Pharmacogenetics of CYP2D6 and tamoxifen therapy: Light at the end of the tunnel?

Accepted Manuscript Title: Pharmacogenetics of CYP2D6 and tamoxifen therapy: light at the end of the tunnel? Author: M.Del Re V. Citi S. Crucitta E. Rofi F. Belcari R.H. van Schaik R. Danesi PII: DOI: Reference:

S1043-6618(16)30221-3 http://dx.doi.org/doi:10.1016/j.phrs.2016.03.025 YPHRS 3112

To appear in:

Pharmacological Research

Received date: Revised date: Accepted date:

24-1-2016 6-3-2016 21-3-2016

Please cite this article as: Re MDel, Citi V, Crucitta S, Rofi E, Belcari F, van Schaik RH, Danesi R.Pharmacogenetics of CYP2D6 and tamoxifen therapy: light at the end of the tunnel?.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2016.03.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pharmacogenetics of CYP2D6 and tamoxifen therapy: light at the end of the tunnel? M. Del Re1, V. Citi1, S. Crucitta1, E. Rofi1, F. Belcari1, R.H. van Schaik2, R. Danesi1 1

Clinical Pharmacology and Pharmacogenetics Unit, Department of Laboratory Medicine,

University Hospital, Pisa, Italy 2

Department of Clinical Chemistry, Erasmus MC, Rotterdam, The Netherlands

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Grafical abstract

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Abstract The clinical usefulness of assessing the enzymatic activity of CYPD6 in patients taking tamoxifen had been longly debated. In favor of preemptive evaluation of phenotypic profile of patients is the strong pharmacologic rationale, being that the formation of endoxifen the major and clinically most important metabolite of tamoxifen, is largely dependent on the activity of CYP2D6. This enzyme is highly polymorphic enzyme for which the activity is largely depending on genetics, but. that can also be inhibited by a number of drugs, i.e. antidepressants, which are frequently used in patients with cancer. Unfortunately, the clinical trials that have been published in that last years are contradicting each other on the association between CYP2D6 and significant clinical endpoints, and for this reason CYP2D6 genotyping is at present not generally recommended. Despite this, the CYP2D6 genotyping test for tamoxifen is available in many laboratories and it may still be an appropriate test to use it in specific cases.

Keywords: pharmacogenetics, breast cancer, CYP2D6, tamoxifen, polymorphisms Introduction Tamoxifen is the most commonly used drug for the treatment of estrogen receptor positive (ER+) breast cancer and is classified as a selective estrogen receptor modulator (SERMs). Nearly 60% of all premenopausal women with breast cancer are diagnosed with a ER+ disease, being candidates for hormonal therapy [1]. Treatment with tamoxifen for at least 5 years is the standard of care, and is associated with an overall positive clinical outcome [2]. Moreover, one-third of early-stage breast cancer patients treated with five-years tamoxifen after surgery relapse within fifteen years, suggesting that benefits from therapy

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is not uniform and the adverse drug reactions ADRs vary considerably between patients [3]. However, substantial interindividual variation exists in steady state levels of tamoxifen and its metabolites following standard dosing. Most of these differences could be linked to tamoxifen’s metabolism. Tamoxifenis a prodrug that is metabolized mainly via cytochrome P450 (CYP) enzymes to a moltitude of active metabolites with variable potencies towards the ER. Understanding the mechanisms of variable response to tamoxifen is one of the major point of interest for clinicians and scientists in order to improve tamoxifen therapy. Also the role of drug-drug interactions that can influence plasma concentrations of endoxifen by inhibition of the cytochrome P450 enzymes, in particular CYP2D6, is a major point of attention. In particular, since the risk of hot flashes is high in women who take tamoxifen, selective serotonin reuptake inhibitors (SSRIs) are frequently prescribed. However, some SSRIs (i.e. paroxetine, fluoxetine) are known to inhibit CYP2D6, thus negatively affecting the metabolic activation of tamoxifen [4]. A good understanding of these interactions, both genetically and environmentally, on CYP2D6 metabolism and their potential impact, are of importance for treating patients effectively with this drug.

