Chirurgische Arbeitsgemeinschaft für Adipositastherapie (CA-ADIP) In Zusammenarbeit mit Deutsche Adipositas-Gesellschaft (DAG) Deutsche Gesellschaft für Psychosomatische Medizin und Psychotherapie Deutsche Gesellschaft für Ernährungsmedizin S3-Leitlinie: Chirurgie der Adipositas
Laboratory testing of cyp2d6 alleles in relation to tamoxifen therapyACMG STANDARDS AND GUIDELINES
American College of Medical Genetics and Genomics laboratory testing of CYP2D6 alleles in relation
to tamoxifen therapy
Elaine Lyon, PhD, FACMG1, Julie Gastier Foster, PhD, FACMG2,3, Glenn E. Palomaki, PhD4, Victoria M. Pratt, PhD, FACMG5, Kristen Reynolds, PhD6, M. Fernanda Sábato, MS7, Stuart A. Scott, PhD, FACMG8 and Patrik Vitazka, MD, PhD7; A working group of the Molecular Genetics Subcommittee on behalf of the American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee Disclaimer: Disease-specific statements are intended to augment the current general ACMG Standards and Guidelines for Clinical Genetic Laboratories.
Individual laboratories are responsible for meeting the Clinical Laboratory Improvement Amendments (CLIA)/College of American Pathologists (CAP) quality assurance standards with respect to appropriate sample documentation, assay validation, general proficiency, and quality control measures. These ACMG Standards and Guidelines are developed primarily as an educational resource for clinical laboratory geneticists, to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines is voluntary and does not necessarily ensure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of al proper procedures and tests or exclusive of other procedures and tests that are reasonably directed toward obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances of the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether it is in conformance with these Standards and Guidelines or not. They are also advised to take notice of the date on which any particular standard or guideline was adopted and to consider other relevant medical and scientific information that becomes available after that date. It would also be prudent for them to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.
Tamoxifen, a widely prescribed drug for the treatment and pre- article describes the clinical significance of variants as well as vention of breast cancer, is metabolized to more potent metabo- important analytical, interpretative, and reporting issues. It is lites by the cytochrome P450 2D6 (CYP2D6) enzyme. Variants in designed to be a guideline for clinical laboratory professionals in the CYP2D6 gene can cause patients to be either intermediate or performing tests and interpreting results with respect to CYP2D6 poor metabolizers, thereby rendering tamoxifen treatment less genetic variants.
effective. Testing for CYP2D6 gene variants is available in Clinical Laboratory Improvement Amendments–certified clinical labora- Genet Med advance online publication 6 September 2012 tories; however, the biological complexity of the variants makes Key Words: CYP2D6; laboratory guidelines; pharmacogenetics;
result interpretation and phenotype prediction challenging. This clinical validity, available platforms, challenges in the interpreta- ACMG Standards and Guidelines Laboratories are beginning to offer pharmacogenetics assays in tion and reporting of complex test results, and the potential for support of continuing efforts toward more personalized medi- clinical utility. Clinical CYP2D6 genotyping can also be ordered ACMG Standards and Guidelines cine. In patients with breast cancer for whom treatment with in the context of other drugs and potential adverse outcomes tamoxifen is being considered, the clinical validity of CYP2D6 (e.g., antidepressants, antipsychotics, and codeine). However, a genotyping is not well established. Nonetheless, the results may detailed description of implementing CYP2D6 genotyping for be helpful in identifying patients who will not respond well to these other medications fal s outside the scope of this article.
the treatment. This article is intended to help clinical laboratory personnel who are offering or intending to offer CYP2D6 geno- mechanism of action and metabolism
typing in patients for whom tamoxifen therapy is being consid- of tamoxifen
ered. The compound tamoxifen belongs to the class of drugs Tamoxifen is one of the most widely used drugs in the treatment known as selective estrogen receptor modulators. Our focus and prevention of estrogen receptor–positive breast cancer. here is on laboratory implementation including analytical and It is a chemical y nonsteroidal agent, the trans-isomer of a Department of Pathology, University of Utah School of Medicine, Molecular Genetics, ARUP Laboratories, Salt Lake City, Utah, USA; 2Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital, Columbus, Ohio, USA; 3Departments of Pathology and Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio, USA; 4Department of Pathology and Laboratory Medicine, Alpert Medical School of Brown University, Women & Infants Hospital, Providence, Rhode Island, USA; 5Department of Molecular Genetics, Quest Diagnostics Nichols Institute, Chantil y, Virginia, USA; 6PGXL Laboratories and Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, Kentucky, USA; 7Division of Molecular Diagnostics, Department of Pathology, Virginia Commonwealth University Medical Center, Richmond, Virginia, USA; 8Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York City, New York, USA. Correspondence: Elaine Lyon ( American College of Medical Genetics and Genomics Submitted 30 July 2012; accepted 30 July 2012; advance online publication 6 September 2012. doi: Genetics in medicine
ACMG STANDARDS AND GUIDELINES
LYON et al Testing for CYP2D6 alleles triphenylethylene derivative. The pharmacology of tamoxifen subfamilies that are designated alphabetically in the order in is thought to be linked to the interaction of the parent drug and which they are described (e.g., *2A, *2B, and *2C). Unlike many its metabolites with the estrogen receptor in both breast and heritable disease mutations, each CYP2D6 allele may include nonbreast tissues. This interaction induces a specific confor- several single-nucleotide polymorphisms — a haplotype, rather mational change in the estrogen receptor, leading to alteration than a single-site mutation. The *1 (wild-type) allele encodes of downstream signaling pathways, and resulting ultimately in a ful y functioning enzyme and is the most commonly occur- transcriptional and posttranscriptional changes in estrogen- ring allele in most populations. Benign allelic variants such as regulated genes, with consequent inhibition of estrogen effects.1 *2 and *35 have normal activity, whereas deleterious variants Although the metabolism of tamoxifen in vivo involves several result in null alleles (no protein expressed or nonfunctional) CYP isoforms, cytochrome P450 (CYP)2D6 predominantly or proteins with altered enzymatic activity. Examples of non- catalyzes the formation of the two most potent metabolites of functional alleles are *5 (gene deletion) and *4 (splice-site vari- tamoxifen: endoxifen and 4-OH tamoxifen ).2 The ant). The *10 and *41 variant alleles have partial activity and are metabolic activity of CYP2D6 is known to be influenced by referred to as decreased-function or reduced-function alleles. the individual's genotype and by environmental factors (e.g., A gene duplication of a functional allele results in increased the ingestion of CYD2D6 inhibitors, which are commonly pre- expression of the active enzyme. Duplication of nonfunctional scribed). Commonly occurring CYP2D6 genetic variants that and reduced-function alleles has also been reported; however, lead to low or absent CYP2D6 activity and/or the direct inhibi- the exact effect of some of these alleles on enzyme activity is tion of CYP2D6 enzyme activity can significantly contribute to difficult to determine.5 A list of variants commonly tested for, interindividual variability in the concentration levels of tamox- with their predicted activities, is shown . It is impor- ifen metabolites.3,4 It has been demonstrated that, despite their tant to note that all CYP2D6 sequence variants in this article are contributory roles, other CYP isoforms appear to be less impor- defined using their common names. They refer to nucleotide tant in tamoxifen metabolism.2 positions, with nucleotide 1 being the A of the ATG-translation initiation codon of GenBank Accession Number M33388 (for more information see The CYP2D6 gene is located at chromosome 22q13.1. No dis- Supplementary Table S1 online describes the common
ease is known to be directly associated with CYP2D6; however, names and the corresponding Human Genome Variant Society when exposed to an exogenous substance, adverse events may nomenclature for the major CYP2D6 alleles.
