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Many antidepressants, atomoxetine, and several antipsychotics are metabolized by the cytochrome P450 enzymes CYP2D6 and CYP2C19, and guidelines for prescribers based on genetic variants exist. Although some laboratories offer such testing, there is no consensus regarding validated methodology for clinical genotyping of CYP2D6 and CYP2C19. The aim of this paper was to cross-validate multiple technologies for genotyping CYP2D6 and CYP2C19 against each other, and to contribute to feasibility for clinical implementation by providing an enhanced range of assay options, customizable automated translation of data into haplotypes, and a workflow algorithm. AmpliChip CYP450 and some TaqMan single nucleotide variant (SNV) and copy number variant (CNV) data in the Genome-based therapeutic drugs for depression (GENDEP) study were used to select 95 samples (out of 853) to represent as broad a range of CYP2D6 and CYP2C19 genotypes as possible. These 95 included a larger range of CYP2D6 hybrid configurations than have previously been reported using inter-technology data. Genotyping techniques employed were: further TaqMan CNV and SNV assays, xTAGv3 Luminex CYP2D6 and CYP2C19, PharmacoScan, the Ion AmpliSeq Pharmacogenomics Panel, and, for samples with CYP2D6 hybrid configurations, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing and Luminex. Agena MassARRAY was also used for CYP2C19. This study has led to the development of a broader range of TaqMan SNV assays, haplotype phasing methodology with TaqMan adaptable for other technologies, a multiplex genotyping method for efficient identification of some hybrid haplotypes, a customizable automated translation of SNV and CNV data into haplotypes, and a clinical workflow algorithm.

BackgroundCYP2D6 and CYP2C19 are cytochrome P450 enzymes involved in the metabolism of many medications from multiple therapeutic classes. Associations between patterns of variants (known as haplotypes) in the genes encoding them (CYP2D6andCYP2C19) and enzyme activities are well described. The genes in fact comprise 21% of biomarkers in drug labels. Despite this, genotyping is not common, partly attributable to its challenging nature (CYP2D6having >100 haplotypes, including those with sequence from an adjacent pseudogene, and gene duplications). We cross-validated different methodologies for identifying haplotypes in these genes against each other.MethodsNinety-two samples with a variety ofCYP2D6andCYP2C19genotypes according to prior AmpliChip CYP450 and TaqManCYP2C19*17data were selected from the Genome-based therapeutic drugs for depression (GENDEP) study. Genotyping was performed with TaqMan copy number variant (CNV) and single nucleotide variant (SNV) analysis, the next generation sequencing-based Ion S5 AmpliSeq Pharmacogenomics Panel, PharmacoScan, long-range polymerase chain reaction (L-PCR) followed by amplicon analysis, and Agena forCYP2C19. Variant pattern to haplotype translation was automated.ResultsThe inter-platform concordance forCYP2C19was high (up to 100% for available data). ForCYP2D6, the IonS5-PharmacoScan concordance was 94% for a range of variants tested apart from those with at least one extra copy of aCYP2D6gene (occurring at a frequency of 3.8%, 33/853), or those with substantial sequence derived from pseudogene, known as hybrids (3%, 26/853).ConclusionsInter-platform concordance forCYP2C19was high, and, moreover, the Ion S5 and PharmacoScan data were 100% concordant with that from a TaqManCYP2C19*2assay. We have also demonstrated feasibility of using an NGS platform for genotypingCYP2D6andCYP2C19, with automated data interpretation methodology. This points the way to a method of makingCYP2D6andCYP2C19genotyping more readily accessible.

Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second‐Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high‐quality, full‐length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene‐locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD‐powered CYP2D6 gene‐variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy‐number variation.

The highly polymorphic CYP2D6 gene locus leads to a wide range of enzyme activity. Since there are limited data for Thai, the major aim was to investigate CYP2D6 genetic variation in a large Thai population (n = 920). CYP2D6 genotyping was performed using four different platforms. Genotype call rates of the Luminex xTAG® and AmpliChip CYP450 test were 96.5% and 87.4%, respectively. Based on Luminex xTAG® data, the most common alleles and genotypes were *1 0 (49.6%), *1 (24.6%), *2 (10.8%), *5 (6.7%), *41 (6.5%) and *1/*10 (23.9%), *10/*10 (21.5%), *2/*10 (9.4%), *5/*10 (6.9%), *10/*41 (5.7%), respectively. Challenges and limitations of the platforms evaluated are discussed. These data add to our knowledge regarding interethnic variability in CYP2D6 activity and contribute to improving drug therapy in the Thai.

Background CYP450 polymorphisms result in variable rates of drug metabolism. CYP drug-drug interactions can contribute to altered drug effectiveness and safety. Study objectives The primary objective was to determine the percentage of emergency department (ED) patients with cytochrome 2C19 (CYP2C19) drug-drug interactions. The secondary objective was to determine the prevalence of CYP2C19 polymorphisms in a US ED population. Methods We conducted a prospective observational study in an urban academic ED with 72,000 annual visits. Drug ingestion histories for the 48 hours preceding ED visit were obtained; each drug was coded as CYP2C19 substrate, inhibitor, inducer, or not CYP2C19 dependent. Ten percent of patients were randomized to undergo CYP2C19 genotyping using the Roche Amplichip. Results A total of 502 patients were included; 61% were female, 65% were white, and median age was 39 years (interquartile range, 22-53). One hundred thirty-one (26.1%) patients had taken at least 1 CYP2C19-dependent home drug. Eighteen (13.7%) patients who were already taking a CYP2C19-dependent drug were given or prescribed a CYP2C19-dependent drug while in the ED. Among the 53 patients genotyped, 52 (98%) were extensive metabolizers and 1 was a poor metabolizer. Conclusions In a population of ED patients, more than a quarter had taken a CYP2C19-dependent drug in the preceding 48 hours, but few were given or prescribed another CYP2C19-dependent drug in the ED. On genotyping analysis, CYP2C19 polymorphisms were uncommon in our cohort. We conclude that changing prescribing practice due to CYP2C19 drug-drug interaction or genotype is unlikely to be useful in most US ED populations.

Background In 2005, the FDA approved the Roche AmpliChip™ for clinical application. The AmpliChip is a microarray chip that has the capability to play an important role in clinical pharmacogenetics. Objective Because of the possible influence the AmpliChip may have on patient medication management, the purpose of the review is to address the available evidence for the AmpliChip's overall performance at three key levels: analytic validity (genotyping accuracy, and prediction of the phenotype from the genotype) and clinical utility. Data sources We searched Medline, Embase and PubMed for studies of the AmpliChip. Limits were English language and 2005 (the year of FDA approval) and onwards, and we corresponded with authors for further papers of interest. Results 17 articles provided data for analysis in this review: 4 involving genotype accuracy, 7 involving genotype to phenotype prediction and 9 involving clinical utility. Conclusion There is limited literature comparing AmpliChip results to gold standard tests and test-retest reliability when assessing genotype accuracy. Also, there is limited literature on the accuracy of AmpliChip predictions of phenotypes from genotypes and minimal evidence with appropriately powered studies whether the AmpliChip genotype to phenotype predictions result in clinical benefit. At all three levels there is significant evidence that the AmpliChip has the potential to be a robust clinical tool. However, more and adequately powered studies are required to determine fully whether the AmpliChip is a clinically effective tool.