Tamoxifen metabolism Tamoxifen is extensively metabolised to more active or inactive products by phase I and II enzymes, including CYP450 enzymes, uridine-5’-diphospho-glucuronosyl-transferases (UGTs), and sulfotransferase 1A1 (SULT1A1). The tamoxifen major primary metabolite, accounting for 90% of tamoxifen oxidation, is N-desmethyltamoxifen, resulting from deemthylation by CYP3A4/5. A small amount of drug is converted to 4-OH-tamoxifen, a minor metabolite but with high anti-estrogenic potential, by CYP2D6. A minority of tamoxifen is inactivated by the UGTs (including UGT1A8, 1A10, 2B7, 2B15, and 2B17

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isoforms) into the glucuronide metabolites and by flavin-containing monooxygenases (FMOs) into the tamoxifen N-oxide. Both N-desmethyl-tamoxifen and 4-OH-tamoxifen are secondarily metabolized to 4-OH-N-desmethyl-tamoxifen (endoxifen), by CYP3A4/5 and CYP2D6, respectively. 4-OH-tamoxifen and endoxifen are important active metabolites, exhibiting similar potency [5]. Both N-desmethyl-tamoxifen and 4-OH-tamoxifen are inactivated by UGT and 4-OH-tamoxifen also by SULT1A1. Endoxifen is than metabolised by CYP3A4/5 to norendoxifen, a potent inhibitor of CYP19A1, which is inactivated by UGTs [6]. Moreover, also CYP1B1, CYP2B6 and CYP2C19 may be the responsible for the conversion of trans-4-OH-tamoxifen to its weakly estrogenic cis-isomer form, a metabolite that could be possibly associated with drug-resistant phenotypes (Fig. 1) [7].

CYP2D6 CYP2D6 accounts for ~2-3% of total liver CYPs and its hepatic protein content varies dramatically among people mainly due to its genetic polymorphisms [8, 9]. CYP2D6 metabolises a large number of drugs (anti-cancer drugs, antidepressants, antiarrhythmics, antipsychotics) in comparison to its relatively minor expression in liver and includes ~15–25% of all clinically used drugs. Although the genetic component has a strong correlation with CYP2D6 enzymatic activity, CYP2D6 can also be inhibited by numerous compounds that bind it with high affinity or that are competing for the enzyme [10]. The CYP2D6 gene is highly polymorphic, with over 105 allelic variants, including single nucleotide polymorphisms (SNPs), gene amplifications, deletions and other structural variants [11], generating absent, non-functional proteins or with decreased activity, or encoding increased protein expression (Table 1). In vitro and in vivo studies largely demonstrated the impact of genetic polymorphisms on CYP2D6 function [12; 13; 14].

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The most frequent inactivating variant among the Caucasian population with an overall frequency of 18%-20% is characterized the splice-site disrupting SNP c.1846G>A, which results in loss of enzyme activity, [12]. The variant CYP2D6*10 is the most common decreased activity allele among the Asian population, with an allele frequency of >50% [15]; the CYP2D6*10 variant gene carries the c.100C>T SNP, that abolishes the PPGP sequence necessary for folding of P450, thus reducing its affinity for substrates [16; 17]. Among the African American population, CYP2D6*17 appears to be the major deficient variant allele, encoding for an altered active site structure, leading to an altered substrate specificity [17; 18]. Some CYP2D6 alleles and their functional activity are reported in Table 1. Based on CYP2D6 genotypes, individuals can be classified into four groups by functional predicted activity classifications: poor metabolizers (PM, two non-functional CYP2D6 alleles), intermediate metabolizers (IM, one functional allele or two reduced function alleles), extensive (/normal) metabolizers (EM, two functional alleles), and ultra-rapid metabolizers (UM, duplication of functional alleles) (Table 1). PMs and IMs have 60% and 74% lower endoxifen concentrations respectively, when compared to women with extensive CYP2D6 metabolism [19]. Due to the lower endoxifen plasma concentrations, PMs possibly benefit less from treatment with tamoxifen. Martinez de Dueñas et al. [2014] evaluated tamoxifen dose adjustment in CYP2D6 PM patients in order to obtain plasma concentrations of endoxifen comparable to patients with extensive CYP2D6 metabolism. CYP2D6 genotype and endoxifen concentration were obained from 249 breast cancer patients in adjuvant treatment with tamoxifen. In PM patients tamoxifen dose was increased to 40 mg and to 60 mg daily for a 4-month period each, and the endoxifen measurements were performed after each dose increase. After the 20 mg tamoxifen standard dose, the baseline endoxifen concentration in EM patients was higher