occur. Alleles for the CYP2D6 gene are defined by combinations of variants found on a chromosome and designated using a star ("*") nomenclature.5 Subsequent variant alleles are numbered Genotypes are used to predict phenotypes of poor metabo- in the order in which they are identified and characterized. lizer (PM), intermediate metabolizer (IM), extensive (nor- Within each "*" allele designation, additional variations linked mal) metabolizer (EM), and ultra-rapid metabolizer (UM). in cis with the defining single-nucleotide polymorphism create Phenotype prediction is dependent on the biallelic expression of alleles: two nonfunctional alleles result in a PM phenotype, whereas two functional alleles, or one functional allele with one decreased-function allele, result in an EM phenotype. IM phenotypes are more difficult to assess, given the lack of con- sensus in this regard. A nonfunctional allele in combination with a decreased-function allele, or two decreased-function Tamoxifen (TAM) CYP2C19, CYP3A)
alleles, have some residual enzyme activity and are considered to be IM phenotypes. A nonfunctional allele in combination with a functional allele may be considered to be an EM phe- notype, consistent with an autosomal recessive inheritance. (CYP2C9 + otherCYP isoforms) However, such genotypes would confer only 50% activity as compared to two copies of functional alleles, and could also be considered as IM phenotypes.6 Another term used for this gen- otype is "heterozygous extensive"7; this nomenclature may fur- ther confuse the issue. The term "heterozygous" simply implies that there are two different alleles present but does not imply any difference in activity, and could therefore be misleading. CYP2D6 gene duplications also result in several possible phe- notype scenarios. Duplication of functional alleles results in figure 1 tamoxifen metabolism. The parent drug tamoxifen (TAM) is
the UM phenotype. However, duplication of nonfunctional converted to the more potent metabolite endoxifen by a complex metabolic route that is ultimately dependent on cytochrome P450 2D6 activity. Source: alleles (e.g., *4) has also been reported. Not all platforms iden- ref. 4. By permission of Oxford University Press.
tify whether a heterozygous duplicated allele is nonfunctional, Genetics in medicine
ACMG STANDARDS AND GUIDELINES
Testing for CYP2D6 alleles LYON et al table 1 Summary of CYP2D6 variants and alleles detected by three commercial platforms
luminex xtag V3
−1584G, 1661G>C, 2850c>t, 4180G>C
−1584G, 1039C>T, 1661G>C, 2850c>t, 4180G>c
100C>T, 1039C>T, 1661G>C, 1846G>a,
100C>T, 1661G>C, 1846G>a, 4180G>C
2850C>T, 4180G>C 1707delt, 1976G>A, 4180G>C
1661G>C, 1758G>t, 2850C>T, 4180G>C
1661G>C, 1758G>t, 2850C>T, 4180G>C
100c>t, 1661G>C, 4180G>C
100c>t, 1039C>T, 1661G>C, 4180G>C
883G>c, 1661G>C, 2850C>T, 4180G>C
883G>c, 1661G>C, 2850C>T, 4180G>C
124G>a, 1661G>C, 2850C>T, 4180G>C
1758G>a, 2850C>T, 4180G>C
1023c>t, 1661G>C, 2850C>T, 4180G>C
1023c>t, 1661G>C, 2850C>T, 4180G>C
1661G>C, 2539-2542delaact, 2850C>T, 4180G>C
1661G>C, 1973insG, 1978C>T, 1979T>C,
2850C>T, 4180G>C 1659G>a, 1661G>C, 2850C>T, 3183G>a,
1659G>a, 1661G>C, 2850C>T, 3183G>a, 4180G>C 1659G>A
−1584C, 31G>a, 1661G>C,
−1584C, 31G>a, 1661G>C, 2850C>T, 4180G>C
2850C>T, 4180G>C 100C>T, 1039C>T, 1661G>C, 4180G>C, gene conversion to CYP2D7 in exon 9
1023c>t, 1661G>C, 1863ins(ttt cGc ccc)2,
2850C>T, 4180G>C 1661G>C, 2850C>T, 2988G>a, 4180G>C
−1584c, 1661G>C, 2850c>t, 4180G>c
Nucleotide changes in bold define the allele.
DF, decreased function; F, functional; IF, increased function; NF, nonfunctional.
and therefore the results of duplication with variants would elements, and genomic variants that alter their functions, are need to be interpreted as "indeterminate" unless familial stud- not yet well understood. The *2 allele is detected by all com- ies are performed for haplotype analysis Also, there mercial platforms and is considered to be functional. The *2A is currently no commercial assay that can detect the number subtype is characterized by the additional promoter variant of duplicated alleles when duplication is present. This too, can −1584G>A, which may affect expression. Increased expression affect the phenotype, because multiduplication (more than two of this allele has been associated with some substrates, but its copies) of a functional allele can lead to ultra-rapid metabo- effect on tamoxifen metabolism is not well understood. Gene lism regardless of the other allele. Laboratories must consider conversion—a mechanism of mutation transfer between the these complexities, the capability of the assay, and the current pseudogene and the gene—may also fail to be analyzed by a state of knowledge, and should provide a clear explanation in given platform. Gene conversions may be difficult to detect and the genotype test report about how a given phenotype predic- this may be a limitation of particular platforms. For example, tion has been arrived at.
the gene-converted *36 nonfunctional allele may mistakenly be There is only limited accuracy in calling an allele normal/ called a *10 decreased-function allele.5 functional on the basis of the nucleotide sequence detected. Critical to phenotype interpretation is a good understand- This is because genotyping is not comprehensive enough to ing of the functional consequences of a given variant as regards identify all currently known sequence variations. Rather, geno- enzyme activity. More specifical y, how does the sequence vari- typing identifies only the variants for which the assay is spe- ant affect the behavior of the enzyme? The National Academy of cifically designed. For example, sequence variants located in Clinical Biochemistry (NACB) Laboratory Medicine Practice the intronic or regulatory regions, which are not ascertained in Guidelines for Pharmacogenetics recommend that pharma- the assay, may also affect gene expression. CYP2D6 regulatory cogenetics assays should include testing for genetic variants Genetics in medicine
ACMG STANDARDS AND GUIDELINES
LYON et al Testing for CYP2D6 alleles allele. For example, the variant 2064G>A (new nomenclature: 1976G>A) has decreased activity toward debrisoquine in vivo but absent activity with bufuralol in vitro.14,15 However, its effect on tamoxifen metabolism remains undetermined.
