregometry (LTA) after stimulation with 1.6 mM of arachidonic acid (AA), (ii) LTA after adenosine diphosphate (ADP) (5, 10, and 20 microM) stimulation, (iii) whole blood aggregometry, (iv) PFA-100, (v) VerifyNow Aspirin; urinary 11 dhTxB2 concentrations were also measured. Eight patients (4 %, 95 % CI: 0.01 to 0.07) were deemed resistant to aspirin by LTA and AA. The prevalence of aspirin resistance varied according to the assay used: 10.3 to 51.7 % for LTA using ADP as the agonist, 18.0 % for whole blood aggregometry, 59.5 % for PFA-100, 6.7 % for VerifyNow Aspirin, and finally, 22.9 % by measuring urinary 11 dhTxB2 concentrations. Results from these tests showed poor correlation and agreement between themselves. The authors concluded that platelet function tests are not equally effective in measuring aspirin's anti-platelet effect and correlate poorly amongst themselves and that the clinical usefulness of the different assays to classify correctly patients as aspirin resistant remains undetermined.
Hedegaard et al (2009) assessed the use of optical platelet aggregation versus thromboxane metabolites in healthy individuals and patients with stable coronary artery disease after low-dose aspirin administration. The authors investigated whether 75 mg of daily non-enteric coated aspirin would completely inhibit the platelet cyclooxygenase-1 activity to a comparable extent in healthy individuals and stable coronary artery disease (CAD) patients. Serum thromboxane B2 (S-TxB2), urinary 11 dhTxB2 (U-TxM) and arachidonic acid-induced optical platelet aggregometry (OPA) were compared in 44 coronary artery disease (CAD) patients on aspirin and in 22 healthy individuals before and after aspirin. Optical platelet aggregometry was performed in duplicate for 4 consecutive days during aspirin treatment after 1 week of treatment. Compliance was optimized by face-to-face interviews and pill counting and confirmed by S-TxB2 measurements. The authors found that aspirin inhibited S-TxB2 in healthy individuals (greater than 99 %; median 1.1 ng/mL, inter-quartile range [IQR] = 0.8;1.9 after aspirin) and in patients, S-TxB2 was reduced to a similar level (0.9 ng/mL (0.7;1.5)). Healthy individuals had a median U-TxM of 278.5 pg/mg creatinine (229.5;380.0) before aspirin and 68.5 pg/mg creatinine (59.0;99.7) on aspirin corresponding to an average 74 % inhibition of the endogenous TxA2 biosynthesis. In patients median U-TxM was 67.5 pg/mg creatinine (54.0;85.5). Seven study participants (11 %) were aspirin low-responders according to OPA, but none had S-TxB2 in the highest quartile. The authors concluded that low-dose aspirin suppressed S-TxB2 to comparable levels in CAD patients and healthy individuals. The authors found that despite an almost complete inhibition of S-TxB2, some participants were low-responders according to OPA. The authors concluded that thorough compliance control and use of thromboxane-specific assays are important when measuring platelet response to aspirin.
While some investigators believe that aspirin resistance can be detected by thromboxane metabolites in urine, other investigators support the view that aspirin resistance can not be defined by the level of serum thromboxane or its urinary metabolites because these measurements do not correlate with the reduction of inhibition of platelet aggregation in response to multiple stimuli as well as various other factors. Investigators have found a number of variables that may impact an individual's response to aspirin, including patient's compliance, dose, smoking, hyperlipidemia, hyperglycemia, acute coronary syndrome, percutaneous revascularization, recent stroke, extracorporeal circulation, heart failure, exercise, circadian rhythm, absorption, concomitant medications, and polymorphisms.
Many issues are yet to be resolved in order to apply the concept of "aspirin resistance" to actual clinical practice. The clinical usefulness of a test that measures thromboxane metabolites in urine has yet to be determined. The relevance of the various ex vivo functional indexes of platelet capacity to in vivo platelet activation and the precise mechanisms underlying aspirin resistance are still largely unknown. Further investigation is needed regarding strategies to identify and treat patients resistant to aspirin.
Genetic Testing for rs3798220 Allele (LPA-Aspirin Check)
Investigators have described a non-synonymous single nucleotide polymorphism (SNP rs3798220) in apo(a) gene (LPA) that has been associated with increased plasma levels of Lp(a) and advanced coronary artery disease. Observational studies have also associated that this polymorphism may identify a subgroup of patients who benefit from chronic aspirin therapy in regard to a reduction in clinical coronary heart disease events, and a subgroup which reveals no benefit and thus an increased risk for gastrointestinal bleeding. A genetic testing for the rs3798220 allele (LPA-Aspirin Check, Celera, Alameda, CA) is commercially available. This SNP, encoding an isoleucine to methionine substitution in the protease-like domain of apo(a) at amino acid 4399 (I4399M), has been associated with both elevated levels of Lp(a) and cardiovascular disease. In the Women's Health Study, carriers of this apolipoprotein(a) variant had elevated Lp(a), doubled cardiovascular risk, and appeared to benefit more from aspirin than non-carriers (Chasman et al, 2009). Chasman and colleagues (2009) investigated whether this allele was associated with elevated Lp(a) and cardiovascular risk in a post hoc analysis of the Women's Health Study, a randomized trial of low-dose aspirin, and whether aspirin reduced cardiovascular risk in minor allele carriers. The investigators determined genotypes of rs3798220 for 25,131 initially healthy Caucasian participants. Among women with successful genotyping, the minor allele of rs3798220 in the LPA gene was carried by 906 (3.6 %) heterozygotes and 15 (0.06 %) homozygotes for a minor allele frequency of 1.9 %. Median Lp(a) levels at baseline were 10.0, 79.5, and 153.9mg/dL for major allele homozygotes, heterozygotes, and minor allele homozygotes, respectively (p < 0.0001). During the 9.9 years of follow-up, minor allele carriers (3.7 %) in the placebo group had 2-fold higher risk of major cardiovascular events than non-carriers (age-adjusted hazard ratio (HR) = 2.21, 95 % CI: 1.39 to 3.52). The investigators found that, among carriers, risk was reduced more than 2-fold by aspirin: for aspirin compared with placebo the age-adjusted HR was 0.44 (95 % CI: 0.20 to 0.94); risk was not significantly reduced among non-carriers (age-adjusted HR = 0.91, 95 % CI: 0.77 to 1.08). The investigators reported that this interaction between carrier status and aspirin allocation was significant (p = 0.048). Shiffman and colleagues (2009) reported data on the interaction of the LPA rs3798220 variant and aspirin use from the Atherosclerosis Risk in Communities (ARIC) study, a prospective cohort study of risk factors for coronary artery disease in 15,792 individuals. The LPA genetic substudy of ARIC included 6752 individuals with data available for LPA genotype and aspirin use, including 221 individuals with the LPA rs3798220 genotype. Among carriers of rs3798220, the risk of cardiovascular events was compared in aspirin users and non-users. The HR for non-aspirin users (n = 168) was elevated at 1.57 but did not reach statistical significance (95 % CI: 0.92 to 2.69), while the HR for users of aspirin was not elevated at 0.86 (95 % CI 0.38 to 1.95). Prospective clinical studies are necessary to determine whether selection of persons for chronic aspirin therapy on the basis of rs2798220 status results in improved clinical outcomes.
Area Under the Curve (AUC)-Targeted 5-Fluorouracil Dosing
Diagnostic tests (e.g., Myriad Genetics OnDose) have been developed to measure colorectal cancer patients’ exposure to 5-fluorouracil (5‑FU), to help oncologists adjust and optimize 5‑FU dosing. This testing is based upon the belief that that 5‑FU chemotherapy dosing can be improved by modulating dose to plasma concentration, specifically an AUC target value, as treatment is delivered. An assessment by the BlueCross BlueShield Association Technology Evaluation Center (TEC, 2010) stated that, given the limitations of the existing evidence, the evidence is insufficient to draw conclusions about the impact of 5‑FU exposure measurement and AUC-targeted dose adjustment on outcomes of patients administered current chemotherapy regimens for colorectal or head and neck cancer.