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(11.30 ng/ml) compared to the PM patients (2.33 ng/ml). The tamoxifen dose increase in PM significantly raised the endoxifen concentration to 8.38 ng/ml and to 9.30 ng/ml, respectively, making it comparable with those observed in EM patients receiving 20 mg of tamoxifen. Moreover, authors say that tamoxifen doses of 40 mg and 60 mg were generally well tolerated, and they did not observe any differences in the incidence of ADRs after increasing the tamoxifen dose [20]. A prospective study of early breast cancer patients using tamoxifen, selected 12 CYP2D6 PM and 12 IM and included them in a one-step tamoxifen dose escalation for 2 months. Endoxifen plasma levels and tamoxifen adverse events were determined at baseline and after 2 months. As a result, in IM (baseline endoxifen: 17.8 nM) dose escalation with 46 mg increased endoxifen to levels comparable with those observed in extensive metabolizers (IM: 30.3 nM vs EM: 33.7 nM). In PM (baseline endoxifen: 8.0 nM) the mean endoxifen level increased from 24 to 81% of the mean concentration in extensive metabolizers, after 90 mg tamoxifen dose escalation (PM: 27.3 nM vs EM: 33.7 nM). Authors concluded that tamoxifen dose escalation significantly increased endoxifen concentrations without increasing side effects, but whether such dose increasing is safe in long-term is uncertain and needs to be explored [21]. Given that the tamoxifen clinical efficacy is affected by a considerable degree on genetic variability in drug-metabolizing enzymes, it has been suggested that the efficacy of tamoxifen therapy depends on an endoxifen-threshold plasma level. Madlensky et al. [2011] identified an endoxifen-treshold suggesting that women in the upper four quintiles of endoxifen had a 26% lower recurrence rate than women in the bottom quintile (lower value treshold: 5.97 ng/mL) [22]. In order to identify patients who are unlikely to reach clinically sufficient endoxifen plasma levels to better personalize the therapy, Hennig et al. [2015] analysed 279 breast cancer patients treated with the standard daily dose of 20 mg

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of tamoxifen. Authors determined CYP2D6 genotypes and their associations with endoxifen-plasma levels, concluding that plasma concentration of five metabolites was significantly correlated with CYP2D6 genotype. But, the mostly unexpected and important result was that in ≈60% of patients, endoxifen plasma levels were below the predefined 5.97 ng/ml level of clinical efficacy, including the 30% of patients with CYP2D6 EM phenotype. Authors explanation about these results is the concomitant use of drugs that can affect CYP2D6 activity, also in EM patients (i.e. SSRIs, expecially paroxetine, strong CYP2D6 inhibitor), concluding that in tamoxifen treated patients the use of strong CYP2D6 inhibitors should be avoided, recommending to perform a direct monitoring of endoxifen plasma concentration to personalize and optimize the treatment [23]. To investigate the role of major CYP2D6 allelic variants, Muroi et al. performed an in vitro functional characterization of 50 CYP2D6 variants. In order to evaluate the enzymatic activities of the CYP2D6 variants they determined the kinetic parameters and intrinsic clearance of Ndesmethyltamoxifen. They generated 50 expression constructs, which were transfected into the COS-7 cell line; unfortunately, the kinetic parameters were determined only for 20 CYP2D6 variants, because for the other 30 the amount of metabolite produced was at or below the detection limit at the lower substrate concentrations, showing no activity or significantly decreased activity with less than 15% activity compared to CYP2D6 wild-type [24]. Finally, also in a recent publication evaluating the effect of tamoxifen dose increment in patients with impaired CYP2D6 activity, authors found that raising the tamoxifen dose to 40 mg significantly increased endoxifen concentrations in IMs and PMs without increasing side effects [25]. In the study, patients were treated with tamoxifen 20 mg once daily for at least 4 weeks and were classified as PM, IM, or EM based on their genotype and considering their comedication. In PM and IM patients, the tamoxifen dose was increased

