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Research Highlights Highlights from the latest articles in CYP2D6 activity and breast cancer outcomes during treatment with tamoxifen Tamoxifen is widely used as an antiestrogen for the treatment of estrogen receptor (ER)-positive breast cancer (BC) with proven efficacy to reduce recurrence and mortality. However, approximately 30–50% of patients receiving adjuvant tamoxifen therapy may end up experiencing a relapse of BC and acquired resistance in metastatic disease [1] . Tamoxifen undergoes extensive hepatic metabolism, being broken down into more potent metabolites including 4-hydroxytamoxifen and endoxifen (4-hydroxy-Ndesmethyltamoxifen). While endoxifen and 4-hydroxytamoxifen have a 50-fold higher affinity for the ER than tamoxifen, endoxifen is the most active because its plasma concentrations may be up to ten-times higher than that of 4-hydroxy-tamoxifen [2] . Tamoxifen is a substrate of CYP3A, CYP2C9 and CYP2D6, and an inhibitor of P-gp [3] . CYP2D6 plays an important role in the formation of endoxifen from tamoxifen. The CYP2D6 gene is highly polymorphic with more than 70 variant alleles identified to date [101] . In Caucasians, 5–10% of the population lacks CYP2D6 enzyme activity owing to the inheritance of two CYP2D6 nonfunctional alleles (e.g., CYP2D6*3, *4, *5 and *6), and these individuals are classified as poor metabolizers. Furthermore, subjects with two functional alleles (e.g., *1 and *2) are classified as extensive metabolizers (EMs), whereas those with more than two functional copies of the gene are known as ultrarapid metabolizers, and can exhibit high enzyme activity [4,5] . Several studies have shown an association between carrying one or two null or reduced activity CYP2D6 alleles and a worse BC outcome than in EMs during adjuvant tamoxifen treatment [6] . However, contradictory findings have also been reported [7,8] . The studies discussed here analyze the influence of CYP2D6 genotypes and/or phenotypes in patients with ER-positive BC to predict outcomes. They include: A matched case–control prospective study of the Austrian Breast and Colorectal Cancer Study Group Trial 8 10.2217/PGS.13.42 © 2013 Future Medicine Ltd

(ABCSG8) that determined the relationship between CYP2D6 genotype and outcomes in both women with earlystage ER-positive BC receiving tamoxifen adjuvant therapy for 5 years and women receiving tamoxifen for 2 years followed by 3 years of anastrozole [9] ; A prospective CYP2D6 phenotyping study that used the 13C-dextromethorphan breath test in patients treated with tamoxifen to predict the rate of BC recurrence based on endoxifen serum levels and CYP2D6 genotypes [10] ; A matched cohort study of patients with early-stage ER-positive BC taking adjuvant tamoxifen that evaluated the influence of the CYP2D6 EM phenotype (using dextrometorphan:dextrorphan urinary ratio) on relapse [11] . According to these studies’ results, it seems that CYP2D6 activity determined either through genotypes or phenotypes is associated with outcome among early-stage ER-positive BC patients using tamoxifen [9,11] . However, the studies genotyping for CYP2D6 did not include or consider the determination of gene multiplication [9,11] , and those phenotyping did not find any ultrarapid metabolizer. Therefore, future studies evaluating the influence of being an ultrarapid metabolizer on BC outcomes d uring treatment with tamoxifen are needed. Overall, the difficulties in conducting genetic biomarker studies relevant to cancer therapy appear related to the selection of end points or the definition of them, which are frequently different across studies and results cannot be compared [12] . Additionally, another critical aspect is the use of randomized trials prospectively designed but retrospectively analyzed, which make necessary the development of large confirmatory studies for making decisions regarding the use of therapeutic agents depending on specific biomarkers [13] . Finally, there does seem to exist a need of a rapid and robust pheno typing method to evaluate the CYP2D6 activity during clinical practice such as the 13 C-dextromethorphan breath test [10] .