To determine if there is adequate evidence that AUC-targeted dose adjustment of 5-FU improves clinical outcomes, the TEC assessment (BCBSA, 2010) focused primarily on the results of 2 randomized, controlled trials, 1 enrolling patients with colorectal cancer (Gamelin et al, 2008) and the other patients with head and neck cancer (Fety et al, 1998).
The randomized controlled trial of AUC-targeted dosing of 5-FU for colorectal cancer reported significantly improved tumor response and a trend toward improved survival using AUC-targeted dosing compared to fixed dosing (Gamelin et al, 2008). The authors of the clinical trial also reported 18 % grade 3 to 4 diarrhea in the fixed-dose arm, which the TEC assessment noted is higher than reported historically, where the rates of grade 3 to 4 diarrhea have ranged from approximately 5 to 7 %. However, the TEC assessment cited an editorial accompanying the study (Wako and McLeod, 2008) that noted that the fixed-dose administration schedule used in this study is “rarely used in current practice in most countries"; the TEC assessment also noted that this administration schedule is absent from current guidelines.
The AUC dose modulation trial in head and neck cancer patients reported overall 5‑FU exposures that were significantly reduced after dose adjustment compared to the fixed-dose arm (Fety et al, 1998). This resulted in reduced toxicity, but no improvement in clinical response. The TEC assessment noted that the dose adjustment method in this trial may have been too complex, as the 12 protocol violations in this treatment arm (of 61 enrolled) were all related to 5‑FU dose adjustment mis-calculations (BCBSA, 2010). The TEC assessment stated: "Because patients with protocol violations were not evaluated in an intention-to-treat analysis, the results do not reflect the 'real world' experience of this study." The TEC assessment also noted that the study used a 2-drug induction regimen, which has now been replaced by a 3-drug induction regimen.
Pharmacogenetic Testing for 5-Fluorouracil Toxicity
Genetic polymorphisms in the genes coding for dihydropyrimidine dehydrogenase (DPYD) and thymidylate synthase (TYMS), key enzymes in 5-FU metabolism, may result in enzyme products with different activity levels, resulting in 5-FU excess, the accumulation of 5-FU anabolic products, and severe toxicity. TheraGuide 5-FU (Myriad laboratories) is a commercially available test that analyzes DNA from peripheral blood cells to fully sequence the DPYD gene (for the 3 most common DYPD polymorphisms and other rare variants) and detect TYMS variants that increase risk for 5-FU toxicity. An assessment by the BlueCross BlueShield Association Technology Evaluation Center (TEC, 2010) found, however, that testing for genetic variants of the genes coding for DPYD and TYMS enzymes has poor predictive value for 5-FU toxicity and no studies have shown that it is useful in directing 5-FU dose alterations to reduce toxicity without adversely impacting tumor response.
The Cobas 4800 BRAF V600 Mutation Test:
B-RAF(V600E) (also known as BRAF) kinase mutations occur in about 8 % of all solid tumors and approximately 50 % to 60 % of melanomas; these mutations are found at exon 15, at a single amino acid residue, usually a substitution for valine by glutamic acid, V599E, now referred to as V600E (Nazarian et al, 2010; Dienstmann and Tabernero, 2011). The quantification of BRAF-mutated alleles in plasma may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy. The development of efficient methods for its detection in free circulating DNA of patients may lead to the improvement of diagnostic and prognostic tools.
Daniotti and colleagues (2007) examined if BRAFV600E represents a detectable marker in the plasma/serum from patients with melanoma. Circulating cell-free DNA was extracted from the serum or plasma of 15 healthy donors and 41 melanoma patients at different clinical stages and obtained either pre-surgery or post-surgery during follow-up. Quantitative analysis showed higher levels of circulating free DNA in patients compared to controls, with the highest levels detected in samples obtained pre-surgery and at stage IV. Four different PCR methods were compared for their capacity to amplify a few copies of BRAFV600E in wild-type DNA. BRAFV600E was detectable in circulating DNA of 12 patients and in none of the controls; only 1 PCR method reproducibly amplified BRAFV600E. Positive samples were obtained from 8/13 patients at stage IV and from 4/24 patients at stage III, but not in 4 patients at stage I to II; 50 % of the positives were obtained pre-surgery and 50 % at follow-up. Correspondence between circulating DNA and related tumors were examined for 20 patients, and a correlation was found for stage IV patients. The authors concluded that this method can be utilized for monitoring the disease in patients with stage IV melanoma, but it appears unsatisfactory for the early detection of melanoma.
Panka et al (2010) stated that the BRAFV600E mutation has been detected in patients with metastatic melanoma, colon, thyroid and other cancers. Recent studies suggested that tumors with this mutation are especially sensitive to BRAF inhibitors, hence the need to reliably determine the BRAF status of tumor specimens. The present technologies used to screen for this mutation fail to address the problems associated with infiltrating stromal and immune cells bearing wild-type BRAFalleles and thus may fail to detect the presence of mutant BRAFV600E tumors. These investigators developed a rapid, inexpensive method that reduces the contamination of wild-type BRAF sequences from tumor biopsies. The protocol involves a series of PCR amplifications and restriction digestions that take advantage of unique features of both wild-type and mutant BRAF RNA at position 600. Using this protocol, mutant BRAFcan be detected in RNA from mixed populations with as few as 0.1 % BRAFV600E mutant cells.
Pinzani and co-workers (2010) proposed an assay based on the use of a locked nucleic acid probe and an allele specific primer to measure plasma-circulating BRAFV600E concentration in patients affected by cutaneous melanoma (n = 55) and non-melanoma skin cancers (n = 13) as well as 18 healthy subjects. The assay is highly sensitive and accurate in detecting down to 0.3 % of mutated allele in plasma. A significant difference between the control group and invasive melanomas (p < 0.01) was evidenced in BRAFV600E concentration, either as relative percentage or absolute values. Receiver operating characteristic curve indicated that BRAFV600E absolute concentration has the maximal diagnostic relevance with 97 % sensitivity and 83 % specificity. Comparison of the results obtained in plasma with those found in the corresponding tissues indicated an 80 % concordance. The authors concluded that the allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAFV600E mutated DNA in plasma.
Pinzani et al (2011) investigated COLD-PCR (co-amplification at lower denaturation temperature-PCR) as a new approach for the pre-analytical enrichment of the BRAFV600E variant in formalin fixed paraffin embedded (FFPE) melanoma tissues. COLD-PCR was used to selectively amplify BRAFV600E minority alleles from mixtures of wild-type and mutated sequences, and from biological samples. The method showed higher specificity than other conventional PCR-based methods in detecting somatic mutations. These investigators used COLD-PCR to increase the theoretical sensitivity of 3 different post-PCR methods: (i) sequencing, (ii) pyro-sequencing, and (iii) HRMA. The gain in sensitivity seems to be more evident for HRMA, which allows the detection of 3.1 % mutated alleles. More than 20 % of patients initially classified negative for BRAFV600E were found positive after COLD-PCR. The authors concluded that COLD-PCR was confirmed as a suitable method for the enrichment of mutated alleles, particularly for samples in which the percentage of tumor cells is very low.
On August 17, 2011, the FDA approved vemurafenib (Zelboraf) for the treatment of patients with unresectable or metastatic melanoma with the BRAFV600E mutation as detected by an FDA-approved test. The approval was based on a randomized, open-label trial in patients with previously untreated metastatic or unresectable melanoma with the BRAFV600E mutation as detected by the cobas 4800 BRAF V600 Mutation Test (Roche Molecular Systems, Inc.). This companion diagnostic test was approved by the FDA concurrently with vemurafenib’s approval. The cobas 4800 BRAF V600 test is a PCR-based test. It has several advantages over the commonly used Sanger sequencing, including greater sensitivity and reliability for detecting mutations and quicker results.