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to 40 mg daily for 4 weeks. Tamoxifen and its metabolites serum concentrations were measured at baseline and 4 weeks after the dose increment. As expected, the median baseline endoxifen concentration was different between the three phenotype subgroups: PMs had a mean of 4.0 mcg/L endoxifen levels, IMs had 8.3 mcg/L, EMs had 11.4 mcg/L (P=0.040). The increase in tamoxifen dose significantly increased the median endoxifen concentrations in 4 out of 7 PMs from 3.8 to 7.8 mcg/L (P = 0.001) and in 12 out of 16 IMs from 9.5 to 17.4 mcg/L (P<0.001), without increasing side effects [25]. While the association between CYP2D6 phenotype and endoxifen plasma concentrations, or the relationship between active tamoxifen metabolite and the occurrence of adverse events has been well addressed in literature, the potential of the genotyping approach to optimize treatment outcome is still on debate. Saladores et al. [2015] conducted a genotype-pharmacokinetic combined analysis in 587 premenopausal patients and clinical outcome evaluation in 306 patients. Germ-line analysis involved CYP3A5, CYP2C9, CYP2C19 and CYP2D6. Improved distant relapse-free survival (DRFS) was associated with the decreasing of N-desmethyltamoxifen/endoxifen ratio and the increasing of CYP2D6 activity score. Low (<14 nM) endoxifen concentrations compared with high (>35 nM) were associated with shorter DRFS, indicating an association of tamoxifen outcome with endoxifen formation/concentrations [26]. Mwinyi et al. [2014] investigated the impact of polymorphisms CYP2D6, CYP2C19, CYP2C9 and CYP2B6 on the relapse-free time (RFT) in 99 patients who had undergone adjuvant tamoxifen therapy. The genotyping analysis comprised CYP2C9*2 and *3, CYP2C19*2, *3, *4, *6 and *17, CYP2B6*6, *7 and *16, CYP2D6*3, *4, *5, *6, *10 and *41 as well as analysis of CYP2D6 gene duplications/amplications. Authors found no significant associations between CYP2C9, CYP2C19 and CYP2B6 genotypes and RFT, while a trend toward a lower relapse rate was observed for individuals carrying allelic combinations that induce an EM or UM

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genotype of CYP2D6 (p = 0.19), concluding that CYP2D6 genotyping has a good predictive value for its activity-score, but common variants in CYP2C9, CYP2C19, CYP2D6, and CYP2B6 do not have a significant impact on the RFT [27]. Making the story more complicated, in 2005 and 2007 two published studies conducted on 226 and 677 breast cancer patients [28; 29] reported a decreased risk of recurrence and a significantly better disease-free survival in patients carriers of the CYP2D6*4 allele. Even if these findings remain of difficult interpretation, these studies support the effectiveness of tamoxifen in intermediate and poor metabolizers patients. However, there are no other studies confirming that kind of favourable impact of the *4 allele [30; 31; 32]. A recent meta-analysis indicated that overall, there is a significant association between CYP2D6 genotype and decerased efficacy of tamoxifen for post menopausal estrogen positive women on 20 mg/tamoxifen (HR 1.25) (33). In that meta-analyiss, it became also clear that it is very difficult to compare different studies. With all the data currently available, new and better designed studies should be done to fully uncover the potential use of CYP2D6 genotyping (34). (Table 2)

Drug-drug interactions One of the major point of debate is to understand if and how the CYP2D6-inhibitors can influence the tamoxifen outcome. Around 80% of tamoxifen-treated women suffer from hot flashes [35]. Thus, the treatment of that ADRs is the reason for co-prescription of other drugs. Standard treatment for hot flashes has been hormone replacement with estradiol or progestational agents, but, recently, several data suggest that antidepressants inhibiting