clinical Validity and clinical utility
Currently, in both premenopausal and postmenopausal patients with invasive breast cancer involving estrogen receptor– positive tumors, long-term therapy with tamoxifen is the standard treat- ment.16 This treatment reduces the recurrence risk by 50%.17 The clinical outcome of interest is recurrence of cancer, but an intermediate end point could be endoxifen levels. Several studies have shown that circulating levels of endoxifen are lower in the presence of fewer functional CYP2D6 alleles.4,18 1846G>A
If lower endoxifen levels are associated with reduced effective- ness of tamoxifen therapy, CYP2D6 genotyping may provide useful information in personalizing cancer treatments. It has figure 2 pedigree showing possible allele patterns for *4 and *10
been suggested that patients with no functional CYP2D6 alleles alleles in parents and child. In the pattern depicted, the child inherits the *4
should be offered alternatives to tamoxifen.19 allele, which is duplicated, from his mother and the *10 allele from his father. Circulating plasma levels of endoxifen are considered The resulting *4/*10 genotype would generate calls for each nucleotide a surrogate outcome for clinical validity, but an analysis of polymorphism as follows: 100C>T (T/T), 1661G>C (C/C), 2850C>T (T/T), and these levels stratified by metabolic phenotype could provide 4180G>C (C/C), and heterozygous for 1846G>A (G/A), plus a duplication.
insight into whether higher levels actual y do translate into improved survival. We extrapolated data from figures and with either (i) a well-defined influence on enzyme function summarized the statistics from two published reports.4,19 In or (ii) a clear relationship between the variant and an observ- both studies, the women were given 20 mg/q.d. of tamoxifen, able influence on drug pharmacokinetics, pharmacodynam- and none were reported as receiving CYP2D6 inhibitors. ics, or toxicology.8 The rationale is that, when there is a clear, For this literature review, PMs were defined in terms of the mechanistic, cause–effect relationship between the variant and presence of two nonfunctional alleles. Although no consen- the end point (e.g., metabolic clearance rate), the phenotype sus exists in this regard, for the purposes of this review we can be predicted with greater reliability. The functional conse- defined IMs as those having two decreased-function al eles, quences of sequence changes are varied. A single variant can one decreased-function allele and one nonfunctional allele, abolish p rotein activity by itself (such as the 1846G>A variant or one nonfunctional al ele and one functional al ele. EMs of *4) or may do so in combination with another single variant. were defined as those having two functional alleles or one Alternatively, a variant such as 100C>T may decrease protein functional allele and one decreased-function allele. Less well- activity without abolishing it, leading to "decreased function" defined or infrequent al ele combinations were omitted from alleles, namely *10. Also, even when a sequence change by itself the analysis (e.g., UMs). The reported mean values and stan- has no effect on the protein, it may be in linkage disequilibrium dard deviations were similar in the two reports, and the indi- with others (as part of a haplotype), thereby affecting protein vidual observations were combined and fitted to a Gaussian activity. The ability to identify the variant that is causal to the distribution after a logarithmic transformation. functional end point is essential to assay design as well as to shows the overlapping distributions of plasma endoxifen assignment of the most appropriate phenotype.
(x-axis) in the PM, IM, and EM groups, both in relative and Other challenges in predicting phenotype from genotype test absolute terms. In the distribution for each of the results involve substrate or drug specificity. Sequence variants three metabolic groups has the same area, so that shape and can change substrate specificity rather than enzymatic activ- position can be directly compared. may be more ity; therefore the metabolism of one drug cannot be assumed relevant to the clinical scenario, in that the prevalence of each to be identical to that of another.9 Some allelic variants (e.g., group is taken into account, with the areas of the distribution CYP2D6*17) are associated with substrate-dependent decreases curves for the PM, IM, and EM groups being in the ratio of in catalytic properties.10–13 Variant proteins may decrease the 1:4:9 ( 7, 29, and 64%, respectively). Of note, many of the incorporation of heme into, or the stability of, the CYP2D6 lower values occurred among patients in the lower tails of the apoprotein (e.g., when conserved elements common to the IM and EM groups, overlapping with the PM group. Possible heme binding site or the planar pocket of CYP are affected).
explanations for this finding are unreported CYP2D6 inhibi- Differences may also exist between in vitro and in vivo mea- tors, other CYP2D6 variants not detected by the assays used, surements. If drugs are also metabolized by other enzymes, in and variants with large effect sizes in other genes involved in vitro experiments may not reflect the real activity of a certain tamoxifen metabolism.
Genetics in medicine
ACMG STANDARDS AND GUIDELINES
Testing for CYP2D6 alleles LYON et al table 2 Detection and false-positive rates, given differ-
ing thresholds of therapeutic endoxifen levels
PM, poor metabolizer.
aProportion of women with "nontherapeutic" circulating endoxifen who Endoxifen plasma concentration (nmol/l) have a CYP2D6-based PM genotype. bProportion of women with "thera-peutic" circulating endoxifen who have a CYP2D6-based PM genotype.
PM as a positive test) along with the false-positive rate (propor- tion of patients with PM genotype having therapeutic levels). Therefore, using 20 nmol/l as the definition of a therapeutic level, identifying the PM genotype by CYP2D6 genotyping has a detection rate of 69%, and a corresponding false-positive rate of between 3 and 4%. That is, 31% of the patients with nontherapeutic levels of endoxifen would not be identified, and 3–4% of those classified as nontherapeutic would actually lie within the therapeutic range. Drug models show that, ulti- mately, only small levels of endoxifen are needed to block all receptor sites,17 implying that the suggested cause–effect rela- tionship between CYP2D6 status and sub-therapeutic endox- ifen concentrations may be only part of the story.