IL28B Polymorphism Genotyping for Interferon Therapy for Hepatitis C:
The IL28B gene is involved with viral resistance and is up-regulated by interferons. IL28B polymorphisms appear to be a strong independent predictor of viral responsiveness. Thus, it is likely that testing for IL28B polymorphisms will be included in making treatment decisions and potentially guiding therapy. Currently, there are no studies evaluating the impact of ILB28B on treatment decisions. Well-designed studies are needed to clarify the role, if any, of IL28B polymorphism genotyping for interferon therapy for hepatitis C.
CYP2D6 Polymorphism and Alzheimer's Disease:
In a multi-center, prospective cohort study, Pilotto et al (2009) evaluated the influence of the single nucleotide polymorphism rs1080985 in the cytochrome P450 2D6 (CYP2D6) gene on the efficacy of donepezil in patients with mild to moderate Alzheimer's disease (AD). A total of 127 white patients with AD according to the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association Work Group criteria were included in this study. Patients were treated with donepezil 5 to 10 mg/daily for 6 months. Cognitive and functional statuses were evaluated at baseline and at 6-month follow-up. Response to therapy was defined according to the National Institute for Health and Clinical Excellence criteria. Compliance and drug-related adverse events were also evaluated. The analyses identifying the CYP2D6 and APOE polymorphisms were performed in blinded fashion. At 6-month follow-up, 69 of 115 patients (60 %) were responders and 46 patients (40 %) were non-responders to donepezil treatment. A significantly higher frequency of patients with the G allele of rs1080985 was found in non-responders than in responders (58.7 % versus 34.8 %, p = 0.013). Logistic regression analysis adjusted for age, sex, Mini-Mental State Examination score at baseline, and APOE demonstrated that patients with the G allele had a significantly higher risk of poor response to donepezil treatment (odds ratio 3.431, 95 % CI: 1.490 to 7.901). The authors concluded that the single nucleotide polymorphism rs1080985 in the CYP2D6 gene may influence the clinical efficacy of donepezil in patients with mild to moderate AD. The analysis of CYP2D6 genotypes may be useful in identifying subgroups of patients with AD who have different clinical responses to donepezil.
In a multi-center prospective cohort study, Seripa et al (2011) evaluated the effect of 16 functional polymorphisms in the CYP2D6 gene on the clinical response to donepezil treatment in patients with mild-to-moderate AD. These researchers evaluated 57 unrelated Caucasians clinically diagnosed as AD according to the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association Work Group criteria. Patients were treated with donepezil (5 to 10 mg/daily) for 6 months. The response to donepezil treatment was evaluated at 6-month follow-up according to the National Institute for Health and Clinical Excellence requirements. The identification of 16 clinically relevant CYP2D6 gene variants was performed by a high-throughput genetic analysis. Thirty-eight of 57 patients (67 %) were responders and 19 patients (33 %) were non-responders to donepezil treatment. A significantly higher frequency of gene variants conferring decreased or absent enzyme activity was observed in responder than in non-responder patients (73.68 % versus 36.84 %; p = 0.005). The presence of gene variants conferring decreased or absent activity of the CYP2D6 enzyme was significantly associated with a clinical response to donepezil treatment (odds ratio = 6.286; 95 % CI: 1.828 to 21.667). The authors concluded that functional polymorphisms in the CYP2D6 gene can influence the clinical efficacy of donepezil. The analysis of CYP2D6 genotypes may be useful in identifying subgroups of AD patients with different clinical response to donepezil treatment.
The Vysis ALK Break Apart FISH Probe Kit:
Crizotinib (Xalkori) is a kinase inhibitor which has recently received accelerated FDA approval for use in locally advanced or metastatic NSCLC that is ALK-positive as detected by an FDA-approved test. The FDA approved the Vysis ALK Break Apart FISH Probe Kit concurrently with crizotinib as a companion diagnostic test designed to detect rearrangements of the ALK gene in NSCLC. FISH refers to fluorescence in-situ hybridization, which has numerous uses, including identify whether too many, or too few, copies of a particular gene are present in the body’s cells or whether certain genes have rearrangements that play an active role in disease progression (Abbott Press Release, 2011).
Oncogenic fusion genes consisting of EML4 and anaplastic lymphoma kinase (ALK) are present in a subgroup of non-small-cell lung cancers, representing 2 to 7 % of such tumors (Kwak et al, 2010). Rodig et al (2010) noted that “crizotinib has been particularly effective against anaplastic large cell lymphoma and non-small cell lung cancer (NSCLC) cell lines that harbor ALK translocations resulting in expression of oncogenic ALK fusion proteins”.
Kwak et al (2011) screened tumor samples from approximately 1500 NSCLC patients for the presence of ALK rearrangements and found 82 patients with advanced ALK-positive disease in their study population. These patients were subsequently enrolled in a clinical trial of crizotinib therapy. At a mean treatment duration of 6.4 months, the overall response rate was 57 %, with an estimated probability of 6-month progression free survival was estimated at study completion to be 72 %. The investigators also noted patients with ALK rearrangements tended to be younger than those without, and most of these patients were light or non-smokers.
The 2010 American Society of Clinical Oncology Annual Meeting included a presentation on crizotinib for the treatment of advanced NSCLC. Stinchcombe et al (2011) summarized the evidence presented and stated that the results of crizotinib in molecularly selected patients with advanced NSCLC whose tumor cells had a novel fusion protein involving ALK reinforces the importance of understanding molecular heterogeneity in this patient population.
GeneSightRx Testing
GeneSightRx, developed by AssureRx Health (Mason, OH), is a technology that measures and analyzes genomic variants affecting the metabolism and response to health medications in patients. These genomic tests were developed to serve as a clinical treatment support tool, supposedly providing objective genetic-based patient information in advance of making a medication decision. Knowing a patient's genetic profile may aid in understanding which medications the patient would metabolize properly and help inform treatment choices unique to each patient. GeneSightRx Psychotropic analyzes 6 genes that may affect a patient’s response to anti-depressant and anti-psychotic medications. Four pharmacokinetic genes from the cytochrome P450 family and 2 pharmacodynamic genes related specifically to the serotonin system are genotyped.
Kirchheiner et al (2010) stated that more than 50 years of pharmacogenetic research have produced many examples of the impact of inherited variability in the response to psychotropic drugs. These successes, however, have as yet failed to translate into broadly applicable strategies for the improvement of individual drug treatment in psychiatry. One important argument against the widespread adoption of pharmacogenetics as a clinical tool is the lack of evidence showing its impact on medical decision making and on risk benefit ratio for the patients. The individual drug metabolizing capacity is assessed by genotyping drug metabolizing enzymes. The potential implications of information gained from genotyping are dose adjustments according to genotype. However, even when the consequences of genotype on pharmacokinetics are significant and well known, as in the case of many tricyclic anti-depressants and several selective serotonin reuptake inhibitors (SSRIs), there is still considerable controversy on whether adjustment of dosage driven by genetic information may improve therapeutic efficacy, and/or adverse events is prevented, to an extent of any practical importance in clinical practice. Different types of pharmacogenetic studies may improve the understanding of the functional consequence of a genetic variant in the clinical setting. The use of intermediate phenotypes instead of broad outcome parameters such as drug response or remission might improve the knowledge on what exactly happens if an individual with a specific genotype takes a certain drug.