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serotonin reuptake and norepinephrine reuptake inhibitor (SNRI) may also be effective [36]. In a double-blind, placebo-controlled, randomised trial the efficacy of venlafaxine was assessed in women assigned to placebo (n=56) or venlafaxine 37.5 mg daily (n=56), 75 mg daily (n=55), or 150 mg daily (n=54). The primary endpoint was to define the average daily hot-flash activity. After week 4 of treatment, median hot flash scores were reduced from baseline by 27%, 37%, 61%, and 61%, respectively in the four groups, confirming that venlafaxine is an effective non-hormonal treatment for hot flashes, though the efficacy must be balanced against the drug's side-effects [37]. Loprinzi et al. [2002] developed a double-blinded, randomized, two-period (4 weeks per period), cross-over trial to study the efficacy of fluoxetine (20 mg/d) for treating hot flashes. The primary end-point was the assesment of hot flashes frequency and hot flash score. Eighty-one randomized women started the protocol therapy and as a result, hot flash scores decreased 50% in the fluoxetine arm versus 36% in the placebo arm, concluding that fluoxetine resulted in an improvement of hot flashes [38]. Also other studies confirmed the efficacy of SSRIs, counting a 50%–65% reduction in the frequency and severity of hot flashes, compared to placebo that was associated with a 27%–38% of reduction [39]. The relevant clinical issue is that, unfortunately, some of the SSRIs often prescribed to alleviate tamoxifen-associated hot flashes, are classified as strong CYP2D6 inhibitors (Table 3), that can affect CYP2D6 activity and endoxifen levels compromising tamoxifen efficacy. On the contrary, a few drugs are known to induce CYP2D6 (i.e., dexamethasone and rifampicin); therefore the ultra metabolizer phenotype is mainly due to genetic factors rather than drug interactions. In a prospective clinical trial, the effects of coadministration of paroxetine on tamoxifen metabolism was tested measuring tamoxifen and its metabolites in plasma of 12 women

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taking adjuvant tamoxifen. CYP2D6 genotype was known and tamoxifen metabolites were measured before and after 4 weeks of coadministered paroxetine. Plasma levels of endoxifen statistically significantly decreased from a mean of 12.4 ng/mL before paroxetine coadministration to 5.5 ng/mL afterward, concluding and suggesting that coadministration of paroxetine decreased the concentration of endoxifen in plasma, and that CYP2D6 genotype and drug interactions should be considered in women treated with tamoxifen [39]. Other studies evaluated the effect of CYP2D6 genotype and concomitant medications on endoxifen plasma concentrations. Borges et al. prospectively enrolled 158 patients with breast cancer who were taking tamoxifen, they performed the genotyping analyses and measured plasma concentrations of tamoxifen and its metabolites at the fourth month of tamoxifen treatment. They found 3 different genotype groups based on the endoxifen/N-desmethyltamoxifen ratio: (1) low ratios composed of patients lacking any functional allele (PM); (2) intermediate ratios represented by patients with 1 active allele (IM); and (3) high ratios composed of patients with 2 or more functional alleles (EM). Interestingly, the mean endoxifen plasma concentration was significantly lower in CYP2D6 extensive metabolizers who were taking potent CYP2D6 inhibitors than in those who were not taking CYP2D6 inhibitors (23.5 +/- 9.5 nmol/L vs 84.1 +/- 39.4 nmol/L, P<0.001), concluding that potent CYP2D6 inhibitors may have an impact on the response to tamoxifen therapy [40]. Jin et al. [2005] publishd in 2005 results obtained from the analysis of the effects of concomitant use of SSRIs in women who take tamoxifen, and the genotypes of genes involved in tamoxifen-metabolism. They analysed data coming from 80 patients who were beginning tamoxifen therapy (20 mg/day) and were genotyped for common alleles of the CYP2D6, CYP2C9, CYP3A5, and SULT1A1 genes. Plasma concentrations of tamoxifen and its metabolites were measured after 1 and 4 months of tamoxifen therapy. Plasma endoxifen concentrations were statistically significantly lower in