Endoxifen plasma concentration (nmol/l) Several studies have examined the relationship between dis- ease recurrence and CYP2D6 genotype, with widely varying figure 3 a graphic representation of the distribution of plasma
results. Some have shown a strong positive association, whereas endoxifen levels in women treated with tamoxifen, stratified by
the three main cytochrome p450 2d6 metabolic phenotypes. The
others have found none; some have even found a negative asso- data sources and analytic methods are described in the text. (a) The relative
ciation with EMs. In postmenopausal patients taking adjuvant distributions (same area under the curve for each distribution) for the tamoxifen, several studies have shown that impaired CYP2D6 poor (PM), intermediate (IM), and extensive (EM) metabolizer phenotypes. metabolism, caused either by genetic variation12,20–22 or by Although the mean levels are clearly different, there is important overlap, enzyme inhibition,11 is a predictor of worse outcomes in breast especially between the IM and EM phenotypes. (b) The same data, but
incorporating the prevalence of each of the phenotypes into the figure (area
cancer, including statistical y significant shorter time to recur- under the distribution represents the proportion of the population with that rence and poorer relapse-free survival. However, overal survival was not found to be significantly different among the various groups. Some subsequent studies of the association between Currently, there is no established "lower limit" of circulat- CYP2D6 status and breast cancer outcomes in tamoxifen-treated ing endoxifen below which the treatment would be ineffec- patients in preventative12 and metastatic23 settings have sup- tive.can be used to help evaluate whether genotyp- ported the consistency of these data. However, other adjuvant ing might be a useful surrogate for a nontherapeutic value of studies have refuted such data with evidence that tamoxifen- circulating endoxifen; for example, if a hypothetical level of treated women with CYP2D6 variants have similar outcomes24 <20 nmol/l is considered nontherapeutic, CYP2D6 genotyping or even better outcomes13,25 than women with normal CYP2D6 would be a screening test to identify patients who are predicted genotypes. Investigators in the Consortium on Breast Cancer to have nontherapeutic endoxifen levels. The outcome is deter- Pharmacogenetics have recently suggested that these studies, in mined by the endoxifen level (e.g., <20 and ≥20 nmol/l), while addition to being confounded by variations in treatment strate- the screening test is CYP2D6 genotype–inferred metabolic phe- gies and settings, were al smal and predominantly retrospective notype (PM = positive; IM and EM = negative). Using the same in nature, resulting in a low confidence level.26 Given this uncer- data as for shows the detection rate (propor- tainty, the Consortium on Breast Cancer Pharmacogenetics tion of all patients having nontherapeutic levels detected using investigators recommend against routine CYP2D6 genotyping Genetics in medicine
ACMG STANDARDS AND GUIDELINES
LYON et al Testing for CYP2D6 alleles for patients who are candidates for tamoxifen, while recogniz- table 3 Commercial platforms for CYP2D6 genotyping
ing that circumstances may exist where such knowledge could be helpful. They do recommend, however, that patients treated Website (accessed 15 february 2009)
with tamoxifen avoid comedications that are known to inhibit CYP2D6 metabolism.
26 Meanwhile, a recent study in >1,300 Autogenomics Cleared patients showed significant disease-free and event-free survival in patients with early-stage breast cancer treated with tamoxifen, and that those with genotype-predicted EM phenotypes had significantly lower recurrence rates and better disease-free and event-free survival than those who were predicted as being IM FDA, Food and Drug Administration.
or PM phenotypes. As with other studies, overal survival was not significantly different among the groups.27 observed across various ethnic groups. Twenty CYP2D6 alleles It is difficult to establish clear evidence of the clinical validity of (*1(presumed),*2ABD,*3,*4ABDJK,*5,*6ABC,*7,*8,*9,*10AB CYP2D6 genotyping in patients for whom tamoxifen treatment ,*11,*15,*17,*19,*20,*29,*35,*36,*40, and *41), seven CYP2D6 is being considered. Studies designed to settle the issue may or duplications (*1XN,*2XN,*4XN,*10XN,*17XN,*35XN, and may not be feasible. If one overarching study is not possible, *41XN), and three CYP2C19 alleles (*1 (presumed),*2, and *3) several studies may be required to achieve a supportive level of are analyzed and reported. The microarray utilizes approxi- evidence.17 There is potential for CYP2D6 genotyping to have mately 240 unique probes to accurately detect each variant with clinical utility, but the current evidence base is heterogeneous a high level of redundancy (>15,000 different oligonucleotide and based on relatively small sample sizes. The 2009 National probes). The probes are synthesized on a glass substrate to ana- Comprehensive Cancer Network breast cancer guidelines do lyze both sense and antisense strands of amplified target DNA.
not include any recommendations concerning treatment modi- fications based on the outcome of CYP2D6 genotyping.16 description of the assay
Although clinical validity in terms of survival has not been The AmpliChip CYP450 Test amplifies the CYP2D6 and established, the strong association of CYP2D6 genotype CYP2C19 genes in two separate reactions that are subse- with endoxifen levels suggests that genotyping is a practical quently pooled after PCR amplification. The reaction contain- approach to predicting tamoxifen metabolizer phenotypes.4,18 ing CYP450 Primer Mix A uses primers that generate ampli- As commercial products become available, clinical laboratories fied product encompassing the promoter region and coding are implementing CYP2D6 genotyping and offering it as a clini- regions of the CYP2D6 gene, and a CYP2D6 gene duplication- cal test for tamoxifen as well as for other CYP2D6-metabolized specific product, when present, in the specimen or control. The reaction containing CYP450 Primer Mix B uses primers that generate amplified product encompassing exon 4 and exon 5 of the CYP2C19 gene, and a CYP2D6 gene deletion-specific Several commercial CYP2D6 genotyping assay platforms are product, when present, in the specimen or control. The pooled available ). Although all of them test for the presence DNA amplicons from the two independent amplification reac- of most common variants, they differ with respect to the range tions are fragmented by DNAse I and alkaline phosphatase; of variants detected. They also differ in how they call alleles and this is done in order to generate smal , single-stranded DNA in whether or not an allele designation is provided as part of fragments with an average size of 50–200 nucleotides, and to the result. Also, for some combinations of alleles, the classifica- destroy the residual deoxyribonucleotide triphosphate from tion into metabolic phenotypes is not yet standardized. Here we the amplification reactions, respectively. The fragmented describe and compare three commercial platform DNA amplicons are subsequently labeled with biotin at their 3′ termini by terminal transferase, using AmpliChip TdT amplichip cyp450 test—background and variants tested
Labeling Reagent as substrate. The biotin-labeled CYP450 The AmpliChip CYP450 Test (manufactured by Roche target DNA fragments are hybridized to the oligonucleotides Molecular Systems, Branchburg, NJ; distributed by Roche on the AmpliChip CYP450 Microarray, using the Affymetrix Diagnostics, Indianapolis, IN) is an oligonucleotide microarray GeneChip Fluidics Station 450Dx (Santa Clara, CA) and hybridization method of identifying an individual's CYP2D6 an AmpliChip CYP450-specific protocol. The hybridized and CYP2C19 genotypes from genomic DNA extracted from a AmpliChip CYP450 Microarray is washed and stained with whole-blood sample. (Note: CYP2C19 has not been noted to be a streptavidin-conjugated fluorescent dye (phycoerythrin). an important pathway in tamoxifen metabolism, and its use in After staining, the AmpliChip CYP450 Microarray is scanned this context will not be specifically addressed). This assay was by an Affymetrix GeneChip Scanner 3000Dx using a laser that cleared for clinical use by the US Food and Drug Administration excites the fluorescent label that is bound to the hybridized (FDA) at the beginning of 2006.28 CYP450 target DNA fragments. The amount of emitted light is The AmpliChip CYP450 Test detects the most common proportional to the amount of bound target DNA at each loca- and phenotypical y relevant CYP2D6 and CYP2C19 variants tion on the probe microarray.