In this study we investigated the role of the polymorphic types of CYP2D6 in the Turkish population for therapeutic efficacy among patients with early stage breast cancer who treated with tamoxifen. CYP2D6 plays an important role in tamoxifen metabolism. CYP2D6 genotypes were determined of 115 patients treated with adjuvant tamoxifen using the AmpliChip CYP450 test. Genotypes were classified by metabolizer groups and evaluated by the diagnostic and clinical characteristics of the patients and the recurrences of the disease. To create an intergroup comparison of the categorical measurements, the Chi-Square test was used. The most common CYP2D6 gene polymorphism was *1/*2 with a percentage of 19.9% (n= 19). In the Turkish population, among patients with early stage breast cancer, the normal metabolizer group was the largest with a percentage of 77.1% (n= 74). The percentage rates were determined to be 11.5% (n= 11) for intermediate metabolizers, 5.2% (n= 5) for ultrarapid metabolizers and 2.1% (n= 2) for poor metabolizers. In patients with early stage breast cancer treated with adjuvant tamoxifen. no significant correlation was detected between CYP2D6 types and recurrence and metastasis rates. This study was the first study to determine CYP2D6 genotypes and characteristics of Turkish patients with early stage breast cancer. Although no correlation was demonstrated between CYP2D6 genotype polymorphism and disease recurrence in the earlier period, long-term follow-ups seem to be warranted.

Genetic polymorphisms in cytochrome P 450 (CYP) enzymes could lead to a phenotype with altered enzyme activity. In pharmacotherapy, genotype-based dose recommendations achieved great importance for several drugs. In our pilot study, we ask if these genetic tests should be applied to forensic problems as a matter of routine. Starting from 2004 through 2008, we screened routine cases for samples where the relation of parent compound to metabolite(s) (P/M ratio), particularly morphine to codeine ratios and diazepam to its metabolites, was noticeable or not consistent with the information provided by the defendants. We found 11 samples with conspicuous results. These were analyzed for polymorphisms of the CYP 2D6 and 2C19 genes using the Roche AmpliChip Cytochrome P450 Genotyping test. If not previously conducted, a general unknown analysis by gas chromatography/mass spectrometry (GC/MS) was additionally carried out. For CYP 2D6, we found two cases with the genotype poor metabolizer (PM), three cases with heterozygote extensive metabolizer genotype classified as an intermediate metabolizer (IM) with probably reduced enzyme activities, but no ultrarapid metabolizer genotype. For CYP 2C19, two cases were characterized as IM phenotypes, with no PM found. Once we achieved no appropriate amounts of DNA, one case was excluded after GC/MS analysis. Only in one case could the polymorphism clearly explain the changes in drug metabolism. More frequently, a drug-drug interaction was thought to have a stronger impact. Additionally, our results suggest that IM genotypes may be more relevant than previously suspected. With respect to the small number of cases in which we thought a genotyping would be helpful, we conclude that the overall relevance of toxicogenetics in forensic problems is moderate. However, in some individual cases, a genotyping may provide new insight.

Genetic factors can result in variance in drug metabolism enzyme function, which is one major mechanism impacting on interindividual variability in response and side effects. We therefore performed a pilot study to investigate genetic variants in the drug metabolizing enzymes CYP2D6 and CYP2C19.We evaluated 35 schizophrenic and 39 obsessive compulsive disorder (OCD) patients treated with various antipsychotics and antidepressants. Patients were assessed for treatment response and side effects. Genotyping for CYP2D6 and CYP2C19 was performed using the AmpliChip(®). Statistical analysis was performed using analysis of variance and Fisher's exact test. Cases of poor metabolizers (PMs) or ultrarapid metabolizers (UMs) were examined in further detail to assess medication outcomes.Statistical analysis identified no overall significant association of CYP2D6 metabolizer status with treatment response or occurrence of side effects. Nonetheless, case reports of PM and UM individuals indicated lack of response and/or occurrence of side effects in most of these patients. A secondary analysis comparing OCD subjects with impaired 2D6 function to extensive metabolizers was significant (p=0.021).Although not conclusive, there was some association between CYP2D6 impaired metabolic status and medication response. Our case reports suggest a potential clinical benefit of CYP genotyping for specific patients. Further validation of CYP2D6 and CYP2C19 testing in prospective, randomized trials is warranted.

Background: CYP2D6 is a major drug-metabolizing enzyme. Polymorphic variation includes copy number variants such as gene deletions, duplications and multiplications of functional and nonfunctional gene units. In this article we describe the first systematic characterization of a CYP2D6*9x2 gene duplication. CYP2D6*9 is an allelic variant conferring reduced enzymatic activity. This novel gene duplication was discovered in two unrelated Spanish psychiatric patients. Both subjects were initially tested with the AmpliChip CYP450 test, which indicated the presence of a duplication and the CYP2D6*9 allele, but did not make a genotype call. The goal of the study was to resolve this issue by characterizing the CYP2D6 gene locus in these patients. Materials & methods: Both individuals and one offspring were regenotyped using our own CYP2D6 genotyping strategy employing long-range PCR and TaqMan-based SNP detection. In addition, gene resequencing and genotyping of duplication-specific long-range PCR products and quantitative gene copy number analysis was applied. Results: The duplication was mapped to the CYP2D6*9 allele and copy number analysis determined a CYP2D6*9x2 gene duplication in all three individuals. Because CYP2D6*9x2 is not recognized by the AmpliChip CYP450 test, this structural arrangement was responsible for the ‘no call’ on the AmpliChip CYP450 test report. Conclusion: The full characterization of this allele will aid in the interpretation of AmpliChip CYP450 test results for clinical and research applications. Original submitted 8 June 2011; Revision submitted 18 July 2011

Polymorphisms of CYP450 metabolizer enzymes and transport proteins play crucial roles in the inter-individual variability of drug efficiency. The aim of our study was to predict the frequency of functional variants of CYP2D6, CYP2C19 and ABCB1 genes in the Hungarian population. One hundred twelve unrelated healthy subjects donated DNA sample in the study. ABCB1 C3435T and G2677T/A single-nucleotide polymorphisms (SNPs) were determined by LightCycler polymerase chain reaction. Because only limited amount of data is available on the rare allelic variants of CYP2D6 in the European populations, our study applied an expanded set of CYP2D6 and CYP2C19 alleles by using AmpliChip test. Our results show that the CYP2D6 phenotypes were 1.9% ultra-rapid metabolizer, 6.5% intermediate metabolizer (IM), 8.3% poor metabolizer (PM) and 83.3% extensive metabolizer (EM), and the CYP2C19 phenotypes were 1.8% PM, 31.2% IM and 67% EM. The prevalence of the commonly observed CYP2D6 and CYP2C19 alleles in our study corresponds with that of other European populations. Nevertheless, our study confirms that extending the CYP2D6 allele set with loss-of-function variants such as CYP2D6*7, *9, *41 is worth considering. Frequency of the wild type ABCB1 3435C was 42.8% whereas the prevelance of 2677 G was 50.4%. Although frequency data of G2677T/A SNP in the European area are limited, some discrepancies with other studies were found. Copyright © 2011 John Wiley & Sons, Ltd.

The role of pharmacogenomics and tamoxifen was investigated by analyzing several polymorphisms of cytochrome P450 and SULT1A1 gene in a nested case control study from the Italian Tamoxifen Prevention Trial. This study included 182 Caucasian subjects, 47 breast cancer (BC) cases and 135 matched controls. We used the AmpliChip CYP450 Test to screen 33 alleles of CYP2D6 and 3 of CYP2C19. One more variant for CYP2C19*17 and two single-nucleotide polymorphisms for the gene SULT1A1 were also performed. By using the AmpliChip CYP450 Test, out of 182 subjects, we identified 8 poor metabolizer (PM), 17 intermediate metabolizer (IM), 151 extensive metabolizer (EM) and 3 ultrarapid metabolizer (UM). PM women allocated to the tamoxifen arm showed a higher risk of developing BC compared to the remaining phenotypes (P=0.035). In an exploratory analysis, among 58 women with a CYP2D6*2A allele, 9 BCs were diagnosed in the placebo arm and only 1 in the tamoxifen arm (P=0.0001). CYP2C19 and SULT1A1 polymorphisms did not show any correlation with tamoxifen efficacy. Tamoxifen showed reduced efficacy in CYP2D6 PMs in the chemoprevention setting. Conversely, the CYP2D6*2A allele may be associated with increased efficacy of tamoxifen. These findings support the relevance of pharmaco-genomics in tailoring tamoxifen treatment.