Fleeman et al (2010) examined if testing for cytochrome P450 (CYP) polymorphisms in adults entering anti-psychotic treatment for schizophrenia leads to improvement in outcomes, is useful in medical, personal or public health decision-making, and is a cost-effective use of health-care resources. The following electronic databases were searched for relevant published literature: Cochrane Controlled Trials Register, Cochrane Database of Systematic Reviews, Database of Abstracts of Reviews of Effectiveness, EMBASE, Health Technology Assessment database, ISI Web of Knowledge, MEDLINE, PsycINFO, NHS Economic Evaluation Database, Health Economic Evaluation Database, Cost-effectiveness Analysis (CEA) Registry and the Centre for Health Economics website. In addition, publicly available information on various genotyping tests was sought from the internet and advisory panel members. A systematic review of analytical validity, clinical validity and clinical utility of CYP testing was undertaken. Data were extracted into structured tables and narratively discussed, and meta-analysis was undertaken when possible. A review of economic evaluations of CYP testing in psychiatry and a review of economic models related to schizophrenia were also carried out. For analytical validity, 46 studies of a range of different genotyping tests for 11 different CYP polymorphisms (most commonly CYP2D6) were included. Sensitivity and specificity were high (99 to 100 %). For clinical validity, 51 studies were found. In patients tested for CYP2D6, an association between genotype and tardive dyskinesia (including Abnormal Involuntary Movement Scale scores) was found. The only other significant finding linked the CYP2D6 genotype to parkinsonism. One small unpublished study met the inclusion criteria for clinical utility. One economic evaluation assessing the costs and benefits of CYP testing for prescribing anti-depressants and 28 economic models of schizophrenia were identified; none was suitable for developing a model to examine the cost-effectiveness of CYP testing. The authors concluded that tests for determining genotypes appear to be accurate although not all aspects of analytical validity were reported. Given the absence of convincing evidence from clinical validity studies, the lack of clinical utility and economic studies, and the unsuitability of published schizophrenia models, no model was developed; instead key features and data requirements for economic modeling were presented. Recommendations for future research cover both aspects of research quality and data that will be required to inform the development of future economic models.
Lohoff and Ferraro (2010) noted that despite continued efforts to optimize pharmacological treatment for individuals with psychiatric disorders, efficacy and tolerability of medication remains highly variable. In addition to clinical heterogeneity, diagnostic uncertainty, environmental, social factors, and genetic factors have been identified as playing an important role in the inter-individual differences in therapeutic and toxic drug effects. This article described recent developments in the field of psychiatric pharmacogenetics and focused on anti-depressant, anti-psychotic, anti-convulsant, and mood-stabilizing drugs. Recent findings from pharmacogenetic clinical trials were reviewed and clinical implications were discussed. The authors concluded that although current data are too sparse to allow the development of guidelines for using pharmacogenetic testing in routine clinical practice, the field of psychiatric pharmacogenetics is rapidly developing and identification of genetic biomarkers that predict therapeutic response and risk of side effects will ultimately help the practitioner to choose effective and safe treatment for patients suffering from psychiatric disorders.
Plesnicar (2010) stated that anti-psychotics are the lodestar in the treatment of schizophrenia despite the variability of the therapeutic response and drug-induced adverse effects (especially extrapyramidal symptoms, gain weight, and metabolic disturbances). More and more data are supporting the notion that genetic factors -- as well as often over-looked personal and environmental factors -- that define the inter-individual differences in pharmacokinetic and pharmacodynamic treatment response. At present, there are no practical pharmakogenetic tests that could be used in everyday clinical practice; however, in the field of psychiatry they are expected within a few years. Pharmacogenetic tests will indubitably become an important tool for personalized prescription.
Gelenberg (2010) noted that variety of American and European guidelines are available for clinicians treating major depressive disorder and depressive subtypes. Major Western guidelines published since 2000 made similar recommendations for all stages of treatment for depression, including a reliance on measurement-based care. First-line treatment is usually a SSRI, psychotherapy, or a combination of pharmacotherapy and psychotherapy. Next-step treatment recommendations are switching or augmentation, depending on patient response to the initial treatment. Maintenance therapy continues the approach that led to remission. The American Psychiatric Association will release a new treatment guideline to offer information on developments made since the last guidelines were published in 2000. Despite progress made during the last decade, no major breakthroughs in the treatment of depression have occurred, and genetic testing developments allowing for personalized care remain the goal of research.
Lewis et al (2011) tested the hypothesis that patients homozygous for the long (insertion) polymorphism of the serotonin transporter (5-HTTLPR) have an increased response to SSRI antidepressants but not to noradrenaline reuptake inhibitors (NARIs) antidepressants. In an individually randomized, parallel-group controlled trial, people meeting criteria for a depressive episode who were referred by their general practitioner were randomized to receive either citalopram (an SSRI) or reboxetine (an NARI). Randomization was by means of a remote automated system accessed by telephone. The main outcome was depressive symptoms, measured by Beck Depression Inventory (BDI) total score 6 weeks after randomization. A total of 298 participants were randomized to receive citalopram and 303 were randomized to reboxetine. At 6 weeks follow-up, complete data were available for 258 participants taking citalopram and 262 taking reboxetine. These invesigators found no evidence to support an influence of 5-HTTLPR on outcome following antidepressant treatment. The interaction term for BDI score at 6 weeks was 0.50 (95 % CI: -2.04 to 3.03, p = 0.70), which indicated that responses to the SSRI and NARI were similar irrespective of 5-HTTLPR genotype. The authors concluded that it is unlikely that the 5-HTTLPR polymorphism alone will be clinically useful in predicting response to antidepressants in people with depression.
In a review and meta-analysis, Biernacka et al (2012) evaluated the evidence for association between the serotonin transporter gene promoter polymorphism (5HTTLPR) and antidepressant induced mania (AIM). Medline up to November 2009 was searched for key words bipolar, antidepressant, serotonin transporter, SLC6A4, switch, and mania. A total of 5 studies have evaluated the SLC6A4 promoter polymorphism and AIM in adults (total n = 340 AIM+ cases, n = 543 AIM- controls). Although a random effects meta-analysis showed weak evidence of association of the S allele with AIM+ status, a test of heterogeneity indicated significant differences in estimated genetic effects between studies. A similar weak association was observed in a meta-analysis based on a subset of 3 studies that excluded patients on mood stabilizers; however the result was again not statistically significant. The authors concluded that there is insufficient published data to confirm an association between 5HTTLPR and antidepressant induced mania. Pharmacogenomic studies of antidepressant induced mania have high potential clinical impact provided future studies are of adequate sample size and include rigorously assessed patient characteristics (e.g., ancestry, rapid cycling, concurrent mood stabilization, and length of antidepressant exposure).
Vetti et al (2010) systematically categorized the experience from routine CYP2D6 genotyping in a diagnostic laboratory. All samples submitted to the authors' laboratory for CYP2D6 genotyping in the period of June 29, 1998 to December 28, 2009 were examined retrospectively. The samples were classified into 3 indication groups based on clinical information given in the request form. All samples, and a control group consisting of 100 healthy blood donors, were tested for the 4 most prevalent non-functional CYP2D6 alleles in the European population, and for ultra-rapid metabolizer-associated duplications of the gene. A total of 325 samples were included. The proportion of ultra-rapid metabolizers was significantly higher in the patient group (4.0 %, p = 0.045) than in the control group (0 %), with the highest proportion among those patients that used a known CYP2D6 substrate. The percentage of poor metabolizers was not significantly higher in the patient group (8.3 %) than in the control group (6.0 %) (p = 0.528). The authors concluded that the CYP2D6 analysis could rarely explain the patients' side effects or lack of drug response, even though the study group was selected because of clinical problems due to drugs they were using. Two explanations may be that the indication(s) for genetic testing is not clearly defined and that the CYP2D6 genotype is only one of many factors that determine individual drug response.
Jurgens et al (2012) examined the clinical impact of CYP2D6 genotype in patients with a diagnosis within the schizophrenic spectrum using medication pattern as proxy for therapeutic and side effect. The study was conducted in patients genotyped during an inpatient stay (n = 576). Continuous antipsychotic, adjuvant, and anticholinergic drug regimens were registered retrospectively in a cross-sectional manner before genotyping. Antipsychotics were divided into CYP2D6 dependent and independent, and dose equivalents were calculated as chlorpromazine equivalents (CPZEq). Poor metabolizers and ultra-rapid metabolizers were treated with significantly higher median CPZEq doses (625.8; inter-quartile range [IQR] 460.4 to 926.7; and 550; IQR, 199.8 to 1,049) than extensive metabolizers (EMs) and intermediate metabolizers (IMs) (384; IQR, 150 to 698; and 446; IQR, 150 to 800) (p = 0.018). Logistic regression showed no association between anticholinergic treatment and CYP2D6 genotype or concomitant treatment with CYP2D6 inhibitors (p = 0.79 and p = 0.46, respectively). The authors concluded that these findings indicated that CYP2D6 genotype has no sufficient clinical impact that poor metabolizers and ultra-rapid metabolizers are easily clinically identified with.