Genetics in medicine
ACMG STANDARDS AND GUIDELINES
Testing for CYP2D6 alleles LYON et al The next step is an ASPE reaction. Each position to be inter- Data analysis is performed using the Affymetrix GeneChip rogated has two primers associated with it, designed to have the Operating Software (GCOSDx Version 1.1.3; Santa Clara, CA) 3′ base specific to either the wild-type or the variant allele. Each and the AmpliChip CYP450_US Data Analysis Software v2.1, allele-specific primer also has a 5′ tag sequence necessary for or by means of the new operating system, Affymetrix Molecular hybridization to the bead array. Allele-specific primer exten- Diagnostics Software for the Affymetrix GeneChip System sion and subsequent incorporation of biotin-deoxyribose cyto- 3000Dx v.2 and the AmpliChip CYP450 Data Analysis Software sine triphosphate occurs only from a perfectly matched 3′ end v3.1. The presence or absence of the particular polymorphisms of the allele-specific primer. The next step is hybridization of listed in determines the CYP2D6 and CYP2C19 allele the ASPE products to the microspheres. Each bead of the array and predicts the enzymatic activity of the gene product. The is composed of red and infrared fluorophores, and is tagged report generated by the CYP450 Data Analysis Software v2.1 with a universal anti-tag sequence complementary to the allele- for each particular sample includes the genotype call for the specific primer tags. The bead-ASPE hybridization products CYP2D6 and CYP2C19 genes and the predicted phenotypes. are incubated with an R-phycoerythrin-strepavidin conjugate The upgraded version of the software (CYP450 Data Analysis reporter solution, and fluorescence intensities are measured Software v3.1) allows independent reporting of either the using the Luminex xMAP IS system. Two lasers are used to CYP2D6 or CYP2C19 genotypes and phenotypes, or both. The detect the unique mixtures of fluorescent signals that identify AmpliChip CYP450 Test classifies individuals into one of four each microsphere and any phycoerythrin signal that may have CYP2D6 phenotypes: UM, EM, IM, and PM, and two CYP2C19 been produced.29 phenotypes: EM and PM. The software also provides a table showing the interrogated alleles, and a site and call list that data analysis
describes the polymorphism site identified by the nucleotide The xTAG data analysis software provides a wild-type (WT), position and base change as well as the polymorphism cal .
heterozygous (HET), or homozygous variant (MUT) cal at each variant position. The software indicates the variants with which advantages of the platform
the identified alleles are commonly associated; however, the As compared to other methods, the Amplichip CYP450 Test information is not al encompassing, given that it assigns neither allows for multiple simultaneous testing of many alleles, and haplotypes nor predicted phenotypes. It is the user who must potentially reduces technician time by automating the process. assign the genotype on the basis of the software-generated cal s It utilizes 240 probes to detect each polymorphism with wild- at each variant position. Although the assay can detect the pres- type and mutant probe sets, thereby reducing the possibility of ence of a duplication, it cannot detect how many copies of the missing a nontargeted mutation and resulting in greater confi- gene are present, and cannot predict which al ele is duplicated in dence in the *1 allele call. Another advantage is the availability a heterozygous sample. This is of particular importance for phe- of software that analyzes the data using specific algorithms for notype designation in situations wherein the heterozygous al eles genotype information and predicted phenotypes. Finally, the include a nonfunctional or decreased-function allele. For exam- assay can be performed with input DNA levels as low as 25 ng ple, in a patient carrying the CYP2D6*1/*4 genotype with gene for CYP2D6 and 2.5 ng for CYP2C19.
duplication, either the *1 or the *4 allele may be the duplicated allele. If the *1 allele is duplicated (*1XN/*4), the patient would luminex—background and variants tested
have two functional copies of the gene (assuming N = 2) and Luminex is a bead array platform based on allele-specific would thereby be predicted to be an EM phenotype. However, primer extension (ASPE) and hybridization to oligonucleotide- if the *4 is the duplicated allele (*1/*4XN), it would continue bound microspheres. The current version of the assay (Version to remain nonfunctional, and the patient would have only one 3) interrogates 22 variants (including 20 nucleotide positions, functional allele; this would result in an IM-to-EM phenotype.
as well as gene deletion and duplication) corresponding to 17 variant alleles. The variants that can be detected using this array advantages of the platform
are *1 (presumed), *2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *12, *14, The Luminex assay is performed in a 96-well format for PCR, *15, *17, *29, *35, and *41. This assay was cleared for clinical use and the plate is transferred directly to the plate reader. Signal by the FDA in August 2010.29 detection is very rapid, with all 96 samples being completed in 10 min. The platform is well suited for high throughput.
description of the assay
The xTAG CYP2D6 assay (Luminex, Austin, TX) consists of autoGenomics—background and variants tested
two separate multiplex PCR reactions with amplicons >1 kilo- The AutoGenomics (Carlsbad, CA) platform is a film-based base in size. The large amplicons are necessary for avoiding microarray tested on the INFINITI Analyzer.30 The analyzer co-amplification of pseudogenes that could interfere with the includes a confocal microscope with two lasers that detect the assay. After PCR, the products are treated with exonuclease I amount of fluorescently labeled target that is bound to each and shrimp alkaline phosphatase to remove unincorporated probe on the microarray. Two separate CYP2D6 assays are deoxyribonucleotide triphosphate and primers, respectively. currently available from AutoGenomics. The CYP2D6T assay Genetics in medicine
ACMG STANDARDS AND GUIDELINES
LYON et al Testing for CYP2D6 alleles includes detection of the *2, *3, *4, *5, *6, *7,*8, *9, *14, *29, and variant, resulting in the *10 decreased-function allele. Although *41 variants. The CYP2D6I assay includes detection of the *2, 1846G>A can exist on a chromosome without 100C>T (*4M), *3, *4, *5, *6, *7, *8, *9, *10, *12, *14, *17, *29, and *41 variants, this allele is rare. When both variants are seen as heterozy- as well as of duplication alleles.