These findings are from a qualitative study examining clinician experiences of employing the AmpliChip® CYP450 test in psychiatric practice. One hundred tests were made available to secondary care mental health service clinicians commencing patient treatment with risperidone across three District Health Boards within New Zealand. Feedback was sought on clinicians’ ( n = 33) experiences of ordering the test and receiving results, utilization of results, and perceived advantages and disadvantages. Difficulties were reported regarding ordering the test and receiving the results; however, real or perceived advantages of employing the test results in practice were widely reported. Analysis of the ways in which the test results were reportedly utilized revealed that they generally played a supporting role with regard to dosing decisions, rather than being the main influence on clinician behaviour. The test itself, and subsequent results, was viewed as potentially useful in facilitating the development of the doctor–patient relationship. Reported disadvantages of the test included potential over-reliance at the expense of clinical wisdom, cost, and challenges inherent in introducing a new clinical procedure into routine practice. These findings indicate that psychiatric clinicians are receptive to employing this test as a clinical support tool if its implementation is carefully considered and economically justifiable.

Introduction:An important technological advance in genetic testing is the DNA microarray, which allows for the simultaneous testing of thousands of DNA sequences. The AmpliChip CYP450 Test employs this microarray technology for cytochrome P450 (CYP) 2D6 and CYP2C19 genotyping. Isoenzymes encoded by these genes are responsible for the metabolism of many widely prescribed drugs. The objectives of this study were to identify CYP2D6 and CYP2C19 alleles and phenotypes in a psychiatric patient population in Kentucky, and to describe practical issues associated with DNA microarray technology.Methods:A total of 4,532 psychiatric patients were recruited from three state hospitals in Kentucky. Whole blood, buccal swabs, or saliva samples were genotyped with the AmpliChip CYP450 Test to derive a predicted phenotype.Results:In this cohort, the overall prevalence of CYP2D6 poor metabolizers was 7.6% (95% CI 7%, 8.3%), 8.2% in the Caucasians (95% CI 7.4%, 9.1%) and 1.8% in the African Americans (95% CI 0.9%, 3.5%). The overall prevalence of CYP2D6 ultrarapid metabolizers was 1.5% (95% CI 1.2%, 1.9%), 1.5% in the Caucasians (95% CI 1.1%, 1.9%) and 2.0% in the African Americans (95% CI 1.1%, 3.7%). The overall prevalence of CYP2C19 poor metabolizers was 2.0% (95% CI 1.8%, 2.7%), 2.2% in Caucasians (95% CI 1.6%, 2.5%) and 4.0% in African Americans (95% CI 2.6%, 6.1%).Conclusion:We also propose a numeric system for expression of CYP2D6 and CYP2C19 enzyme activity to aid clinicians in determining treatment strategy for patients receiving therapeutics that are metabolized by the CYP2D6 or CYP2C19 gene products.

This article focuses on the first generation of pharmacogenetic tests that are potentially useful in psychiatry. All pharmacogenetic tests currently on the market, or soon to be marketed in psychiatry, for which some information has been published in peer-reviewed journal articles (or abstracts), were selected. Five pharmacogenetic tests are reviewed in detail: the Roche AmpliChip CYP450 Test, the Luminex Tag-It Mutation Detection Kit, the LGC clozapine response test, the PGxPredict: Clozapine test, and the Genomas PhyzioType system. After reviewing these tests, three practical aspects of implementing pharmacogenetic testing in psychiatric clinical practice are briefly reviewed: (1) the evaluation of these tests in clinical practice, (2) cost-effectiveness, and (3) regulatory oversight. Finally, the future of these and other pharmacogenetic tests in psychiatry is discussed.

Microarray-based clinical tests have become powerful tools in the diagnosis and treatment of diseases. In contrast to traditional DNA-based tests that largely focus on single genes associated with rare conditions, microarray-based tests are ideal for the study of diseases with underlying complex genetic causes. Several microarray based tests have been translated into clinical practice such as MammaPrint and AmpliChip CYP450. Additional cancer-related microarray-based tests are either in the process of FDA review or under active development, including Tissue of Tumor Origin and AmpliChip p53. All diagnostic microarray testing is ordered by physicians and tested by a Clinical Laboratories Improvement Amendment-certified (CLIA) reference laboratory. Recently, companies offering consumer based microarray testing have emerged. Individuals can order tests online and service providers deliver the results directly to the clients via a password-protected secure website. Navigenics, 23andMe and deCODE Genetics represent pioneering companies in this field. Although the progress of these microarray-based tests is extremely encouraging with the potential to revolutionize the recognition and treatment of common diseases, these tests are still in their infancy and face technical, clinical and marketing challenges. In this article, we review microarray-based tests which are currently approved or under review by the FDA, as well as the consumer-based testing. We also provide a summary of the challenges and strategic solutions in the development and clinical use of the microarray-based tests. Finally, we present a brief outlook for the future of microarray-based clinical applications.

Oncology pharmacogenomics has seen a great deal of progress in the past 10 years. The release of the Human Genome Project data and the availability of fast, affordable genotyping platforms has allowed the field to expand and has provided invaluable data for pharmacogenomics research. The introduction of US FDA-approved targeted therapy (trastuzumab), package insert changes (irinotecan and tamoxifen) and the initiation of a genotype-guided clinical trial for cancer therapy (TYMS TSER in rectal cancer), along with panels of DNA and expression markers (Roche AmpliChip(®) and Oncotype Dx™ panel) are paving the way towards the integration of pharmacogenomics into clinical practice.

Polymorphic enzyme cytochrome P450 (CYP) 2C19 is expressed not only in the liver but also in the brain and mediates the biotransformation of 5-hydroxytriptamine (5-HT). We investigated possible association between genetic polymorphism of CYP2C19 and individual personality traits, possibly influenced by neurotransmitters. Mentally and physically healthy Japanese subjects were enrolled in this study (n = 352). Temperament and Character Inventory (TCI) and CYP2C19 genotyping were performed in all subjects. We detected CYP2C19*2 and *3 (http://www.imm.ki.se/CYPalleles/) using Amplichip CYP450 DNA tip. The number of genotypes classified as homozygous extensive metabolizer (EM), heterozygous EM, and poor metabolizer were 113, 181, and 58, respectively. Significant difference was found in TCI score in harm avoidance (HA; F = 3.138, P < 0.05). Post hoc analysis showed that TCI score in harm avoidance in homozygous EM was significantly lower than that in heterozygous EM (P < 0.05) or PM (P < 0.05). In sub-item analyses, HA3 (shyness with strangers, P < 0.01) and HA1 (anticipatory worry, P < 0.05) of TCI scores were significantly different among CYP2C19 genotypes. Meanwhile, there were no differences in TCI scores of novelty seeking (NS; F = 0.350, n.s.), reward dependence (RD; F = 1.080, n.s.), or persistence (P; F = 0.786, n.s.) among CYP2C19 genotypes. This study demonstrated that a significant association between CYP2C19 activity and HA is present in Japanese.

Aim: The aim of this study was to investigate the concordance between the novel GenoChip CYP2D6 macroarray and the AmpliChip, which is considered the gold standard in CYP2D6 genotyping. Materials & methods: Germline DNA of 200 patients was genotyped with both the AmpliChip and the GenoChip CYP2D6 macroarray. Results: One hundred and ninety-eight samples (99%) showed concordance. In two discordant samples the AmpliChip identified a *41 allele while the GenoChip CYP2D6 macroarray did not. Sanger sequencing showed that the 2988G>A mutation and thus the *41 allele was not present in both samples. Conclusion: We conclude that that the GenoChip CYP2D6 macroarray is a valid method for detecting genetic variants of CYP2D6 in a Caucasian population. Original submitted 10 November 2014; Revision submitted 23 February 2015

We investigated the accumulation of aberrant CYP2D6 genotypes and predicted metabolizer phenotypes (ultrarapid metabolizer, intermediate metabolizer and poor metabolizer) potentially affecting the antidepressant treatment response in depressive patients indicated for electroconvulsive therapy (ECT) compared with patients with a single episode of depression. Seventy-six Dutch White patients with unipolar or bipolar treatment-resistant depression who underwent ECT were genotyped using the Amplichip CYP450 Test for CYP2D6. Two hundred and eight patients with a single episode of unipolar or bipolar depression were used as controls. No difference was observed in the prevalence of CYP2D6 phenotypes (poor metabolizer, intermediate metabolizer, extensive metabolizer and ultrarapid metabolizer) between the ECT and the control patients (5.3, 38.7, 56.0 and 0.0% vs. 6.4, 51.0, 42.6 and 0.0%, respectively). The types of depression (odds ratio = 0.33, P = 0.018) and age (odds ratio = 1.55 for a 10-year increase, P < 0.001), but not CYP2D6 phenotype or activity score were associated with the response to antidepressant treatment. In conclusion, preemptive genotyping for CYP2D6 currently appears to have no clinical implications in treatment-resistant depressive patients indicated for ECT.