Platelet Reactivity/Function Testing (VerifyNow P2Y12 Assay)
Clinical evidence has been controversial regarding the influence of clopidogrel on treatment platelet reactivity and ischemic outcomes. Brar et al (2011) systematically evaluated the significance of platelet reactivity on clopidogrel treatment on adverse cardiovascular events using a collaborative meta-analysis using patient-level data for the VerifyNow P2Y12 assay (Accumetrics, San Diego, CA). MEDLINE, Scopus, and the Cochrane library databases were searched through January 2010. A database containing individual patient-level time-to-event data was generated from identified studies. The primary outcome of interest was a composite of death, myocardial infarction (MI), or stent thrombosis (STh). Secondary outcomes included the incidence of: (i) death; (ii) MI; and (iii) STh. A total of 6 studies with 3,059 patients were included. In each study, clopidogrel responsiveness was assessed using the same point-of-care assay after percutaneous coronary intervention (PCI). The primary endpoint occurred more frequently in higher quartiles of P2Y(12) reaction unit (PRU) values: quartile I, 5.8 %; quartile II, 6.9 %; quartile III, 10.9 %; quartile IV, 15.8 % (p < 0.001). Taking quartile I as referent, the hazard ratios (HRs) for the primary endpoint were as follows: quartile II, HR: 1.13 (95 % CI: 0.72 to 1.78; p = 0.60); quartile III, HR: 1.82 (95 % CI: 1.20 to 2.75; p = 0.005); quartile IV, HR: 2.62 (95 % CI: 1.78 to 3.87; p < 0.001). On a continuous scale, every 10-U increase in PRU was associated with a significantly higher rate of the primary endpoint (HR: 1.04; 95 % CI: 1.03 to 1.06; p < 0.0001). According to receiver-operating characteristic (ROC) curve analysis, a PRU value of 230 appeared to best predict death, MI, or STh (p < 0.001). A PRU value greater than or equal to 230 was associated with a higher rate of the composite primary endpoint (HR: 2.10; 95 % CI: 1.62 to 2.73; p < 0.0001), as well as the individual endpoints of death (HR: 1.66; 95 % CI: 1.04 to 2.68; p = 0.04), MI (HR: 2.04; 95 % CI: 1.51 to 2.76; p < 0.001), and STh (HR: 3.11; 95 % CI: 1.50 to 6.46; p = 0.002). The authors concluded that in this collaborative meta-analysis, the level of on-treatment platelet reactivity according to the P2Y(12) assay is associated with long-term cardiovascular events after PCI, including death, MI, and STh.
Gurbel and Tantry (2011) noted that platelet-mediated thrombosis is a dreaded clinical event and is the primary cause of acute coronary syndromes (ACS) and post-PCI ischemic events. There has been a long-standing interest in the ex-vivo quantification of platelet reactivity to assess the risk of thrombosis. Early studies demonstrated platelet activation and heightened platelet reactivity in ACS and after PCI. However, a demonstration that heightened reactivity actually precipitated the ischemic event was lacking. Knowledge of platelet receptor physiology and the advent of novel inhibitors have significantly advanced the field. The P2Y12 receptor has been shown to play a pivotal role in the amplification of platelet activation by multiple agonists and its inhibition has resulted in improved clinical outcomes. The most widely used drug to block P2Y12 receptor, clopidogrel, is associated with resistance in selected patients and these patients have been shown to be at increased risk for post-PCI ischemic event occurrence in multiple studies. Importantly, a threshold of high platelet reactivity has been demonstrated, and beyond this threshold ischemic events occur precipitously. Based on the current evidence, it is rational to quantify the intensity of the ADP-P2Y12 interaction in the patient at the greatest risk for thrombosis -- the PCI patient. However, there is only evidence from small clinical trials demonstrating the clinical efficacy of changing an anti-platelet regimen based on an ex-vivo platelet function measurement. Moreover, there are numerous patients with vulnerable coronary anatomy that have not yet experienced plaque rupture; the prognostic role of a measurement of platelet reactivity in the latter group has never been studied. The authors stated that large-scale trials are ongoing that will investigate the role of personalized anti-platelet therapy in the PCI patient.
Holmes et al (2011) appraised evidence on the association of CYP2C19 genotype and clopidogrel response through systematic review and meta-analysis. PubMed and EMBASE from their inception to October 2011 were searched. Studies that reported clopidogrel metabolism, platelet reactivity or clinically relevant outcomes (cardiovascular disease [CVD] events and bleeding), and information on CYP2C19 genotype were included. These researchers extracted information on study design, genotyping, and disease outcomes and investigated sources of bias. They retrieved 32 studies of 42,016 patients reporting 3,545 CVD events, 579 STh, and 1,413 bleeding events. Six studies were randomized trials ("effect-modification" design) and the remaining 26 reported individuals exposed to clopidogrel ("treatment-only" design). In treatment-only analysis, individuals with 1 or more CYP2C19 alleles associated with lower enzyme activity had lower levels of active clopidogrel metabolites, less platelet inhibition, lower risk of bleeding (relative risk [RR], 0.84; 95 % CI: 0.75 to 0.94; absolute risk reduction of 5 to 8 events per 1,000 individuals), and higher risk of CVD events (RR, 1.18; 95 % CI: 1.09 to 1.28; absolute risk increase of 8 to 12 events per 1,000 individuals). However, there was evidence of small-study bias (Harbord test p = 0.001). When analyses were restricted to studies with 200 or more events, the point estimate was attenuated (RR, 0.97; 95 % CI: 0.86 to 1.09). In effect-modification studies, CYP2C19 genotype was not associated with modification of the effect of clopidogrel on CVD end points or bleeding (p > 0.05 for interaction for both). Other limitations included selective outcome reporting and potential for genotype mis-classification due to problems with the * allele nomenclature for cytochrome enzymes. The authors concluded that although there was an association between the CYP2C19 genotype and clopidogrel responsiveness, overall there was no significant association of genotype with cardiovascular events.
Varenhorst et al (2011) noted that insufficient platelet inhibition is a major determinant of STh, although the etiology is multi-factorial. These investigators examined on-clopidogrel platelet reactivity in patients with previous angiographically confirmed STh, MI, and controls. Using the Swedish Coronary Angiography and Angioplasty Registry, these researchers identified patients with angiographically confirmed STh (n = 48) or MI (n = 30) while on dual anti-platelet therapy within 6 months of PCI and matched control patients (n = 78). On-clopidogrel platelet reactivity was measured with VerifyNow P2Y12 and vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay. The mean PRU was higher (246.8 +/- 75.9 versus 200.0 +/- 82.7, p = 0.001) in STh patients compared with controls. The optimal cut-off for STh was 222 PRU or higher (area under the curve 0.69, p < 0.0001) in a ROC analysis. The cut-off level resulted in 70.2 % sensitivity and 67.3 % specificity. There was no significant difference in mean PRU but a higher device-reported percent inhibition (45.1 +/- 23.8 versus 32.1 +/- 23.2, p = 0.04) in patients with MI compared with controls. Results with the VASP phosphorylation assay were not related to the occurrence of STh or MI. The authors concluded that STh was associated with high on-clopidogrel platelet reactivity measured with VerifyNow (cut-off level of PRU greatr than or equal to 222), but spontaneous MI in stented patients on clopidogrel treatment was not. There was, however, a substantial overlap in on-clopidogrel platelet reactivity between patients with and without on-treatment STh questioning the clinical use of platelet function testing to identify patients at high-risk for STh.