gotes, the probable genotype is *4 heterozygous (one copy of a nonfunctional allele) given that 1846G>A and 100C>T are description of the assay
presumed to be on the same chromosome. Some instruments DNA is first PCR-amplified with primers specific to the CYP2D6 may call each DNA variant separately, without combining vari- locus, designed to avoid amplification of the pseudogenes. After ant combinations into a haplotype. Without haplotyping, one PCR, the products are treated with exonuclease I/shrimp alka- copy each of 100C>T and 1846G>A could be misinterpreted line phosphatase to remove excess primer. The treated products as being *4/*10 compound heterozygous (one copy of a non- are then loaded onto the INFINITI Analyzer. ASPE allows for functional allele and one copy of a decreased-function allele). labeling and signal amplification, and the fluorescently labeled A presumed *4/*10 combination would appear as two cop- ASPE products are hybridized to the BioFilmChip microarray. ies of 100C>T (homozygous T/T) with only the polymorphic The labeled products bind specifically to their complementary nucleotide T called and not C, and with one copy of 1846G>A microarray locations. The microarray is scanned by the internal (heterozygous GA). In addition, *2 is defined by 2850C>T and confocal microscope 4180G>C, and is associated with an EM phenotype. The defin- ing variant in *41 is 2988G>A with 2850C>T and 4180G>C in the haplotype, and is associated with decreased enzymatic The fluorescence levels detected by the microscope are converted activity. When all three variants are present in the heterozygous into genotype data by the Qmatic software (AutoGenomics, state, the correct star allele genotype is a *41 heterozygote. If Carlsbad, CA). As with similar ASPE-based methods, the 2988G>A is heterozygous G/A, 2850C>T is homozygous T/T, specific allele that is duplicated cannot be determined in a and 4180G>C is homozygous C/C, the correct star allele geno- heterozygous sample. Allelic ratios are determined, and the type is a *2/*41 compound heterozygote. When using platforms software calls homozygous mutation (M), wild-type (W), or that identify a star allele for each nucleotide change, labora- heterozygous (H) for each allele.
tories will need to assign the correct haplotypes for the allele assignments ().
advantages of the platform
Occasional y, combinations of variants are detected that are The platform includes liquid handling, and therefore all post- not consistent with published alleles. In addition, if a dupli- PCR steps are automated. The platform is suited for low-to- cation is detected with a nonfunctional or decreased-function moderate throughput, with a capacity to analyze 24 samples at allele, some of the assays will be unable to determine which of the alleles (the functional allele or the nonfunctional/ decreased-function allele) is duplicated. In both cases, fam- ily studies may help determine the haplotype (chromosome Analyses for multiple variants pose complexities, and there are phasgives an example of a family study and shows several challenging issues with respect to all these platforms. the allelic patterns for a *4/*10 duplication in the proband and Some of the complexities that may affect interpretation are dis- each parent.
As with many genetic tests, not all variants are detected by Although the entire industry uses "haplotype/star nomencla- assays developed (commercial y or by laboratories) for tar- ture" when calling CYP2D6 genotyping results, this standard geted mutation panels. In the case of rare alleles that the assay practice is misleading because haplotyping assays, in contrast is not designed to detect, the software of the assay may default to genotyping assays, are intended to look at a "larger pic- to an allele that is most genetical y similar, or call as "no vari- ture" of haplotypic block segregation. The assays described in ant detected." For example, a platform may not be designed the CYP2D6 genotypes section are genotyping assays, and yet to detect the allele defining variant 2573insC that is present alleles are often defined in terms of the combination of vari- in the CYP2D6*21B allele, predicted to be a loss-of-function ants in the haplotype Although molecular haplo- allele. In specimens containing this rare insertion allele, it typing techniques have been described, they are complex and may be identified as the closely related CYP2D6*2 allele, a rarely used in clinical molecular laboratories. Instead, variants normal-function allele. Another example: the *41 allele has are detected individual y and then, not always correctly, com- been recently described as a decreased-function allele defined bined for a presumed haplotype and allele on the basis of pub- by the 2988G>A variant. Assays that do not detect this variant lished data. For example, the nonfunctional *4 allele is defined will make a call based on the combined presence of other vari- by several variants, including the 100C>T and 1846G>A. The ants in the haplotype, namely, 1584C, 1661G>C, 2850C>T, and 1846G>T variant, characteristic for the *4 allele, causes a splice- 4180G>C, which are also found in *2 subtypes.
site change; this change results in a frameshift that destroys the Although CYP2D6 variants are found in all ethnic groups, activity of the enzyme. The 100C>T variant may also be present some alleles are seen more frequently in specific populations. without the accompanying 1846G>A change. This is a milder For example, *3 is more prevalent in Caucasian populations Genetics in medicine
ACMG STANDARDS AND GUIDELINES
Testing for CYP2D6 alleles LYON et al than in Asian or African populations, whereas *10 is more interprets "no variant allele is detected" as a *1 or normal al ele. prevalent in Asian populationsummarizes allele fre- It may therefore be required that this nomenclature be used quencies of the more common variants in Caucasian, Asian, when seeking FDA clearance for an assay.
and African-American populations.
Although the use of standard nomenclature is ideal, com- mon names (such as the star nomenclature) may be more read- ily recognized. For example, standard nomenclature of the *4 Because the reason for testing is to predict the metabolic phe- allele is based on the presence of the 1846G>A defining vari- notype on the basis of the presence or absence of the nucleotide ant, although other haplotypic variants could also be present. variation, laboratory reports should give the predicted pheno- A *4/*41 genotype, when expressed in standard nomenclature, type while reporting the result, along with details of the alleles may appear as (depending on the platform used): detected. When none of the targeted variants is detected, this is CYP2D6: 100C>T, 1661G>C, 1846G>A, 4180G>C/1661G>C, consistent with the *1 or normal allele. However, because not al 2850C>T, 2988G>A, 4180G>C.
possible variants are included in the testing panel, rare variants Using only allele-defining variants, the simplest form that are present may go undetected. For this reason, the result in standard nomenclature would appear as CYP2D6: is more accurately phrased as "none of the targeted variants 1846G>A/2988G>A.
detected." However, laboratory reports often use the terminol- Because laboratories use different platforms and may detect ogy "Negative" or "*1" to indicate this situation. The Amplichip different variants, the report should include all the variants that platform that was cleared by the FDA for commercial use have been tested for, the methodology, the details of the assay, and its limitations. The NACB's Laboratory Medicine Practice Guidelines8 are in agreement in recommending that the list of table 4 CYP2D6 allele frequencies by race
nucleotide variants tested and detected be included in the report in the event that nomenclature systems change over time. The College of American Pathologists Molecular Pathology labora- tory checklist also requires the reporting of all gene loci tested CYP2D6 reports should have an interpretative component, which may include additional recommendations such as use of alternative therapies, altered dosage, avoidance of known CYP2D6 inhibitors, and other confounding clinical factors, if known. The CAP Molecular Pathology checklist requires that a test report include not only the variants tested for and detected, but also the analytic and clinical interpretation (if appropriate) of the results (MOL.36000). The interpretative report should effectively convey the result and its clinical ramifications to a "nonexpert physician."31 NACB guidelines concur that the information provided in the interpretative report should be "useful for guiding therapeutic management and decision making."8 The inclusion of suggestions for patient-specific dos- ages is not encouraged in reports because laboratories typically do not have relevant information (such as the patient's height and weight and concomitant medications) that can affect dos- ing. However, the reports could indicate whether lower or higher doses, or alternative drugs, may be considered.