Pedro Dorado*1, Eva Peñas-Lledó1 & Adrián LLerena1,2 CICAB, Clinical Research Centre, Extremadura University Hospital & Medical School, Badajoz 06080, Spain 2 CIBERSAM, Instituto de Salud Carlos III, Madrid, Spain *Author for correspondence: Tel.: +34 924 218 040 Fax: +34 924 289 675 [email protected] 1

Financial & competing interests disclosure The study has been partly supported by the Institute of Health Carlos III-FIS and the European Union (FEDER) Grants PI10 /02010, PI10 /02758, CP06/00030 (PD), from Gobierno de Extremadura BS10023. It was coordinated in the RIBEF (Red Iberoamericana de Farmacogenética y Farmacogenómica; www.ribef.com) network. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Pharmacogenomics (2013) 14(6), 603–606

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News & Views – Research Highlights including gene multiplication of worldwide potential use. Biotechniques 39(10 Suppl.), S571–S574 (2005). 5

References 1

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Schroth W, Antoniadou L, Fritz P et al. Breast cancer treatment outcome with adjuvant tamoxifen relative to patient CYP2D6 and CYP2C19 genotypes. J. Clin. Oncol. 25(33), 5187–5193 (2007). Coezy E, Borgna JL, Rochefort H. Tamoxifen and metabolites in MCF7 cells: Correlation between binding to estrogen receptor and inhibition of cell growth. Cancer Res. 42(1), 317–323 (1982). Desta Z, Ward BA, Soukhova NV, Flockhart DA. Comprehensive evaluation of tamoxifen sequential biotransformation by the human cytochrome P450 system in vitro: prominent roles for CYP3A and CYP2D6. J. Pharmacol. Exp. Ther. 310(3), 1062–1075 (2004). Dorado P, Cáceres M, Pozo-Guisado E, Wong ML, Licinio J, Llerena A. Development of a PCR-based strategy for CYP2D6 genotyping

LLerena A, Dorado P, Peñas-Lledó EM. Pharmacogenetics of debrisoquine and its use as a marker for CYP2D6 hydroxylation capacity. Pharmacogenomics 10(1), 17–28 (2009).

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Higgins MJ, Stearns V. Pharmacogenetics of endocrine therapy for breast cancer. Annu. Rev. Med. 62, 281–293 (2011).

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Rae JM, Drury S, Hayes DF et al. CYP2D6 and UGT2B7 genotype and risk of recurrence in tamoxifen-treated breast cancer patients. J. Natl Cancer Inst. 104(6), 452–460 (2012).

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Regan MM, Leyland-Jones B, Bouzyk M et al. CYP2D6 genotype and tamoxifen response in postmenopausal women with endocrine-responsive breast cancer: the Breast International Group 1–98 Trial. J. Natl Cancer Inst. 104(6), 441–451 (2012). Goetz MP, Suman VJ, Hoskin TL et al. CYP2D6 metabolism and patient outcome in the Austrian Breast and Colorectal Cancer Study Group Trial (ABCSG) 8. Clin. Cancer Res. 19(2), 500–507 (2013).

10 Opdam FL, Dezentje VO, den Hartigh J et al.

The use of the (13)C-dextromethorphan breath test for phenotyping CYP2D6 in breast cancer patients using tamoxifen: association with CYP2D6 genotype and serum endoxifen levels. Cancer Chemother. Pharmacol. 71(3), 593–601 (2013). 11 Trojan A, Vergopoulos A, Breitenstein U et al.

CYP2D6 phenotype indicative for optimized antiestrogen efficacy associates with outcome in early breast cancer patients. Cancer Chemother. Pharmacol. 71(2), 301–306 (2013). 12 Newman WG, Flockhart D. Breast cancer

pharmacogenomics: where we are going. Pharmacogenomics 13(6), 629–631 (2012). 13 Kelly CM, Pritchard KI. CYP2D6 genotype as

a marker for benefit of adjuvant tamoxifen in postmenopausal women: lessons learned. J. Natl Cancer Inst. 104(6), 427–428 (2012).