In a randomized, double-blind, active-control trial (Gauging Responsiveness with A VerifyNow assay-Impact on Thrombosis And Safety [GRAVITAS]), Price et al (2011) evaluated the effect of high-dose compared with standard-dose clopidogrel in patients with high on-treatment platelet reactivity after PCI. A total of of 2,214 patients with high on-treatment reactivity 12 to 24 hours after PCI with drug-eluting stents at 83 centers in North America between July 2008 and April 2010 were included in this study. Patients received high-dose clopidogrel (600-mg initial dose, 150 mg daily thereafter) or standard-dose clopidogrel (no additional loading dose, 75 mg daily) for 6 months. The primary end point was the 6-month incidence of death from cardiovascular causes, non-fatal MI, or STh. The key safety end point was severe or moderate bleeding according to the Global Utilization of Streptokinase and t-PA for Occluded Coronary Arteries (GUSTO) definition. A key pharmacodynamic end point was the rate of persistently high on-treatment reactivity at 30 days. At 6 months, the primary end point had occurred in 25 of 1,109 patients (2.3 %) receiving high-dose clopidogrel compared with 25 of 1,105 patients (2.3 %) receiving standard-dose clopidogrel (hazard ratio [HR], 1.01; 95 % CI: 0.58 to 1.76; p = 0.97). Severe or moderate bleeding was not increased with the high-dose regimen (15 [1.4 %] versus 25 [2.3 %], HR, 0.59; 95 % CI: 0.31 to 1.11; p = 0.10). Compared with standard-dose clopidogrel, high-dose clopidogrel provided a 22 % (95 % CI: 18 % to 26 %) absolute reduction in the rate of high on-treatment reactivity at 30 days (62 %; 95 % CI: 59 % to 65 % versus 40 %; 95 % CI: 37 % to 43 %; p < 0.001). The authors concluded that among patients with high on-treatment reactivity after PCI with drug-eluting stents, the use of high-dose clopidogrel compared with standard-dose clopidogrel did not reduce the incidence of death from cardiovascular causes, non-fatal MI, or STh.
Sharma et al (2012) noted that the substantial reduction in ischemic events provided by the dual anti-platelet regimen with aspirin and clopidogrel is well-documented in patients with ACS and patients undergoing PCI. Recently the variable response to the anti-platelet agents has received considerable attention after several "boxed warnings" on clopidogrel. This led to intense controversy on pharmacokinetic, pharmacodynamic, and pharmacogenomic issues of anti-platelet drugs, especially clopidogrel. Research use of platelet function testing has been successfully validated in identifying new anti-platelet drugs like prasugrel and ticagrelor. These platelet function assays are no longer regarded just as a laboratory phenomenon but rather as tools that have been shown to predict mortality in several clinical trials. It is believed that suboptimal response to an anti-platelet regimen (pharmacodynamic effect) may be associated with cardiovascular, cerebrovascular, and peripheral arterial events. There has been intense controversy about this variable response of anti-platelet drugs and the role of platelet function testing to guide anti-platelet therapy. While the importance of routine platelet function testing may be uncertain, it may be useful in high-risk patients such as those with diabetes mellitus, diffuse 3-vessel coronary artery disease, left main stenosis, diffuse atherosclerotic disease, and those with chronic renal failure undergoing PCI. It could also be useful in patients with suspected pharmacodynamic interaction with other drugs to assure the adequacy of platelet inhibition. The authors stated that "While we wait for definitive trials, a predictive prognostic algorithm is necessary to individualize antiplatelet therapy with P2Y12 inhibitors based on platelet function assays and genetic testing".
Yu et al (2012) confirmed the predictive cut-off values for PRU and aspirin reaction units (ARU) and evaluated the clinical impact of VerifyNow® assays. From November 2007 to October 2009, a total of 186 eligible patients were prospectively recruited. Post-treatment platelet reactivity was measured by VerifyNow® assays within 12 to 24 hours after intervention, followed by standard dual maintenance dose therapy for 1 year. All patients had scheduled clinical follow-ups at 1, 3, 6, and 12 months. The rate of low- responders to clopidogrel, aspirin, and both drugs were 41.4 %, 10.2 %, and 3.8 %, respectively. The predictive factors for low-responsiveness to clopidogrel (PRU greater than or equal to 240) were female sex, age, and non-use of cilostazol medication in uni-variate analysis and age greater than or equal to 65 years and non-use cilostazol in the multi-variate analysis. The predictors of low-responsiveness to aspirin (ARU greater than or equal to 550) were male sex and age in both uni-variate and multi-variate analyses. There was no significant difference in the clinical event rate with a cut-off value of PRU greater than or equal to 240 or ARU greater than or equal to 550 for 30 days and 1-year (p > 0.05). The authors concluded that hypo-responsiveness to anti-platelet agents (namely aspirin and clopidogrel) was identified in about 50 % of the patients. The cut-off point of PRU greater than or equal to 240 or ARU greater than or equal to 550 did not confer predictive value for 30-day or 1-year clinical event rates in patients who had undergone PCI with drug-eluting stents.
Kim et al (2012) stated that although adjunctive cilostazol to dual anti-platelet therapy can reduce the risks of clinical events after PCI, whether genetic polymorphism can influence the pharmacodynamics of this regimen has not been evaluated. In this study, a total of 127 patients treated with PCI and taking triple anti-platelet therapy (greater than or equal to 1 month) were enrolled. Platelet reactivity was assessed by conventional aggregometry and the VerifyNow P2Y12 assay. High on-treatment platelet reactivity (HPR) was defined as 5 µm ADP-induced maximal platelet reactivity (Agg(max)) greater than 46 %. CYP3A5*3, CYP2C19*2/*3 and ABCB1 3435C > T were genotyped. CYP3A5*3 and ABCB1 3435C > T variants did not affect the anti-platelet effect of triple anti-platelet therapy. For non-carriers, 1 and 2 carriers of the CYP2C19 loss-of-function (LOF) allele, Agg(max) consecutively increased after the addition of 5 µm [mean (95 % CI): 24.6 % (20.8 to 28.5 %) versus 28.7 % (25.4 to 32.0 %) versus 32.3 % (25.8 to 38.7 %), p = 0.062, respectively] and 20 µm ADP [34.2 % (29.3 to 39.0 %) versus 41.7 % (37.8 to 45.6 %) versus 44.9 % (37.9 to 51.9 %), p = 0.007, respectively]. Likewise, late platelet reactivity and PRU proportionally changed according to the number of CYP2C19 LOF alleles. High on-treatment platelet reactivities were observed in 9.2 % of subjects: 6.3 %, 7.4 % and 20.0 % with 0, 1 and 2 carriers of CYP2C19 LOF allele(s) (p = 0.099). In multi-variate analysis, carriage of 2 CYP2C19 LOF alleles was a significant predictor for the prevalence of HPR (odds ratio 5.78, 95 % CI: 1.21 to 27.78, p = 0.028). The authors concluded that among PCI-treated patients, the effect of triple anti-platelet therapy is influenced by the CYP2C19 LOF allele. They stated that its clinical benefit needs to be validated according to the CYP2C19 metabolic phenotype in future clinical trials.
O'Connor et al (2012) noted that clopidogrel used in conjunction with aspirin has a central role in the treatment of patients with an ACS and/or undergoing PCI. The pharmacokinetic and pharmacodynamic responses to this drug are highly variable leaving up to 1/3 of patients with inadequate platelet inhibition or HPR, and subsequent increased ischemic cardiovascular events. Genetic variability in drug absorption and metabolism is a key factor responsible for the inefficient generation of the active drug metabolite. The 2-step hepatic cytochrome P450 (CYP)-dependant oxidative metabolism of the pro-drug appears to be of particular importance. Pharmacogenomic analyses have identified LOF variant alleles of CYP 2C19 and specifically the 2C19*2 allele, to be the predominant genetic mediators of the anti-platelet effect of clopidogrel. Carriers were have been shown to have lower active metabolite levels of clopidogrel, higher platelet reactivity and associated poorer outcomes. Rapid and accurate point-of-care genetic tests to identify these alleles are currently in development but several questions about the role of such testing remain such as patient selection and whether personalized treatment based on genotype has a positive impact on clinical outcome. At present, genetic testing can not be recommended in routine clinical practice due to insufficient prospective data. However, the significant body of research published to date suggests a likely role when used in combination with platelet function analysis in ACS patients undergoing stenting who have other known risk factors for recurrent ischemic events.