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PM and IM (20.0 nM and 43.1 nM, respectively) than in EM (78.0 nM). Twenty-four patients were taking CYP2D6 inhibitors and among them who were EM, the mean plasma endoxifen concentration was 58% lower than that for those who were not assuming CYP2D6 inhibitors (38.6 nM vs 91.4 nM). Moreover, the plasma endoxifen concentration was reduced substantially in subjects who took paroxetine and less reduced in women taking venlafaxine. No effect was counted for CYP2C9, CYP3A5 or SULT1A1 genetic variations on plasma concentrations of tamoxifen or its metabolites [41]. These results suggest us that the switch from strong inhibitors, like paroxetine, to weak inhibitors is strongly supported in patients taking tamoxifen. Binkhorst et collegaues enrolled 10 breast cancer patients who were treated with tamoxifen in combination with strong CYP2D6inhibitors (paroxetine or fluoxetine) for at least 4 weeks. Patients were than switched to treatment with escitalopram or venlafaxine and pharmacokinetic blood sampling was performed over 24 h before and after the switch. Endoxifen plasma concentrations were 3fold higher on escitalopram treatment than during paroxetine or fluoxetine coadministration (median 387 nM vs 99.2 nM) [42]. It seemed that the percentage of co-prescription for strong CYP2D6 inhibitors decreased over time and increased for weak CYP2D6 inhibitors. That is also demonstrated in a Belgian analysis that evaluated the evolution of coprescriptions of tamoxifen and CYP2D6 inhibitors in Belgium including women with at least one tamoxifen registration between January 2006 and December 2009. During the period, the co-prescription of fluoxetine and paroxetine decreased, while the co-prescription of venlafaxine increased over time as well. General practitioners, followed by psychiatrists, internists (including oncologists), and gynaecologists are the major prescribers and, interestingly, gynaecologists and psychiatrists prescribed more venlafaxine and less paroxetine than general practitioners and internists, suggesting the need to disseminate the knowledge of drug-drug interactions to all medical professionals to minimize the risk of

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deleterious drug interactions that may reduce the effectiveness of treatment with tamoxifen [43] (Table 4).

Tamoxifen transport (OATP) and inactivation (UGT) The organic anion-transporting polypeptide 1B1 (OATP1B1), encoded by SLCO1B1 gene, is one of the main hepatic transmembrane transporter protein, and is highly expressed in the sinusoidal membrane of human hepatocytes. It is very well known that OATP1B1 is an highly polymorphic transporter and the c.521T>C (rs4149056) and c.388A>G (rs2306283) polymorphisms are the most frequent [44]. In particular, the OATP1B1 c.521C allele is associated with decreased OATP1B1 transport activities [45]. Wlcek et colleagues in 2008 demonstrated in vitro experiments that OATP1B1 expression can be detected in breast cancer cell lines and normal breast tissue samples and, interestingly, the expression of OATP2B1, OPATP3A1 and OATP4A1 was higher in non-malignant specimens than in tumor tissue samples [46]. Theese findings lead to ask if OATP genes plays a role in drug therapy, however, only few studies are available on the association of OATP1B1 polymorphisms with treatment response and/or patient OS for breast cancer patients treated with tamoxifen. A study published by Muto et al. examined the expression of OATP8/SLCO1B3 in 102 cases of breast carcinoma using immunohistochemistry and correlated the findings with various clinicopathological parameters in order to examine the possible

biological

and

clinical

significance

of

the

transporter.

Founding

that

OATP8/SLCO1B3 immunoreactivity significantly reduced breast cancer recurrence and improved patient’s prognosis [47]. In 2011, Justenhoven et al. studied the potential functions of 31 polymorphisms of OATPs and pregnane X receptor in breast cancer risk, funding no associations [48]. Zhang et al. evaluated the association of CYP2D6*10 (c.100C>T and c.1039C>T), OATP1B1 c.388A>G and c.521T>C polymorphisms on