The p53 tumor suppressor gene (TP53) is reported to be mutated in nearly half of all tumors and plays a central role in genome integrity. Detection of mutations in p53 can be accomplished by many assays, including the AmpliChip p53 Research Test. The AmpliChip p53 Research Test has been successfully used to determine p53 status in hematologic malignancies and fresh frozen solid tissues but there are few reports of using the assay with formalin fixed, paraffin-embedded (FFPE) tissue. The objective of this study was to describe analytical performance characterization of the AmpliChip p53 Research Test to detect p53 mutations in genomic DNA isolated from archival FFPE human ovarian tumor tissues. Method correlation with sequencing showed 96% mutation-wise agreement and 99% chip-wise agreement. We furthermore observed 100% agreement (113/113) of the most prevalent TP53 mutations. Workflow reproducibility was 96.8% across 8 samples, with 2 operators, 2 reagent lots and 2 instruments. Section-to-section reproducibility was 100% for each sample across a 60 μm region of the FFPE block from ovarian tumors. These data indicate that the AmpliChip p53 Research Test is an accurate and reproducible method for detecting mutations in TP53 from archival FFPE human ovarian specimens.

Methods for Cytochrome P450-2D6 (CYP2D6) genotyping are often time-consuming and laborious, which can restrict their use in pretherapeutic screening programs. Gene chip technology could overcome this problem. The aim of this study was to evaluate CYP2D6 genotyping by a new improved gene chip compared to a PCR-RFLP method. AmpliChip CYP450 GeneChip(R) (AmpliChip) is a microarray hybridization method for genotyping CYP2D6 and CYP2C19. One hundred fifty-nine DNA samples were genotyped both by AmpliChip as well as by PCR-RFLP and, where applicable, by a SNaPshot technique which detects single nucleotide polymorphisms based on the single base extension principle. In 152 of the 159 samples, CYP2D6 genotypes determined with the AmpliChip were in accordance with the results of PCR-RFLP. All seven discrepant samples had gene duplications and were subjected to SNaPshot analysis. SNaPshot results concurred with those of the AmpliChip for six out of seven samples. In the one divergent result, DNA sequencing confirmed that the AmpliChip had assigned the correct genotype. In conclusion, AmpliChip is a highly reliable method for CYP2D6 genotyping that allows the correct determination of all relevant CYP2D6 alleles in one single run. It therefore represents a very efficient and fast method, offering new perspectives for the application of pharmacogenetics in clinical medicine.

Genotype/phenotype analysis with human hepatocytes has identified a new inactive CYP2D6 allele, CYP2D6*56. Cryopreserved human hepatocytes from 51 livers were evaluated for CYP2D6 activity with dextromethorphan as the probe substrate. Hepatocyte lots that lacked CYP2D6 activity were further evaluated for CYP2D6 expression and known genetic variations, including CYP2D6*2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *14, *15, *17, *18, *19, *20, *25, *26, *29, *30, *35, *40, *41, *43, and various multiple copy CYP2D6 alleles (*1xn, *2xn, and *4xn) by the AmpliChip CYP450 prototype microarray (Roche Molecular Systems, Inc., Branchburg, NJ). Two discrepancies were uncovered between the CYP2D6 genotype and activity by this approach. In one sample, a previously unreported 3201C 224 T transition in exon 7 resulted in Arg344(CGA) being replaced by a stop codon (TGA), resulting in a CYP2D6 enzyme lacking the terminal 153 amino acids. This allele was given the designation of CYP2D6*56 and the GenBank accession number DQ282162. The lack of CYP2D6 activity in cryopreserved hepatocytes and microsomes found in the second sample, despite a normal level of CYP2D6 expression and a genotype (*10/*1) predictive of normal CYP2D6 activity, was attributed to enzyme inactivation by an unknown metabolite. The identification and characterization of the CYP2D6*56 allele indicates that commercial cryopreserved human hepatocytes may provide a valuable means to rapidly identify genetic variations with functional relevance. This integrated approach of identifying alleles and examining allele relationships to gene expression and function could be of tremendous value to understanding the mechanism responsible for functional differences in gene variation. The commercial availability of human cryopreserved hepatocytes also makes this potential readily available to any who are interested in it, not just those with access to private liver banks.

The clinical importance of genetic polymorphisms in drug metabolism is well-known in clinical pharmacotherapy. The first widely available pharmacogenomic microarray technology approved by the Food and Drug Administration as a medical device to clinically genotype genetic polymorphisms in drug metabolism is now available with the launch of AmpliChip technology. This readily accessible clinical microarray test allows the genotyping of cytochrome (CYP) P-450 2D6 and 2C19 and marks a milestone in the epoch of evidence based personalized medicine. Many commonly used drugs are substrates for CYP2D6 and CYP 2C19 and hence may potentially demonstrate phenotypic differences as poor, intermediate, extensive, and ultrarapid metabolizers. These phenotypic variations could lead to expressed differences in pharmacotherapeutic patient outcomes. AmpliChip currently allows for testing of multiple alleles (31) in a single assay. Other technologies for pharmacogenomics are on the horizon. This article reviews the importance of polymorphic enzymes and genotyping as to how genetic polymorphisms alter pharmacotherapy and the emergence of a plethora of technologies that may become routinely available for clinical pharmacogenomic testing in the near future.

The AmpliChip CYP450 Test, which analyzes patient genotypes for cytochrome P450 (CYP) genes CYP2D6 and CYP2C19, is a major step toward introducing personalized prescribing into the clinical environment. Interest in adverse drug reactions (ADRs), the genetic revolution, and pharmacogenetics have converged with the introduction of this tool, which is anticipated to be the first of a new wave of such tools to follow over the next 5-10 years. The AmpliChip CYP450 Test is based on microarray technology, which combines hybridization in precise locations on a glass microarray and a fluorescent labeling system. It classifies individuals into two CYP2C19 phenotypes (extensive metabolizers [EMs] and poor metabolizers [PMs]) by testing three alleles, and into four CYP2D6 phenotypes (ultrarapid metabolizers [UMs], EMs, intermediate metabolizers [IMs], and PMs) by testing 27 alleles, including seven duplications. CYP2D6 is a metabolic enzyme with four activity levels (or phenotypes): UMs with unusually high activity; normal subjects, known as EMs; IMs with low activity; and PMs with no CYP2D6 activity (7% of Caucasians and 1-3% in other ethnic groups). Levels of evidence for the association between CYP2D6 PMs and ADRs are relatively reasonable and include systematic reviews of case-control studies of some typical antipsychotics and tricyclic antidepressants (TCAs). Evidence for other phenotypes is considerably more limited. The CYP2D6 PM phenotype may be associated with risperidone ADRs and discontinuation due to ADRs. Venlafaxine, aripiprazole, duloxetine, and atomoxetine are newer drugs metabolized by CYP2D6 but studies of the clinical relevance of CYP2D6 genotypes are needed. Non-psychiatric drugs metabolized by CYP2D6 include metoprolol, tamoxifen, and codeine-like drugs. CYP2C19 PMs (3-4% of Caucasians and African Americans, and 14-21% of Asians) may require dose adjustment for some TCAs, moclobemide, and citalopram. Other drugs metabolized by CYP2C19 are diazepam and omeprazole. The future of pharmacogenetics depends on the ability to overcome serious obstacles, including the difficulties of conducting and publishing studies in light of resistance from grant agencies, pharmaceutical companies, and some scientific reviewers. Assuming more studies are published, pharmacogenetic clinical applications may be compromised by economic factors and the lack of physician education. The combination of a US FDA-approved test, such as the AmpliChip CYP450 Test, and an FDA definition of CYP2D6 as a 'valid biomarker' makes CYP2D6 genotyping a prime candidate to be the first successful pharmacogenetic test in the clinical environment. One can use microarray technology to test for hundreds of single nucleotide polymorphisms (SNPs) but, taking into account the difficulties for single gene approaches such as CYP2D6, it is unlikely that very complex pharmacogenetic approaches will reach the clinical market in the next 5-10 years.