Mallouk et al (2012) performed a systematic review to estimate the prevalence of poor biological response to clopidogrel and investigated the factors known to modulate this. An exhaustive search was performed. A total of 171 publications were identified, providing data for 45,664 subjects. The estimated prevalence of poor biological response to clopidogrel ranged from 15.9 % to 49.5 % according to the platelet function assay employed. The assays most frequently used were light transmittance aggregometry (LTA), the vasodilator-stimulated phosphoprotein (VASP) assay and the Verifynow® assay. For all these assays, higher cut-off values were associated with a lower prevalence of poor biological response to clopidogrel. However, when choosing a fixed cut-off point for each assay, the prevalence of poor biological response to clopidogrel was highly variable suggesting that other factors could modulate poor biological response to clopidogrel. Finally, none of the studied factors could apparently explain the variability of poor biological response to clopidogrel. This meta-analysis showed that the prevalence of poor biological response depends on the assay employed, the cut-off value and on various unidentified additional factors.
A draft AHRQ report (2012) found evidence that high on-clopidogrel platelet reactivity is associated with an increased risk of adverse cardiovascular outcomes for at least some of the available assays. The report stated that the strength of evidence regarding these prognostic effects is low because of concerns regarding selective outcome reporting and the relatively small number of studies reporting clinical outcomes. The report concluded that strength of evidence regarding the use of platelet reactivity testing to guide anti-platelet treatment selection is insufficient, because studies reporting on clinical outcomes are few, have diverse designs, and included heterogeneous populations.
In summary, there is currently insufficient evidence to support the use of platelet reactivity/function testing for patients who have undergone after PCI. The incorporation of platelet reactivity/function testing into clinical practice awaits the results of ongoing clinical studies where treatment is changed based on platelet reactivity/function testing data.
Beta adrenergic receptor genotyping (ADRB2) for treatment-resistant asthma
ADRB2 (beta adrenergic receptor genotyping) is a genetic test used for asthma patients with poor symptom control. The target for beta2-agonist asthma medication is the B2-adrenergic receptor. The gene for the B2-adrengergic receptor is ADRB2. It is believed that a variation at one location of this gene may predict therapeutic responses to beta2-agonists. The Arg/Arg homozygous genotype at amino acid position 15 may indicate a need for a change in medication.
The textbook Cleveland Clinic: Current Clinical Medicine (2010) states that, in the presence of a polymorphism, the acute bronchodilator response to a β agonist, or protection from a bronchoconstrictor, may be affected. Studies indicate that in patients with Arg16Arg variant, the resulting β2-adrenergic receptor is resistant to endogenous circulating catecholamines (i.e., receptor density and integrity are preserved), with a subsequent ability to produce an acute bronchodilator response to an agonist). There are conflicting data regarding whether Arg/Arg homozygotes are prone to experience reflex morbidity with inhaled LABA, but the weight of evidence, particularly from more-recent studies, indicates that response to LABA when used in combination with ICS does not vary based on β2-adrenergic genotypes at codon 16.
In 2010, Bleecker and colleagues examined whether the response to salmeterol alone or in combination with an inhaled corticosteroid is influenced by beta- receptor polymorphisms. Subjects using only as-needed albuterol were screened and completed two sequential open-label run-in periods (8 wk on as-needed albuterol; 8 wk on as-needed ipratropium). Five hundred forty-four subjects were randomized by Arg16Gly genotype to salmeterol alone or with fluticasone propionate for 16 weeks. Change from baseline in morning peak expiratory flow was the primary endpoint. Lung function responses were sustained over treatment and no statistically significant changes from baseline between genotypes within treatments were observed. Overall mean changes in morning peak flow for salmeterol with fluticasone propionate were 32.6 L/min (Arg/Arg vs. Gly/Gly, 95% confidence interval [CI], -6.3, 22.1), 25.9 L/min (Arg/Arg vs. Arg/Gly, 95% CI, -7.1, 21.3), and 24.9 L/min (Arg/Gly vs. Gly/Gly, 95% CI, -13.0, 14.6), and for salmeterol alone were 19.4 L/min (Arg/Arg vs. Gly/Gly, 95% CI, -1.7, 21.4), 24.6 L/min (Arg/Arg vs. Arg/Gly, 95% CI, -13.0, 10.6), and 12.4 L/min (Arg/Gly vs. Gly/Gly, 95% CI, -0.2, 22.3) for Arg/Arg, Arg/Gly, and Gly/Gly genotypes, respectively. Other measures of asthma control showed similar responses. The results showed no evidence of a pharmacogenetic effect of beta-receptor variation on salmeterol response.
Basu and colleagues (2009) investigated whether the presence of Arg16 allele of the adrenergic beta(2)-receptor agonist gene (ADRB2) predisposed to exacerbations in young asthmatic patients taking regular salmeterol. Arg/Gly status at position 16 of ADRB2 was assessed in 1182 asthmatic patients. Asthma exacerbations, use of beta-agonists and other medications over the previous 6 months, and lung function were also studied. An increased risk of exacerbations per copy of he Arg16 allele was observed in asthmatic patients, regardless of treatment regimen (odds ratio [OR], 1.30; 95% CI, 1.09-1.55; P = .003). This appeared to be largely due to exposure to beta(2)-agonists because the risk of exacerbations observed in patients with the Arg16 allele was only observed in those receiving daily inhaled long- or short-acting beta(2)-agonist treatment (OR, 1.64; 95% CI, 1.22-2.20; P = .001). In contrast, there was no genotypic risk for exacerbations in patients using inhaled beta(2)-agonists less than once a day (OR, 1.08; 95% CI, 0.85-1.36; P = .525). The Arg16 genotype-associated risk for exacerbations was significantly different in those exposed to beta(2)-agonists daily versus those that were not (test for interaction, P = .022). The authors concluded that Arg16 genotype of ADRB2 was associated with exacerbations in asthmatic children and young adults exposed daily to beta(2)-agonists, regardless of whether the exposure is to albuterol or long-acting agonists, such as salmeterol.
In a 2008 article, Martin and associates evaluated the influence of single nucleotide polymorphisms in the beta(2)-adrenoceptor gene, on the response to inhaled beta(2)-agonists in children with acute asthma. One hundred and forty-eight children with acute asthma were recruited and genotyped for beta(2)Arg16Gly and beta(2)Gln27Glu. For Gln27Glu, individuals Gln27Gln took longest to stretch out to 1, 2 and 4 hourly beta(2)-agonists, followed by heterozygotes who were intermediate and Glu27Glu who responded most rapidly (1 hourly: 2.6 hr vs. 2.0 vs. 1.4, p = 0.02; 2 hourly: 10.6 hr vs. 10.7 vs. 6.8, p = 0.07; 4 hourly: 29.8 hr vs. 28.5 vs. 24.3, p = 0.30). The authors reported that the ability to prospectively identify children who respond less effectively to beta (2)-agonists during an acute asthma attack has the potential to allow the generation of genotype-specific treatment pathways.
In 2008, Giubergia et al assessed the frequency of beta2-adrenergic receptor (beta2-AR) polymorphisms in asthmatic children from Argentina, and evaluated their influence on bronchodilator desensitization to albuterol over a 4-week treatment. beta2-AR genotypes were determined in 117 children with asthma and 101 of them were under 4 weeks treatment with albuterol. Spirometric changes in FEV(1) were recorded at the beginning (day 1) and at the end of the study (day 30) and compared to genotypes at position 16 and 27 of the receptor. The frequency of the polymorphisms was calculated in all population. The presence of glutamine at position 27 (Gln27) was significantly more frequent in this Argentinean study population than in other Caucasian populations. The homozygosity for Gln27 polymorphism was associated to a desensitization of the receptor with a decline in the bronchodilator response to albuterol after chronic use.