Website 101 The Human Cytochrome P450 (CYP) Allele

Nomenclature Committee. www.cypalleles.ki.se/cyp2d6.htm (Accessed 14 February 2013)

CYP2D6 genotypes and disease-free survival among women treated with tamoxifen Evaluation of: Goetz MP, Suman VJ, Hoskin TL et al. CYP2D6 metabolism and patient outcome in the Austrian Breast and Colorectal Cancer Study Group Trial (ABCSG) 8. Clin. Cancer Res. 19(2), 500–507 (2013). The purpose of this study was to elucidate the relationship between CYP2D6 genotype and tamoxifen efficacy, because there is still controversy given the heterogeneity on the reported associations between CYP2D6 metabolism and clinical outcomes [1] . Retrospective data from randomized clinical trials of women treated with tamoxifen monotherapy for early-stage estrogen receptor (ER)-positive breast cancer (BC) have shown an association between CYP2D6 genotype and diseasefree survival [2,3] . However, other recent analyses of a subset of patients enrolled in prospective adjuvant clinical trials did not show this association between CYP2D6 604

genotype and disease-free survival [4,5] . Most interestingly, both studies showed a substantial departure from Hardy–Weinberg equilibrium for the most frequently inactive CYP2D6 allele (*4) [6] . The participants (319 and 560 cases and controls, respectively) were postmenopausal women with ER-positive BC randomized to tamoxifen for 5 years (arm A; 166 cases) or tamoxifen for 2 years followed by anastrozole for 3 years (arm B; 153 cases) included in the prospective, multicenter, randomized, open-label trial ABCSG8. The major result of this study was that CYP2D6 poor metabolism (two null CYP2D6 alleles) was associated with a higher probability of an early disease event in women treated with tamoxifen for 5 years but not in patients treated with sequential tamoxifen followed by anastrozole. This result is in keeping with the hypothesis that CYP2D6 mutation may affect the clinical outcomes of women treated with tamoxifen but not of those treated with anastrozole, as CYP3A Pharmacogenomics (2013) 14(6)

and not CYP2D6 are the major CYP450 isoforms involved in the metabolism of anastrozole [7] . The authors conclude that prospective studies are needed to determine whether altering the dose, the duration or choice of adjuvant hormonal therapy based on CYP2D6 genotype or the pharmacologic monitoring of endoxifen levels will improve the clinical outcomes of postmenopausal women with early-stage ER-positive BC.

References 1

Sideras K, Ingle JN, Ames MM et al. Coprescription of tamoxifen and medications that inhibit CYP2D6. J. Clin. Oncol. 28(16), 2768–2776 (2010).

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Goetz MP, Rae JM, Suman VJ et al. Pharmacogenetics of tamoxifen biotransformation is associated with clinical outcomes of efficacy and hot flashes. J. Clin. Oncol. 23(36), 9312–9318 (2005).

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Schroth W, Goetz MP, Hamann U et al. Association between CYP2D6

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Research Highlights – polymorphisms and outcomes among women with early stage breast cancer treated with tamoxifen. JAMA 302(13), 1429–1436 (2009).

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Brauch HB, Schroth W, Ingle JN, Goetz MP. CYP2D6 and tamoxifen: awaiting the denouement. J. Clin. Oncol. 29(34), 4589–4590 (2011).

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Regan MM, Leyland-Jones B, Bouzyk M et al. CYP2D6 genotype and tamoxifen response in postmenopausal women with

Kamdem LK, Liu Y, Stearns V et al. In vitro and in vivo oxidative metabolism and glucuronidation of anastrozole. Br. J. Clin. Pharmacol. 70(6), 854–869 (2010).

News & Views

endocrine-responsive breast cancer: the Breast International Group 1–98 Trial. J. Natl Cancer Inst. 104(6), 441–451 (2012).