The US FDA has granted market approval for the first pharmacogenetic test using a DNA microarray, the AmpliChip CYP450, which genotypes cytochrome P450 (CYP)2D6 and CYP2C19. The test uses software to predict phenotypes and tests for 27 CYP2D6 alleles, including the deletions and duplications, and three CYP2C19 alleles. Other DNA microarray platforms are being developed for CYP testing, but none have been completely developed or approved by the FDA to date. The differences between an implementation of pharmacogenetic tests centered on the individual and implementation using a public health approach are discussed. In this review, the major obstacles to the wide implementation of pharmacogenetic testing in the clinical environment are summarized.

A year after FDA first cleared a drug-metabolizing-enzyme genotyping system for marketing, clinicians have three such systems available to help optimize patients’ medication therapies. First came the AmpliChip CYP450 Test for determining the genotype of a patient’s cytochrome P-450 isoenzyme (

Pharmacogenetics has arrived in clinical psychiatric practice with the FDA approval of the AmpliChip CYP450 Test that genotypes for two cytochrome P450 2D6 (CYP2D6) and 2C19 (CYP2C19) genes. Other pharmacogenetic tests, including those focused on pharmacodynamic genes, are far from ready for clinical application. CYP2D6 is important for the metabolism of many antidepressants and antipsychotics, and CY2C19 is important for some antidepressant metabolism. Poor metabolizers (PMs), lacking the enzyme, account for up to 7% of Caucasians for CYP2D6 and up to 25% of East Asians for CYP2C19. Patients having three or more active CYP2D6 alleles (up to 29% in North Africa and the Middle East), are called CYP2D6 ultra-rapid metabolizers (UMs). CYP2D6 phenotypes (particularly PMs) are probably important in patients taking tricyclic antidepressants (TCAs), venlafaxine, typical antipsychotics, and risperidone. The CYP2C19 PM phenotype is probably important in patients taking TCAs and perhaps citalopram, escitalopram, and sertraline. On the basis of the literature and the authors' clinical experience, the authors provide provisional recommendations for identifying and treating CYP2D6 PMs, CYP2C19 PMs, and CYP2D6 UMs. The next few years will determine whether CYP2D6 genotyping is beneficial for patients taking the new drugs aripiprazole, duloxetine, and atomoxetine. Practical recommendations for dealing with laboratories offering CYP2D6 and CYP2C29 genotyping are provided.

Objective: The cytochrome P450 2D6 (CYP2D6) enzyme metabolizes risperidone. CYP2D6 poor metabolizers have no CYP2D6 activity (7% of whites and 1%-2% of other races). This study tested whether the CYP2D6 poor metabolizer phenotype was associated with adverse drug reactions (ADRs) and discontinuation due to ADRs. Method: Adult inpatients and outpatients were recruited from July 2000 to March 2003 including (1) 325 who were stabilized on risperidone therapy and classified as either expressing moderate-to-marked ADRs (22%, 73/325) or not (78%, 252/325) and (2) 212 who discontinued risperidone and were classified as discontinued due to ADRs (38%, 81/212) or for other reasons (62%, 131/212). Genetic tests were performed by allele-specific polymerase chain reaction and/or by the AmpliChip CYP450 microarray system for up to 34 separate CYP2D6 alleles. Two logistic regression models with dependent variables (moderate-to-marked ADRs while taking risperidone and risperidone discontinuation due to ADRs) were evaluated with respect to the CYP2D6 phenotype. Results: The odds ratios (ORs) and 95% confidence intervals (CIs) for the CYP2D6 poor metabolizer phenotype in the univariate analyses and after correcting for clinical variables were (1) OR = 3.1 (CI = 1.4 to 7.0) and 3.4 (CI = 1.5 to 8.0) for moderate-to-marked ADRs on risperidone and (2) OR = 3.0 (CI = 0.85 to 10.6) and 6.0 (CI = 1.4 to 25.4) for discontinuation due to ADRs. Conclusions: The CYP2D6 poor metabolizer phenotype appears to be associated with risperidone ADRs and discontinuation due to ADRs; however, this finding requires further study in larger patient populations. The CYP3A5 and p-glycoprotein exon 21 and 26 genotypes were not significantly associated with risperidone response.

Pharmacogenetics has assumed increasing importance with the developing concepts of personalized medicine. There is a need to determine the metabolic status of an individual when using drugs, the actions of which are influenced by drug-metabolizing enzymes. Cytochrome P450 (CYP) and its variants, particularly CYP2D6 and CYP2C19, play a role in the metabolism of approximately 25% of all prescription drugs. This review covers the role of the CYP system not only in the metabolism of drugs but also in the pathophysiology of disease. Various technologies for the assessment of CYP status are described, with the focus on AmpliChip CYP450 (Roche Molecular Diagnostics, Alameda, CA, USA), the first approved microarray molecular diagnostic test for the analysis of 29 polymorphisms and mutations of the CYP2D6 gene, and two polymorphisms of the CYP2C19 gene. It combines Roche's PCR technology with the GeneChip microarray system (Affymetrix, Santa Clara, CA, USA). Examples of numerous drugs that are metabolized by the CYP system are listed, and categories of antidepressants, antipsychotics, immunosuppressive and anticancer drugs are described to illustrate the role of testing for CYP polymorphisms in the therapeutic use of these drugs. CYP testing has applications in toxicology and absorption, distribution, metabolism and excretion (ADME) profiling as a guide to drug development. AmpliChip CYP450 may be used in conjunction with pharmacotherapy to guide decision making about selection of drugs and dosage. The test is not a solitary tool to determine optimum drug dosage, but is meant for use along with clinical evaluation and other methods for the selection of the treatment that is best suited for an individual patient. AmpliChip CYP450 is the first DNA microarray test to be cleared by the US FDA, and its clearance paves the way for similar microarray-based diagnostic tests to be developed in the future. This will facilitate the development of personalized medicine.

Genetic factors contribute to the phenotype of drug response. We systematically analyzed all available pharmacogenetic data from Medline databases (1970-2003) on the impact that genetic polymorphisms have on positive and adverse reactions to antidepressants and antipsychotics. Additionally, dose adjustments that would compensate for genetically caused differences in blood concentrations were calculated. To study pharmacokinetic effects, data for 36 antidepressants were screened. We found that for 20 of those, data on polymorphic CYP2D6 or CYP2C19 were found and that in 14 drugs such genetic variation would require at least doubling of the dose in extensive metabolizers in comparison to poor metabolizers. Data for 38 antipsychotics were examined: for 13 of those CYP2D6 and CYP2C19 genotype was of relevance. To study the effects of genetic variability on pharmacodynamic pathways, we reviewed 80 clinical studies on polymorphisms in candidate genes, but those did not for the most part reveal significant associations between neurotransmitter receptor and transporter genotypes and therapy response or adverse drug reactions. In addition associations found in one study could not be replicated in other studies. For this reason, it is not yet possible to translate pharmacogenetic parameters fully into therapeutic recommendations. At present, antidepressant and antipsychotic drug responses can best be explained as the combinatorial outcome of complex systems that interact at multiple levels. In spite of these limitations, combinations of polymorphisms in pharmacokinetic and pharmacodynamic pathways of relevance might contribute to identify genotypes associated with best and worst responders and they may also identify susceptibility to adverse drug reactions.