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paraffin blocks obtained from surgery, with OS in 296 hormone receptor positive breast cancer patients after adjuvant tamoxifen therapy. They found that there was a significant difference in OS between OATP1B1 wild-type and the mutant genotype carrier the C allele, suggesting that the OATP1B1 c.521T>C polymorphism may be an independent prognostic marker for breast cancer patients using tamoxifen therapy [49]. Given that also SULT1A and UGT play a role in the tamoxifen metabolism and transport, in 2013 Fernández-Santander studied the relationsheep between concentrations of tamoxifen metabolites and CYP2D6, CYP3A4, CYP3A5, SULT1A1, SULT1A2 and SULT1E1 genotypes in 135 patients with ER+ breast cancer. As results of the CYP2D6 analysis, EM showed significantly higher endoxifen levels compared to IM or PM patients. Regarding the transporter, while 4-hydroxy-tamoxifen and endoxifen are substrates of the SULT1A2 enzyme, patients carriers of the SULT1A2*2 and SULT1A2*3 alleles had higher plasma levels of both metabolites, suggesting a possible role for SULT1A2 in maintaining optimal plasma concentration for tamoxifen active metabolites [50]. In 2015 Romero-Lorca et al. examined the correlations between the plasma concentrations of tamoxifen's glucuronide metabolites and genotypes of UGT1A4 (p.Pro24Thr), UGT1A4 (p.Leu48Val), UGT2B7

(p.His268Tyr),

UGT2B15

(p.Asp85YTyr),

UGT2B15

(p.Lys523Thr)

and

UGT2B17del in 132 patients with ER+ breast cancer under tamoxifen treatment. They found that patients carrying the UGT1A4_48Val, UGT2B7_268Tyr or with wild type genotypes for UGT2B17_del and UGT2B15_523Lys could be the best candidates for a good response to tamoxifen therapy in terms of eliciting effective plasma active tamoxifen metabolite levels [51].

Available clinical guidelines

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Even if many research efforts have been done in order to clarify if the CYP2D6 genotyping shoud be introduced in clinical practice and whether patients with low activity CYP2D6 genotypes receive less benefit from tamoxifen treatment [52], results of these studies are controversial [53] and CYP2D6 genotyping is not recommended [54]. To date, the FDA says that tamoxifen is a substrate of CYP3A, 2C9 and 2D6, but does not recommend a pharmacogenetic test for CYP2D6 [55]. Apparently, there is a request, presumably also patient driven, for CYP2D6 testing for tamoxifen since this test is offered by a number of laboratories in US and Europe. Also the National Comprehensive Cancer Network (NCCN), because of the limited and conflicting evidences coming from 2 major published studies (BIG 1-98 and ATAC trial) does not recommend CYP2D6 genotype testing to personalize endocrine therapy [56], although these latter studies were later heavily critized for the results produced (o.a. Ratain CPT 2014, Brauch JCO). The Commettee of the American Society of Clinical Oncology (ASCO) encourages caution with concurrent use of CYP2D6 inhibitors because of the drug-drug interactions, but find the available data on CYP2D6 pharmacogenetics insufficient to recommend testing as a tool to determine an adjuvant endocrine strategy [57]. Also the Dutch Pharmacogenetics Working Group suggests using an aromatase inhibitor for PM and IM patients or avoiding the concomitant use of CYP2D6 inhibitors in IM subjects, if the CYP2D6 genotype of the individual is known [58]. The analysis of the existing literature regarding the clinical importance of CYP2D6 genotyping and the outcome of breast cancer is still unclear and contradictory due to the differencies across the studies in terms of data collection, analysis and interpretation. Many aspects that may influence these results should be analysed in the future: the first one is the difference in the genotyping procedure between trials; i.e., in some studies the CYP2D6 genotyping was performed on DNA obtained from paraffinembedded cancer tissue and not on blood-or buccal-derived germinal DNA. It is well

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known that somatic DNA frequently shows “loss of heterozygosity” and can differ from germinal DNA. Also for that reason, results obtained from the ATAC and BIG 1–98 trials beckoned intense criticism because data were obtained from formalin-fixed paraffinembedded tissue, and because they reported a substantial deviation from Hardy Weinberg Equilibrium (HWE), used to signal genotyping errors. Interestingly, the well known chromosomal instability in breast cancer tissue has led to concern about genotyping errors. Moreover, the differencies in CYP2D6 genotyping because of the chromosomal instability, could also lead to a misclassification to a smaller arm of intermediate metabolizers (IM) in favour of extensive metabolizers (EM), thereby confounding research results [59]. In conclusion, there is a significant pharmacologic rationale to support a relevant role of tamoxifen metabolism in drug efficacy, also considering that low endoxifen levels following standard dose tamoxifen in many studies is the only independent predictor factor of therapy failure [60].