C-dextromethophan breath test for phenotyping in breast cancer patients

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Evaluation of: Opdam FL, Dezentje VO, den Hartigh J et al. The use of the (13)C-dextromethorphan breath test for phenotyping CYP2D6 in breast cancer patients using tamoxifen: association with CYP2D6 genotype and serum endoxifen levels. Cancer Chemother. Pharmacol. 71(3), 593–601 (2013). CYP2D6 and CYP3A play a crucial role in the activation of tamoxifen into this most abundant active metabolite [1] . Dextromethorphan (DM) is a known CYP2D6 and CYP3A phenotyping probe that can be used as a simple and safe drug for monitoring combined CYP3A and CYP2D6 activity [2] . DM is metabolized to dextrorphan through O-demethylation by CYP2D6, to 3-methoxymorphinan through N-demethylation by CYP3A and to 3-hydroxy morphinan through N,O-didemethylation by both enzymes [2] . The most common procedure is to administer the phenotyping probe orally and collect all urine produced over a specified time interval, usually 4–12 h. Pheno type status is determined through the use of urinary metabolite ratios and the antimode is used for phenotype assignment. Using DM as a phenotyping probe, poor metabolizer status is calculated for metabolite ratios >0.3. However, the current protocols involve a urine collection after 4–12 h and are not useful as a rapid routine method. future science group

A 13C-dextromethorphan breath test (DM-BT) has been developed to rapidly and selectively assess CYP2D6 phenotype [3] . The DM-BT is based on the principle that CYP2D6-mediated O-demethylation cleaves a 13CH3 group that enters the body’s one carbon pool to be ultimately eliminated as 13CO2 in expired air. The concentration of 13CO2 and 12CO2 in the exhaled breath samples can be measured by infrared spectrometry. In the study of Leeder et al., DM-BT correlated well with urinary 13C-DM:dextrorphan ratio among 30 healthy subjects, and was proposed as an alternative way to phenotype CYP2D6 activity [3] . The aim of this study was to explore the utility of the DM-BT in clinical practice, to evaluate CYP2D6 phenotype and to predict serum steady-state endoxifen levels in a group of breast cancer patients using tamoxifen. The present study has been the first prospective phenotyping study, in which the DM-BT has been used to correlate CYP2D6 phenotype with endoxifen serum levels in breast cancer patients who used tamoxifen in the adjuvant setting. The main result of the study was that CYP2D6 phenotype determined by the DM-BT explained variation in serum steady-state endoxifen levels by 47.5%. In addition, the endoxifen plasma levels in extensive metabolizers (two CYP2D6 active genes) were significantly higher than in heterozygous extensive metabolizers (one CYP2D6 active gene), intermediate www.futuremedicine.com

metabolizers (two CYP2D6 alleles with decreased activity) and poor metabolizers (zero CYP2D6 active genes), which was similar to previous results [4,5] . The authors claimed that in the studied population there was a wide representation of CYP2D6 phenotypes. In spite of that fact, no patients with an ultrarapid metabolism or individuals with more than two CYP2D6 active genes were found. Therefore, future studies including ultrarapid metabolizers are needed. Furthermore, large clinical studies focus on efficacy and safety of increased tamoxifen dosing as well as a large Phase III clinical trial evaluating the efficacy and safety of orally administered endoxifen should be explored. Finally, the authors asserted that during clinical practice, the CYP2D6 phenotype DM-BT might be more useful than the CYP2D6 genotype, because the usual co-medications could alter the phenotype.

References 1

Desta Z, Ward BA, Soukhova NV, Flockhart DA. Comprehensive evaluation of tamoxifen sequential biotransformation by the human cytochrome P450 system in vitro: prominent roles for CYP3A and CYP2D6. J. Pharmacol. Exp. Ther. 310(3), 1062–1075 (2004).

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Yu A, Haining RL. Comparative contribution to dextromethorphan metabolism by cytochrome P450 isoforms in vitro: can dextromethorphan be used as a dual probe for both CYP2D6 and CYP3A activities? Drug Metab. Dispos. 29(11), 1514–1520 (2001).