Rationale: Cytochrome P450 enzymes are important in the metabolism of antidepressants. The highly polymorphic nature of these enzymes has been linked to variability in antidepressant metabolism rates, leading to hope regarding the use of P450 genotyping to guide treatment. However, evidence that P450 genotypic differences underlie the variation in treatment outcomes is inconclusive. Objectives: We explored the links between both P450 genotype and serum concentrations of antidepressant with antidepressant side effects, using data from the Genome-Based Therapeutic Drugs for Depression Project (GENDEP), which is a large (n = 868), pharmacogenetic study of depressed individuals treated with escitalopram or nortriptyline. Methods: Patients were genotyped for the enzymes CYP2C19 and CYP2D6, and serum concentrations of both antidepressant and primary metabolite were measured after 8 weeks of treatment. Side effects were assessed weekly. We investigated associations between P450 genotypes, serum concentrations of antidepressants and side effects, as well as the relationship between P450 genotype and study discontinuation. Results: P450 genotype did not predict total side effect burden (nortriptyline: n = 251, p = 0.5638, β = -0.133, standard error (SE) = 0.229; escitalopram: n = 340, p = 0.9627, β = -0.004, SE = 0.085), study discontinuation (nortriptyline n = 284, hazard ratio (HR) = 1.300, p = 0.174; escitalopram n = 376, HR = 0.870, p = 0.118) or specific side effects. Serum concentrations of antidepressant were only related to a minority of the specific side effects measured: dry mouth, dizziness and diarrhoea. Conclusions: In this sample where antidepressant dosage is titrated using clinical judgement, P450 genotypes do not explain differences between patients in side effects with antidepressants. Serum drug concentrations appear to only explain variability in the occurrence of a minority of specific side effects., SCOPUS: ar.j, info:eu-repo/semantics/published

A Han Chinese patient failed CYP2D6 genotype analysis with the AmpliChip CYP450 Test™. The CYP2D6 gene locus of the patient and her son were extensively genotyped including copy number variation and gene resequencing. Two SNPs were discovered on the patient’s CYP2D6*1 allele, -498C>A and 1661G>C, while the son’s CYP2D6*1 allele had -498C>A only. AmpliChip failure was attributed to the presence of a CYP2D6*1 allele carrying the 1661G>C SNP. Functional analyses of -498C>A did not reveal altered activity in vitro or in vivo suggesting that both novel CYP2D6*1 subvariants are functional. The implementation of pharmacogenetics-guided drug therapy relies on accurate clinical-grade genotype analysis. Although the AmpliChip is a reliable platform, numerous allelic (sub)variants and gene arrangements are not detected or may trigger no calls. While such cases may be rare, the clinical/genetic testing community must be aware of the challenges of CYP2D6 testing on the AmpliChip platform and implications regarding accuracy of test results.

TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.

Background CYP2D6, a major drug-metabolizing enzyme, is encoded by a highly polymorphic and complex gene locus. We have identified a patient who failed to produce a CYP2D6 genotype with the AmpliChip P450 Test (AmpliChip), whereas his CYP2C19 genotype was readily determined. The aim of this investigation was to fully characterize the patient's CYP2D6 gene locus to resolve the AmpliChip no-call. Methods The case, a brother, and son were genotyped with the AmpliChip and subsequently characterized using long-range (XL)-PCR coupled with TaqMan assay technology. Copy number variation was assessed by XL-PCR and quantitative PCR. Selected XL-PCR products were sequenced. Results The AmpliChip also produced a no-call for the son; the brother produced a result. The two alleles of the case were subsequently found to carry additional gene units that likely caused the AmpliChip no-calls. One was characterized as a CYP2D6*68+*4 tandem (CYP2D6*68 is a hybrid gene composed of 2D6 and 2D7), the other as a rare CYP2D6*13+*2 tandem (CYP2D6*13 is a 2D7/2D6 hybrid formerly known as CYP2D6*77). A novel CYP2D6*2 subvariant was identified in the son; the brother also carried the CYP2D6*68+*4 tandem. Conclusions The implementation of pharmacogenetics-guided drug therapy relies on accurate clinical-grade genotype analysis. Although the AmpliChip is deemed to be a reliable platform, numerous more recently discovered allelic variants and gene arrangements are not detected or trigger no-calls. Although such cases may be rare, the clinical/genetic testing community must be aware of the challenges of CYP2D6 testing on the AmpliChip platform and implications regarding accuracy of test results.

Tamoxifen is metabolized into endoxifen, a potent antagonist of the estrogen receptor, in part through cytochrome p450 (CYP) 2D6. Genotypic variation in CYP2D6 affects endoxifen levels, and some have argued that patients who do not efficiently metabolize tamoxifen might wish to consider alternative hormonal treatments. This study evaluated an algorithm in which endoxifen levels and CYP2D6 genotypes were used to make hormonal therapy recommendations for patients on adjuvant tamoxifen for breast cancer. Patients with stage I–III breast cancer who had been taking adjuvant tamoxifen for 8–56 weeks were eligible. At enrollment, baseline whole blood and serum were sent for genotyping by Amplichip and endoxifen measurement, respectively, and endoxifen levels were also measured 3 weeks later. Results were returned to oncologists along with an algorithm-generated treatment recommendation. The algorithm recommended that participants with poor metabolizer genotype and/or baseline endoxifen level

Background Estrogen receptor-positive breast cancer tumors depend on estrogen signaling for their growth and replication and can be treated by anti-estrogen therapy with tamoxifen. Polymorphisms of the CYP2D6 and CYP2C19 genes are associated with an impaired response to tamoxifen. The study objective was to investigate the impact of genetic polymorphisms in CYP2D6 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Spanish women with estrogen receptor-positive breast cancer who were candidates for tamoxifen therapy. Methods: We studied 90 women with estrogen receptor-positive breast cancer, using the AmpliChip CYP450 test to determine CYP2D6 and CYP2C19 gene variants. Plasma levels of tamoxifen and its metabolites were quantified by high-performance liquid chromatography. Results The CYP2D6 phenotype was extensive metabolizer in 80%, intermediate metabolizer in 12.2%, ultra-rapid metabolizer in 2.2%, and poor metabolizer in 5.6% of patients, and the allele frequency was 35.0% for allele *1, 21.0% for *2, and 18.9% for *4. All poor metabolizers in this series were *4/*4, and their endoxifen and 4-hydroxy tamoxifen levels were 25% lower than those of extensive metabolizers. CYP2C19*2 allele, which has been related to breast cancer outcomes, was detected in 15.6% of the studied alleles. Conclusion CYP2D6*4/*4 genotype was inversely associated with 4-hydroxy tamoxifen and endoxifen levels. According to these results, CYP2D6 and CYP2C19 genotyping appears advisable before the prescription of tamoxifen therapy.

Tamoxifen, a prodrug used for adjuvant breast cancer therapy, requires conversion to the active metabolite endoxifen through CYP 2D6. We aimed to construct an algorithm to predict endoxifen concentrations based on a patient’s CYP 2D6 genotype, demographic factors, and co-medication use. Eighty-eight women enrolled in the UCSF TamGen II study and 81 women enrolled in a prospective study at Dana-Farber Cancer Institute were included in this analysis. All the women had been on tamoxifen for at least 3 months before blood collection. Demographic information included the patient’s age, race/ethnicity, body mass index (where available), and self-reported and measured medications and herbals that affect 2D6 activity. DNA was extracted and genotyped for 2D6 (Amplichip, Roche Diagnostics). An activity score was calculated based on genotypes and adjusted for use of medications known to inhibit 2D6. Serum was tested for tamoxifen and metabolite concentrations and for the presence of drugs by liquid chromatography/mass spectrometry. Univariate and multivariate regression analysis were computed for age, body mass index, ethnicity, and adjusted activity score to predict tamoxifen metabolite concentrations in the training data-set of UCSF patients, and the resulting algorithm was validated in the Dana-Farber patients. For the training set, the correlation coefficient (r2) for log endoxifen and N-desmethyltamoxifen:endoxifen ratio to activity score, age, and race, were 0.520 and 0.659, respectively; 0.324 and 0.567 for the validation; and 0.396 and 0.615 for both the datasets combined. An algorithm that incorporates genotype and demographic variables can be used to predict endoxifen concentrations for women on tamoxifen therapy. If endoxifen levels are confirmed to be predictive of tamoxifen benefit, then this algorithm may be helpful to determine which women warrant endoxifen testing.