The NACB also suggests that, in order to provide optimal interpretative guidance, the report should include, when pos- sible, specific information relating to the medications involved in the clinical situation. This implies that when a laboratory knows that CYP2D6 genotyping has been ordered for tamox- References used, geography covered: Bradford 2002 (worldwide summary),37 ifen therapy, interpretative information specific to tamoxifen Xie 2001 (worldwide summary),38 Zhou 2009 (limited information),39 should be provided. The rationale for this recommendation is Sistonen 2007 (worldwide summary of sometimes "distinct" populations).40a that the accuracy of phenotypic prediction is dependent on the Allele frequency data were grouped into ≥10%, 1-9%, and <1% categories. Not all the alleles were tested in the various studies. bOrder of preference: variants detected as well as on the drug substrate in question. data from the United States, from North America, from Northern Europe, In addition, drug–gene and drug–enzyme interactions can dra- other. cOrder of preference: data from the United States, from North America, matical y alter the phenotype. For example, a patient on tamox- from Asia. dOrder of preference: data from the United States, from North America, from Africa.
ifen therapy who is also prescribed a strong CYP2D6 inhibitor Genetics in medicine
ACMG STANDARDS AND GUIDELINES
LYON et al Testing for CYP2D6 alleles (e.g., paroxetine) may be at increased risk for tamoxifen treat- pharmacogenetics module that includes CYP2D6 is adminis- ment failure because of inability to produce adequate levels of tered by the CAP as part of a joint program with the American endoxifen. Even a patient who would be predicted to be an EM College of Medical Genetics and Genomics (ACMG). The chal- on the basis of genotype could appear as a phenocopy of a PM lenge assesses both analytical and interpretative aspects of test- because of the strong inhibition of CYP2D6 by paroxetine. In ing of three samples, twice a year. Although this challenge is not this example, the patient's genotype would not be the best pre- yet graded, the responses are summarized, along with a discus- dictor of phenotype.
sion of the intended responses to help educate participants.36 A "nonexpert physician" may not ful y appreciate these con- founding issues unless they are included in the genotyping report. Perhaps the most significant limitation to providing Laboratories should understand the challenges involved such patient-specific information, however, is the fact that labo- in establishing the clinical validity of CYP2D6 testing for ratories rarely know what drugs are involved in the clinical sce- patients for whom tamoxifen therapy is being considered. nario, and have limited means of gaining such information. An Laboratories that are planning to offer CYP2D6 testing alternative approach could be to consider inclusion of general- should give adequate consideration regarding the platform ized interpretative components in the reports, mentioning how to be used and the alleles to be tested for. Although a variety these confounding factors could potential y affect the pheno- of platforms are available to fit individual laboratory work- type status of a patient. Examples of CYP2D6 genotype reports flows, each has technical challenges beyond those that are in the recommended format32 are given in Appendix A online.
typical for molecular genetics assays, and it is essential that a laboratory that intends to offer the test must understand these challenges. Interpretation is not straightforward, and Well-characterized reference materials (RMs) are fundamen- laboratories should be familiar with allele nomenclature tal to laboratory quality assurance programs, internal quality and haplotypes to properly interpret the results. The assay assurance activities such as quality control, test development/ must be validated and controlled for with available reference validation, and external assessment by proficiency testing (PT). material, and routinely checked through proficiency testing. The selection of appropriate material is based on the needs of Reports should accurately reflect the genotypes as well as the assay, test methodology, and availability. To improve the the predicted phenotypes so as to help clinicians understand public availability of characterized RMs, the Centers for Disease the clinical ramifications of the results, but always within the Control and Prevention, in partnership with the clinical test- context of the known limitations of the assay.
ing community, has established the Genetic Testing Reference Materials Coordination Program.33 Its goal is to improve the SuPPLEMEnTARy MATERiAL supply of publicly available and well-characterized genomic Supplementary material is linked to the online version of the pa- DNA that can be used as reference materials for PT, quality con- trol test development/validation, and research studies. In con- junction with the Association for Molecular Pathology and the DiSCLoSuRE College of American Pathologists (CAP), the Genetic Testing The following authors direct laboratories that perform clinical test- ing for CYP2D6 al eles: E.L., V.M.P., K.R., and S.A.S. The other Reference Materials Coordination Program coordinated the authors declare no conflict of interest.
characterization of 107 DNA samples for clinically relevant polymorphisms for pharmacogenetic loci including CYP2D6.34 REFEREnCES Other loci that have been characterized include CYP450 (2C9, 1. Lau CK, Subramaniam M, Rasmussen K, Spelsberg TC. Rapid induction of the 2C19, 3A4, and 3A5), VKORC1, UGT1A1, MTHFR, NAT2, c-jun protooncogene in the avian oviduct by the antiestrogen tamoxifen. Proc EPXH1, ABCB1, HLA B, and KIF6. These DNA samples are Natl Acad Sci USA 1991;88:829–833.
2. Desta Z, Ward BA, Soukhova NV, Flockhart DA. Comprehensive evaluation available from the Coriell Cell Repositories (Camden, NJ).
of tamoxifen sequential biotransformation by the human cytochrome P450 Several other types of RMs for CYP2D6 testing are available system in vitro: prominent roles for CYP3A and CYP2D6. J Pharmacol Exp Ther for public use, in addition to the cell lines and genomic DNAs 3. Stearns V, Johnson MD, Rae JM, et al. Active tamoxifen metabolite plasma described here. Currently, there are FDA-cleared products that concentrations after coadministration of tamoxifen and the selective have the following characterized alleles: *1, *2M, *3A, *4A, *5, serotonin reuptake inhibitor paroxetine. J Natl Cancer Inst 2003;95:1758– *6B, *17, *29, *41, and *2AxN.35 There are also Research Use 4. Jin Y, Desta Z, Stearns V, et al. CYP2D6 genotype, antidepressant use, and Only products that have the following characterized alleles: *1 tamoxifen metabolism during adjuvant breast cancer treatment. J Natl Cancer (presumed), *2A, *4, *4A, *5, *6B, *7, *9, *10B, *17, *35, *41, *1XN, and *4XN. The RMs have been validated by bidirectional 5. Ingelman-Sundberg M, Daly AK, Nebert DW (eds). The Human Cytochrome P450 (CYP) Allele Nomenclature Database. 2009. . Accessed 26 October 2010.
6. Kirchheiner J, Nickchen K, Bauer M, et al. Pharmacogenetics of antidepressants and antipsychotics: the contribution of allelic variations to the phenotype of For quality control and assurance purposes, and as part of CLIA drug response. Mol Psychiatry 2004;9:442–473.
7. Eap CB, Lima CA, Macciardi F, Woggon B, Powell K, Baumann P. Steady state certification, laboratories participate in proficiency testing. A concentrations of the enantiomers of mianserin and desmethylmianserin Genetics in medicine
ACMG STANDARDS AND GUIDELINES
Testing for CYP2D6 alleles LYON et al in poor and in homozygous and heterozygous extensive metabolizers of 23. Lim HS, Ju Lee H, Seok Lee K, Sook Lee E, Jang IJ, Ro J. Clinical implications debrisoquine. Ther Drug Monit 1998;20:7–13.
of CYP2D6 genotypes predictive of tamoxifen pharmacokinetics in metastatic 8. Valdes RV, Payne D, Linder MW, et al. Guidelines and Recommendations for breast cancer. J Clin Oncol 2007;25:3837–3845.