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Leeder JS, Pearce RE, Gaedigk A, Modak A, Rosen DI. Evaluation of a [13C]dextromethorphan breath test to assess CYP2D6 phenotype. J. Clin. Pharmacol. 48(9), 1041–1051 (2008).

treatment. J. Natl Cancer Inst. 97(1), 30–39 (2005). 5

Jin Y, Desta Z, Stearns V et al. CYP2D6 genotype, antidepressant use, and tamoxifen metabolism during adjuvant breast cancer

Borges S, Desta Z, Li L et al. Quantitative effect of CYP2D6 genotype and inhibitors on tamoxifen metabolism: implication for optimization of breast cancer treatment. Clin. Pharmacol. Ther. 80(1), 61–74 (2006).

Impact of the CYP2D6 phenotype on the outcome of breast cancer patients treated with tamoxifen Evaluation of: Trojan A, Vergopoulos A, Breitenstein U et al. CYP2D6 phenotype indicative for optimized antiestrogen efficacy associates with outcome in early breast cancer patients. Cancer Chemother. Pharmacol. 71(2), 301–306 (2013). Multiple retrospective clinical studies examining the effect of CYP2D6 pharmacogenetic testing on both pre- and post-menopausal women have reported conflicting results [1–3] . It appears that the utility of CYP2D6 pharmacogenetic testing remains uncertain for patient care, and that genotyping–phenotyping studies or phenotyping studies only could be more helpful. In this study the authors investigated whether disease-free survival (DFS) interval in early-stage estrogen receptor (ER)-positive breast cancer (BC) patients using adjuvant tamoxifen therapy, who were identified as CYP2D6 extensive metabolizers (EMs) upon their dextromethorphan:dextrorphan excretion ratio, would differ when compared with matched controls. The present study is a matched cohort study that determined the impact of an

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optimal EM CYP2D6 phenotype on the outcome of patients with invasive BC receiving a minimum of 12 months of adjuvant tamoxifen therapy as part of their antihormonal treatment. Patients who had a diagnosis of early-stage ER-positive BC were recruited. In addition, a control group of patients without CYP2D6 phenotype assessment was evaluated. Survival ana lysis, including a subset analysis for the pathological degree of ER expression, was designed and performed accordingly for patients who underwent regular 3‑month clinical follow-up. From a total of 70 patients with ER-positive BC who were successfully CYP2D6 phenotyped, 59 (84%) were identified as CYP2D6 EMs, whereas the remaining 11 were identified as poor metabolizers and excluded from further analysis due to inferior dextromethorphan:dextrorphan excretion ratios. Clinical and survival data were also available from a cohort of 148 (not phenotyped) matched controls. The main result was that the DFS of patients who were identified as EMs did not differ significantly from controls. However, when the patients with ER expression ≤20% were excluded from the analysis, DFS was associated with a more favorable outcome. Therefore, the authors suggested the

Pharmacogenomics (2013) 14(6)

existence of a positive association between being an extensive CYP2D6 metabolizer and outcome among early-stage ER-positive BC patients using tamoxifen. However, this suggestion appears very speculative since a significant relationship between CYP2D6 activity and DFS was not found. In addition, this study had several limitations. For example, approximately 50% of the patients had often received aromatase inhibitors sequentially to tamoxifen, which may complicate the interpretation of the results. Other limiting factors of the present study were its retrospective design, a small sample size without a preceding power calculation, a lack of endoxifen levels and a potential selection bias.

References 1

Higgins MJ, Stearns V. Pharmacogenetics of endocrine therapy for breast cancer. Annu. Rev. Med. 62, 281–293 (2011).

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Regan MM, Leyland-Jones B, Bouzyk M et al. CYP2D6 genotype and tamoxifen response in postmenopausal women with endocrine-responsive breast cancer: the Breast International Group 1–98 Trial. J. Natl Cancer Inst. 104(6), 441–451 (2012).

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