Recent studies have shown that CYP2D6 acts at critical steps for endogenous morphine biosynthesis. The present study assessed the contribution of CYP2D6 genetic polymorphisms, smoking, and other factors on acute severe postoperative pain (linear analog pain scores ≥8). Methods. Two hundred thirty-six female patients were found to have adequate information in a previously developed female surgical patient database to be included in this current analysis. Multiple logistic regression analysis was used to assess the predictors for acute severe postoperative pain. DNA had been previously extracted from blood samples in all patients and was genotyped by the Amplichip to determine the specific CYP2D6 genotypes. Results. It was noted that the incidence of acute severe postoperative pain (linear analog pain scores ≥8) was more frequent in patients with the CYP2D6 poor metabolizer (PM) genotype, 71%, compared with 28% in intermediate metabolizers (IMs), 26% in extensive metabolizers (EMs), and 27% in ultrarapid metabolizers (UMs). The overall association between metabolizer groups and severe postoperative pain was significant ( P = 0.023). PMs were significantly more likely to suffer from severe postoperative pain than IMs, EMs, and UMs ( P = 0.007, 0.002, and 0.050, respectively). There were no significant differences among IMs, EMs, and UMs. Additionally, it was noted that there was an increased frequency of acute severe postoperative pain in smokers vs nonsmokers ( P = 0.014). Conclusion. This study demonstrated that female patients possessing the PM genotype of CYP2D6 and patients who smoke had a higher incidence of acute severe postoperative pain.

of these materials is meticulously controlled by regulatory authorities such as the US FDA and the EMA. To date, no such regulation exists within the field of genomics. While some assays such as the AmplichipTM (Roche, Almere, The Netherlands) or the invader UGT1A1 molecular assay have been approved by the FDA, the majority of tests lack such an approval [4]. In addition, the table which translates genotypes to phenotypes of the Amplichip array is not included in the FDA approval of the product. Here we discuss current developments and challenges for developing a genome that is suitable for clinical use.

The primary objective of this chapter is to provide an overview of the clinical applications of psychiatric pharmacogenomic testing. Essentially, two current primary objectives of testing exist. The first is to identify medications for which a patient is at increased risk for developing side effects or adverse events. The second is to identify medications that are less likely to be effective. Ultimately, the objective of psychiatric pharmacogenomics will be to identify psychotropic medications that have a high probability of achieving a therapeutic response for an individual patient based on the identification of specific genetic variations (1). Although clinical psychiatric pharmacogenomic testing was being provided at some academic centers before 2004, the introduction of the AmpliChip and its approval by the Food and Drug Administration in 2004 was an important milestone in the clinical adoption of genetic testing that was designed to provide guidance in the selection and dosing of psychiatric medications. Over the next 5 years, many academic medical centers introduced clinical pharmacogenomic testing of drug-metabolizing enzyme genes as a component of the clinical evaluation of patients with atypical responses to medication. As the cost of psychiatric pharmacogenomic testing decreases, there is a growing expectation that genotyping patients before initiating treatment with psychotropic medication will be a cost-effective clinical strategy to minimize adverse events.

Abstract 675 Background: Activation of the p53 pathway by inhibition of its negative regulator MDM2 has been proposed as a novel strategy for cancer therapy. We report the final results of the Phase 1 leukemia trial of RG7112, a small molecule antagonist of MDM2 which activates p53 by disrupting the p53-MDM2 interaction. Methods: The trial was designed as a multi-center, open-label Phase 1 MAD study of patients with relapsed/refractory leukemia treated with escalating doses of single agent RG7112 in two strata: A:AML, ALL, CML-BC; and B:CLL and S-CLL. RG7112 was administered orally daily for 10 days followed by 18 days of rest. Primary objectives were to determine MTD and DLT; secondary objectives to assess PK, PD and clinical responses. Enrollment is complete. 4 patients remain on study, and final data is being compiled. Peripheral blood was collected for PK and biomarkers pre-treatment, at multiple time points during the first 48 hours, and on d10. Bone marrow was collected pre-treatment, d10 and d28. Blood and marrow were purified by MACS sorting (CD19, 33 or 34 Ab). AmpliChip analysis of p53 mutational status (single nucleotide alterations exons 2–11) was performed. MIC-1 (macrophage inhibitory cytokine-1), a p53 transcriptional target, was analyzed in serum by ELISA as a pharmacodynamic marker of p53 activation. Baseline MDM2 mRNA levels, and changes from baseline, as a marker of p53 activation and feedback on MDM2, were measured by RT-PCR. A panel of 24 genes was assessed serially by TLDA (q-PCR). Results: 116 patients (84 AML,10 ALL, 2 CML, 20 CLL/sCLL) were treated with RG7112 doses ranging from 20 – 2430 (1215 BID) mg/m2/day × 10 days. The dose-exposure relationship (Cmax, AUC, and Ctrough) was approximately linear with a median half-life of 1–1.5 days. 43 patients with acute leukemia were dosed at the MTD of 1500 mg BID (flat dose) × 10 days. Major adverse events(> 20%) were nausea, diarrhea and vomiting. Other major (>10%) AEs were complications of leukemia (e.g. neutropenic fever). Clinical activity was noted during dose escalation (ASH 2011), including apoptosis in AML and CLL, CR in an AML patient (360mg/m2) stable for 6 cycles (10 months) and one patient with CLL/Richter's transformation with stable disease (40 mg/m2) for >25 cycles. New data focuses on AML patients treated at MTD. This cohort received a median of 3 prior treatments, 16 were refractory to last treatment. 17 (40%) had evidence of hematologic improvement (greater than 50% drop in peripheral and/or BM blasts by d28). Of the 31 patients who completed cycle 1 dosing, 5 patients (16%) had a CR (2 CR, 2 CRi. 1 CR without recovery). Two CR patients have proceeded to allotransplant. All CR patients' blasts were p53wt. Of the 12 additional patients with single-agent hematologic activity, 3 had biochemical evidence of tumor lysis syndrome, 10 had p53status evaluable, and mutations were detected in 3: (exon 7 missense, exon 9 frameshift, intron 7 splice). p53 activation by RG7112 was demonstrated by MIC-1 induction in serum. MIC-1 levels were elevated at RG7112 plasma concentrations >2 μg/mL or AUC24h >50 μg · h/mL and were highly correlated (0.5629) with exposure to RG7112 (AUC24h) at steady state following a sigmoidal Emax model. Preliminary data review suggests a positive correlation between exposure to RG7112 and changes in MDM2 gene expression at day 2. In addition, q-PCR analysis demonstrated exposure-dependent induction of key p53 transcriptional targets including MDM2, p21, and PUMA. p53 protein increase by WB was demonstrated at the MTD in selected patients. These findings are consistent with the proposed mechanism of action of RG7112. Conclusions: We report evidence of single agent clinical activity in patients with leukemia treated with RG7112, including 6 AML patients with CR, 5 of 31 (16%) at the MTD, and additional patients with significant decreases in blasts. Concentration-related pharmacodynamic biomarker activity of the p53 pathway was demonstrated by increases in MIC-1 levels, and induction of p53 target genes including mdm2, p21 and PUMA. 4 patients remain on treatment and final results will be reported. We provide proof of mechanism and evidence for single-agent clinical activity of MDM2 antagonism in leukemia by demonstrating complete remission, lysis of leukemic blasts and activation of p53 pathway targets in leukemic cells from patients treated with RG7112. Clinical studies of RG7112 in combination are underway. Disclosures: Andreeff: Hoffmann-La Roche: Research Funding; Karyopharm Therapeutics: Unrestricted gift, Unrestricted gift Other. Yee:Roche: Membership on an entity's Board of Directors or advisory committees. Assouline:Roche: travel funding Other. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy; Ariad: Consultancy. Drummond:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Geho:Roche: Employment. Blotner:Roche: Employment. Cheng:Roche: Employment. Vassilev:Roche: Employment. Ding:Roche: Employment. Zhi:Roche: Employment. Middleton:Roche: Employment. Nichols:Roche: Employment.