Laboratory Analysis and Application of Pharmacogenetics to Clinical Practice: 24. Nowell SA, Ahn J, Rae JM, et al. Association of genetic variation in tamoxifen- DRAFT. National Academy of Clinical Biochemistry Laboratory Medicine metabolizing enzymes with overall survival and recurrence of disease in breast cancer patients. Breast Cancer Res Treat 2005;91:249–258.
25. Wegman P, Vainikka L, Stål O, et al. Genotype of metabolic enzymes and the October 2010.
benefit of tamoxifen in postmenopausal breast cancer patients. Breast Cancer 9. Wang B, Yang LP, Zhang XZ, Huang SQ, Bartlam M, Zhou SF. New insights into the structural characteristics and functional relevance of the human 26. Hayes DF, Stearns V, Rae J, Flockhart D; Consortium on Breast Cancer cytochrome P450 2D6 enzyme. Drug Metab Rev 2009;41:573–643.
Pharmacogenomics. A model citizen? Is tamoxifen more effective than aromatase 10. Goetz MP, Rae JM, Suman VJ, et al. Pharmacogenetics of tamoxifen inhibitors if we pick the right patients? J Natl Cancer Inst 2008;100:610–613.
biotransformation is associated with clinical outcomes of efficacy and hot 27. Schroth W, Goetz MP, Hamann U, et al. Association between CYP2D6 flashes. J Clin Oncol 2005;23:9312–9318.
polymorphisms and outcomes among women with early stage breast cancer 11. Goetz MP, Knox SK, Suman VJ, et al. The impact of cytochrome P450 2D6 treated with tamoxifen. JAMA 2009;302:1429–1436.
metabolism in women receiving adjuvant tamoxifen. Breast Cancer Res Treat 28 AmpliChip CYP450 Test for in vitro diagnostics use [package insert]. Roche Molecular Systems I: Branchburg, NJ, Catalog Number 04591445001-06. 2006.
12. Bonanni B, Macis D, Maisonneuve P, et al. Polymorphism in the CYP2D6 29. Luminex xTAB CYP2D6 kit v.3. http://www.luminexcorp.com/prod/groups/public/ tamoxifen-metabolizing gene influences clinical effect but not hot flashes: documents/lmnxcorp/306-cyp2d6-brochure.pdf. Accessed 21 August 2012.
data from the Italian Tamoxifen Trial. J Clin Oncol 2006;24:3708–9; author 30. Autogenomics. INFINITI CYP450 2D6I Assay application notes, 16 April 2009. . Accessed 28 September 13. Wegman P, Elingarami S, Carstensen J, Stål O, Nordenskjöld B, Wingren S. Genetic variants of CYP3A5, CYP2D6, SULT1A1, UGT2B15 and tamoxifen 31. Molecular Pathology Checklist. College of American response in postmenopausal patients with breast cancer. Breast Cancer Res Accessed 27 October 2010.
14. Daly AK, Leathart JB, London SJ, Idle JR. An inactive cytochrome P450 32. Lubin IM, McGovern MM, Gibson Z, et al. Clinician perspectives about molecular CYP2D6 allele containing a deletion and a base substitution. Hum Genet genetic testing for heritable conditions and development of a clinician-friendly laboratory report. J Mol Diagn 2009;11:162–171.
15. Marez D, Legrand M, Sabbagh N, et al. Polymorphism of the cytochrome P450 33. Centers for Disease Control. Genetic Testing Reference Materials Coordination CYP2D6 gene in a European population: characterization of 48 mutations Program (GeT-RM) – and 53 alleles, their frequencies and evolution. Pharmacogenetics 1997; . Accessed 31 December 2008.
34. Pratt VM, Zehnbauer B, Wilson JA, et al. Characterization of 107 genomic DNA 16. The National Comprehensive Cancer Network reference materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1: Accessed 29 October 2010.
a GeT-RM and Association for Molecular Pathology collaborative project. J Mol 17. Lash TL, Lien EA, Sørensen HT, Hamilton-Dutoit S. Genotype-guided tamoxifen therapy: time to pause for reflection? Lancet Oncol 2009;10:825–833.
35. Decisive Accessed 29 June 18. Borges S, Desta Z, Li L, et al. Quantitative effect of CYP2D6 genotype and inhibitors on tamoxifen metabolism: implication for optimization of breast 36. CAP Reference Resources and Publications, cancer treatment. Clin Pharmacol Ther 2006;80:61–74.
19. Goetz MP, Kamal A, Ames MM. Tamoxifen pharmacogenomics: the role of CYP2D6 as a predictor of drug response. Clin Pharmacol Ther 2008;83:160– Accessed 19 February 2011.
20. Kiyotani K, Mushiroda T, Sasa M, et al. Impact of CYP2D6*10 on recurrence-free 37. Bradford LD. CYP2D6 allele frequency in European Caucasians, Asians, Africans survival in breast cancer patients receiving adjuvant tamoxifen therapy. Cancer and their descendants. Pharmacogenomics 2002;3:229–243.
38. Xie HG, Kim RB, Wood AJ, Stein CM. Molecular basis of ethnic differences 21. Ingle JN, Twito DI, Schaid DJ, et al. Combination hormonal therapy with in drug disposition and response. Annu Rev Pharmacol Toxicol 2001;41: tamoxifen plus fluoxymesterone versus tamoxifen alone in postmenopausal women with metastatic breast cancer. An updated analysis. Cancer 39. Zhou SF. Polymorphism of human cytochrome P450 2D6 and its clinical significance: Part I. Clin Pharmacokinet 2009;48:689–723.
22. Schroth W, Antoniadou L, Fritz P, et al. Breast cancer treatment outcome with 40. Sistonen J, Sajantila A, Lao O, Corander J, Barbujani G, Fuselli S. CYP2D6 adjuvant tamoxifen relative to patient CYP2D6 and CYP2C19 genotypes. J Clin worldwide genetic variation shows high frequency of altered activity variants and no continental structure. Pharmacogenet Genomics 2007;17:93–101.
Genetics in medicine
Rev Esp Sanid Penit 2012; 14: 50-61 F Arnau-Peiró, J García-Guerrero, A Herrero-Matías, JC Castellano-Cervera, EJ Vera-Remartínez, V Jorge-Vidal, S. Arques-Egea, C Iranzo-Tatay Descripción de la consulta psiquiátrica en centros penitenciarios de la Comunidad Valenciana Descripción de la consulta psiquiátrica en centros penitenciarios de la Comunidad Valenciana