In 2007, Bleeker et al investigated whether beta2-adrenergic receptor (ADRB2) polymorphisms affect response to longacting beta2-agonists in combination with inhaled corticosteroids. Asthmatics were stratified by ADRB2 genotype in two studies to assess the effects of inhaled corticosteroids plus longacting beta2-agonists on asthma exacerbations. In study 1 (double-blind), 2250 asthmatics were randomly assigned to budesonide plus formoterol maintenance and reliever therapy, fixed-dose budesonide plus formoterol, or fixed-dose fluticasone plus salmeterol for 6 months. Study 2 (open-label) consisted of 405 asthmatics and compared an adjustable regimen of budesonide plus formoterol with fixed-dose budesonide plus formoterol and fixed-dose fluticasone plus salmeterol for 7 months. The relation between ADRB2 polymorphism, severe asthma exacerbations, and other asthma outcomes was analysed. Primary endpoints for studies 1 and 2 were severe asthma exacerbation and asthma control as assessed by measures of exacerbations, respectively. In study 1, Gly16Arg genotype had no effect on the percentage of participants with severe exacerbations across all treatment groups (99 [12%] of 833 Gly/Gly, 110 [11%] of 1028 Gly/Arg, and 32 [9%] of 361 Arg/Arg participants). Secondary endpoints, including forced expiratory volume in 1 s, peak expiratory flow, use of as-needed medication, and number of nights with awakenings were similar between genotype groups. No relation was recorded between ADRB2 haplotype and primary and secondary endpoints. In study 2, the frequency of asthma exacerbations (15 [9%] of 168 Gly/Gly, 13 [8%] of 169 Gly/Arg, and 6 [9%] of 67 Arg/Arg participants) and other study endpoints were closely similar for all ADRB2 genotypes.
Hawkins and colleagues (2006) sought to identify ADRbeta2 polymorphisms and haplotype structure in white and African American subjects and to test for genotype and haplotype association with asthma phenotypes. A 5.3-kb region of ADRbeta2 was resequenced in 669 individuals from 429 whites and 240 African Americans. A total of 12 polymorphisms, representing an optimal haplotype tagging set, were genotyped in whites (338 patients and 326 control subjects) and African Americans (222 patients and 299 control subjects). A total of 49 polymorphisms were identified, 21 of which are novel; 31 polymorphisms (frequency > 0.03) were used to identify 24 haplotypes (frequency > 0.01) and assess linkage disequilibrium. Association with ratio (FEV1/FVC)2 for single-nucleotide polymorphism +79 (p < 0.05) was observed in African Americans. Significant haplotype association for (FEV1/FVC)2 was also observed in African Americans. The authors concluded, “these data suggest that the length of a poly-C repeat (+1269) in the 3' untranslated region of ADRbeta2 may influence lung function, and may be important in delineating variation in beta-agonist responses, especially in African Americans.”
Litonjua (2006) writes the gene that encodes the beta2-adrenergic receptor (ADRB2) is one of the most studied candidate genes in asthma. Candidate gene association studies of ADRB2 and asthma have been dominated by analyses of the two common non-synonymous coding single nucleotide polymorphisms, Arg16Gly and Glu27Gly. Published studies have yielded inconsistent results. Three recent meta-analyses on the effects of these two polymorphisms have found no associations with asthma, although there were suggestions of associations with other asthma-related phenotypes, such as nocturnal asthma and asthma severity. Other recent studies have investigated other single nucleotide polymorphisms in this gene (i.e. single nucleotide polymorphisms in the promoter region and other single nucleotide polymorphisms in the coding region). These analyses have investigated the association between these single nucleotide polymorphisms (and haplotypes of these polymorphisms) and asthma-related phenotypes such as lung function, airways hyperresponsiveness, and response to a bronchodilator, and have suggested that certain regions of the gene may be associated with different phenotypes. Results from these studies, however, have also been inconsistent. Polymorphisms of ADRB2 are not major risk factors for the development of asthma. These polymorphisms are likely to be important, however, in determining drug response. Future studies need to fully characterize all of the variation in the gene and perform comprehensive association studies. Finally, interactions between ADRB2 and other genes in the beta-agonist pathway are an important and active area of research that will shed more light on inter-individual differences in drug response.
In 2005, Taylor et al measured bronchodilator response in patients with asthma stratified by ADRB2 haplotype after eliminating the confounding effect of prior drug treatment with inhaled beta2-agonists and corticosteroids. ADRB2 haplotype was determined in 176 patients with asthma, of whom 161 harbored the six most common combinations. There were no significant differences in bronchodilator response (% improvement in FEV(1)) with respect to any of the major ADRB2 haplotypes or genotypes. The authors concluded, “genetic variation of the ADRB2 does not influence the immediate response to inhaled beta2-agonist. The confounding effect of tolerance resulting from regular beta2-agonist use must be controlled when assessing the pharmacogenetic influences on clinical outcomes with beta2-agonists.”
According to Johnson (2006), the presence of a particular form of the B2-receptor, which might influence clinical efficacy of regular B2-agonists, is of increasing interest in predicting good and poor responders to therapy.
MGMT Gene Methylation Assay for Gliomas
Glioblastomas are among the most aggressive of all known human tumors. The median survival times remain in the 12 to15-month range despite aggressive surgery, radiation, and chemotherapy. Through molecular and genetic profiling efforts, underlying mechanisms of resistance to these therapies are becoming better understood. Resistance to alkylating agents via direct DNA repair by O(6)-methylguanine methyltransferase (MGMT) has been investigated as a barrier to successful treatment. Assessment of MGMT status could help identify gliomas patients more likely to respond to chemotherapy or to benefit from MGMT depletion strategies. Strategies to overcome MGMT-mediated chemoresistance are being actively investigated.
Current guidelines from the National Comprehensive Cancer Network (NCCN, 2012) include recommendations for the use of the MGMT gene methylation assay in assessing likelihood of response to temozolomide in glioblastoma. The guidelines recommend use of temozolomide if methylguanine methyl-transferase [MGMT] promoter methylation is positive in persons with glioblastoma.
In a review, Hegi, et al. (2008) stated that one strategy to overcome MGMT-mediated chemoresistance includes treatment with nontoxic pseudo-substrate inhibitors of MGMT, such as O(6)-benzylguanine, or RNA interference-mediated gene silencing of MGMT. However, the author reported that systemic application of MGMT inhibitors is limited by an increase in hematologic toxicity. Another strategy, the author explained, is to deplete MGMT activity in tumor tissue using a dose-dense temozolomide schedule. The author stated that these alternative schedules are well tolerated; however, it remains unclear whether they are more effective than the standard dosing regimen or whether they effectively deplete MGMT activity in tumor tissue. The author noted that not all patients with glioblastoma having MGMT promoter methylation respond to alkylating agents, and even those who respond will inevitably experience relapse.
Data from a retrospective study reported by Cancer Care Ontario (2006) suggested that newly diagnosed malignant glioma patients who had undergone surgery and external beam radiotherapy had a greater benefit from temozolomide treatment if their tumors had MGMT promoter methylation versus patients with unmethylated MGMT tumors. However, it should be noted that these studies were performed retrospectively and prospective validation is required before MGMT methylation can be used for clinical stratification purposes.
Idbaih, et al. (2007) reviewed recent studies on molecular markers including MGMT in the treatment of glioma and found evidence that MGMT inactivation is a prognostic marker and predictor of chemosensitivity in gliomas. The author stated, "Although such markers remain to be formally validated by ongoing and planned prospective trials, it is likely that they will soon become essential for optimizing treatment decisions."
Mutation Testing in the BRAF Gene (V600E or V600K):
On May 29, 2013, the FDA approved 2 new drugs -- dabrafenib (Tafinlar) and trametinib (Mekinist), for patients with metastatic or unresectable melanoma. Tafinlar, a BRAF inhibitor, is approved to treat patients with melanoma whose tumors express the BRAF V600E gene mutation. Mekinist, a MEK inhibitor, is approved to treat patients whose tumors express the BRAF V600E or V600K gene mutations. Approximately 50 % of melanomas arising in the skin have a BRAF gene mutation. Tafinlar and Mekinist are being approved as single agents, not as a combination treatment. Furthermore, the FDA approved Tafinlar and Mekinist with a genetic test called the THxID BRAF test, a companion diagnostic that will help determine if a patient’s melanoma cells have the V600E or V600K mutation in the BRAF gene.
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