Genotyping arrays assess the sample DNA for the presence or absence of specific sequences or mutations (or copy number variations) and can be conveniently divided into “simple” and “complex” arrays. Simple arrays, typically with less than 100 spots or “features”, can characterise a pathogen (e.g. an HPV subtype) or assess for a few mutations in a patient’s DNA (e.g. screen for common CF mutations). A number of such tests are currently marketed; they are relatively affordable but in competition with even less expensive PCR-based assays. Complex arrays, in contrast, contain many thousands of oligonucleotides and can allow detailed characterisation of several genes (as in the Roche AmpliChip test), provide a one-step screen for a large number (hundreds) of recessive diseases or ensure detailed HLA characterisation for transplant purposes (among others). Genotyping array tests generally have clear clinical correlates and enjoy a relatively straightforward approval path, but suffer from strong competition both at the low end (by PCR-based approaches) and at the high end of complexity (by new-generation sequencing).

To determine rates of pathologic complete response (pCR) and near-complete response (npCR) in operable early-stage breast cancer using neoadjuvant capecitabine plus docetaxel, with or without trastuzumab, and investigate biomarkers of pathologic response. Women with operable early-stage breast cancer were enrolled in a multicenter study of neoadjuvant therapy for four 21-day cycles with capecitabine 825 mg/m(2) plus docetaxel 75 mg/m(2) if human epidermal growth factor receptor 2 (HER2)-negative, and additionally, a standard trastuzumab dose if HER2-positive. Primary endpoint was rate of pCR and npCR. Secondary endpoints were potential associations between response and TP53 mutational analysis using the AmpliChip TP53 assay or immunohistochemical (IHC) staining, and genomic subtyping using the PAM50 assay. In patients who completed treatment and surgery, pCR and npCR rates were 15.8% in patients with HER2-negative and 50% in patients with HER2-positive tumors. Stratified by genomic subtype, patients of HER2-enriched subtype had the best response (72.2%), and luminal A (9.1%) and B (4.8%) subtypes, the poorest. Of 147 patients tested for TP53 mutations using the AmpliChip assay, 78 variants were detected; 55 were missense. Response rate among TP53-mutated patients was 30%, significantly higher than TP53 wild-type patients (10%; P = 0.0032). Concordance between AmpliChip mutation status versus TP53 IHC staining was 65%, with AmpliChip status predictive of response and IHC status not predictive. Capecitabine plus docetaxel in HER2-negative, and with trastuzumab in HER2-positive patients, provided a good response rate with four cycles of non-anthracycline-containing therapy. TP53 mutational analysis and genomic subtyping were predictive.

Given that TP53 alterations predict prognosis and response to therapy in chronic lymphocytic leukemia (CLL), screening for TP53 mutations has an increasing role in patient management. TP53 direct sequencing is a time-consuming method, while the AmpliChip p53 Research Test is a novel non time-consuming microarray-based resequencing assay and queries Exons 2–11. We evaluated the impact of TP53 mutations on clinical outcome by analyzing 98 untreated CLL using the AmpliChip p53 Research Test and direct sequencing and performed microarrays analysis on TP53 mutated and/or deleted cases. The AmpliChip p53 Research Test detected 17 mutations in 14 patients (17.3%); a significant association between TP53 mutations and del(17p) was recorded. From a clinical standpoint, a higher percentage of mutation was found in CLL with unfavorable outcome (17.2% vs. 7.1% in progressive vs. stable cases). Detection of TP53 mutations by the AmpliChip p53 Research Test was associated with a significantly worse survival (P = 0.0002). Comparison of the array and direct sequencing tests showed that the p53 Research Test detected more mutations, although it failed to identify two microdeletions. Finally, microarrays analysis showed a more distinctive signature associated with del(17p) than with TP53 mutations, likely due to a concomitant gene dosage effect. The AmpliChip p53 Research Test is a straightforward method that bears prognostic value. This study confirms a high percentage of TP53 mutations in CLL with unfavorable outcome and a significant association between TP53 aberrations and del(17p). Finally, specific gene expression profiles are recognized for TP53 alterations. © 2011 Wiley-Liss, Inc.

Purpose: This study aimed to validate matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS)/Taqman copy number assay (CNA) CYP2D6 genotyping by AmpliChip CYP450 Test for the prediction of tamoxifen metabolizer phenotypes in breast cancer, and to investigate the influence of CYP2D6 variant coverage on genotype-phenotype relationships and tamoxifen outcome. Experimental Design: Hormone receptor–positive postmenopausal breast cancer patients (n = 492) treated with adjuvant tamoxifen, previously analyzed by MALDI-TOF MS/CNA, were reanalyzed by AmpliChip CYP450 Test and validated by independent methods. Cox proportional hazard ratios (HR) were calculated for recurrence of poor (PM) relative to extensive metabolizer (EM) phenotypes with increasing numbers of CYP2D6 variants. Kaplan-Meier distributions were calculated for different phenotype classifications. Results: Concordance was 99.2% to 99.5% for CNA and 99.8% to 100% per CYP2D6 allele (*3, *4, *5, *9, *10, and *41). The prevalence of predicted phenotypes was 1.2% for ultrarapid metabolizer (UM), 37.2% for EM without variant, 43.5% for heterozygous EM, 9.7% for intermediate metabolizer (IM), and 8.3% for PM. Approximately, one third of patients were misclassified based on a *4 analysis only, but inclusion of all reduced-function alleles increased the PM-associated HR from 1.33 (P = 0.58) to 2.87 (P = 0.006). Kaplan-Meier analyses showed highest and lowest clinical benefit for UM and PM with respect to both the AmpliChip-based and a redefined phenotype assignment. The latter revealed significant allele–dose-dependent associations (P = 0.011) and largest effect size (HRPM_EM = 2.77; 95% confidence interval, 1.31-5.89). Conclusions: MALDI-TOF MS/CNA is suitable for accurate CYP2D6 genotyping. For tamoxifen pharmacogenetics, broad CYP2D6 allele coverage is recommended to reduce phenotype misclassification. Classification based on refined EM and reduced-function metabolizers is advisable. Clin Cancer Res; 16(17); 4468–77. ©2010 AACR.

Background: The deprivation gap for breast cancer survival remains unexplained by stage at presentation, treatment, or co-morbidities. We hypothesised that p53 mutation might contribute to the impaired outcome observed in patients from deprived communities.\ud methods: p53 mutation status was determined using the Roche Amplichip research test in 246 women with primary breast cancer attending a single cancer centre and related to deprivation, pathology, overall, and disease-free survival.\ud results: p53 mutation, identified in 64/246 (26%) of cancers, was most common in 10 out of 17 (58.8%) of the lowest (10th) deprivation decile. Those patients with p53 mutation in the 10th decile had a significantly worse disease-free survival of only 20% at 5 years (Kaplan–Meier logrank χ2=6.050, P=0.014) and worse overall survival of 24% at 5 years (Kaplan–Meier logrank χ2=6.791, P=0.009) than women of deciles 1–9 with p53 mutation (c.f. 56% and 72%, respectively) or patients in the 10th decile with wild-type p53 (no disease relapse or deaths).\ud conclusion: p53 mutation in breast cancer is associated with socio-economic deprivation and may provide a molecular basis, with therapeutic implications, for the poorer outcome in women from deprived communities.