Conclusions In summary, the personalization of treatment requires a patient-specific approach that should take into consideration the many factors that ultimately affect drug efficacy, including genetic and non-genetic elements is possible for tamoxifen therapy with respect to CYP2D6. Besides the potential (but still debated) contribution of genotyping, also direct endoxifen measurements or administration of endoxifen instead of tamoxifen are options to cirucmvent this issue. Talking about drug interaction on the CYP2D6 pathway, it is

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obvious that such a complex heterogeneity cannot be addressed in clinical studies. Therefore, the physician should consider the potential impact of multi-pharmacologic treatments on tamoxifen activation, and, at least in cohorts of patients at major risk of subotimal exposure to endoxifen, decide to consult the laboratory specialist on the appropriateness of the CYP2D6 genotyping to make every possible effort to avoid less tnan optimal treatment.

The authors declare no conflict of interest.

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López

JF,

et

al.

Hot

flushes.

Lancet.

2002

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48. Justenhoven C, Schaeffeler E, Winter S, et al. Polymorphisms of the nuclear receptor pregnane X receptor and organic anion transporter polypeptides 1A2, 1B1, 1B3, and 2B1 are not associated with breast cancer risk. Breast Cancer Res Treat. 2011 Jan;125(2):563-9 49. Zhang X, Pu Z, Ge J, et al. Association of CYP2D6*10, OATP1B1 A388G, and OATP1B1 T521C polymorphisms and overall survival of breast cancer patients after tamoxifen therapy. Med Sci Monit. 2015 Feb 21;21:563-9 50. Fernández-Santander A, Gaibar M, Novillo A, et al. Relationship between genotypes Sult1a2 and Cyp2d6 and tamoxifen metabolism in breast cancer patients. PLoS One. 2013 Jul;8(7):e70183 51. Romero-Lorca A, Novillo A, Gaibar M, et al. Impacts of the Glucuronidase Genotypes UGT1A4, UGT2B7, UGT2B15 and UGT2B17 on Tamoxifen Metabolism in Breast Cancer Patients. PLoS One. 2015 Jul;10(7):e0132269 52. 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 2012; 104: 441–51 53. Hertz DL, McLeod HL, Irvin WJ. Tamoxifen and CYP2D6: Acontradiction of data. Oncologist 2012; 17: 620–30 54. Hertz DL, Snavely AC, McLeod HL, et al. In vivo assessment of the metabolic activity of CYP2D6 diplotypes and alleles. Br J Clin Pharmacol. 2015 Nov;80(5):1122-30 55. www.fda.gov 56. http://www.nccn.org 57. Burstein HJ, Griggs JJ, Prestrud AA, Temin S. American society of clinical oncology clinical practice guideline update on adjuvant endocrine therapy for women with hormone receptor-positive breast cancer. J Oncol Pract. 2010 Sep;6(5):243-6 58. Swen JJ, Nijenhuis M, de Boer A, et al. Pharmacogenetics: from bench to byte-an update of guidelines. Clin Pharmacol Ther. 2011 May;89(5):662-73 59. Johnson JA, Hamadeh IS, Langaee TY. Loss of heterozygosity at the CYP2D6 locus in breast cancer: implications for tamoxifen pharmacogenetic studies. J Natl Cancer Inst. 2015 Jan;107(2) 60. Fox P, Balleine R, Lee C, et al. Dose escalation of tamoxifen in patients with low endoxifen level: evidence for therapeutic drug monitoring - The TADE Study. Clin Cancer Res. 2016 Feb. pii: clincanres.1470.2015

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61. Kimura S, Umeno M, Skoda RC, et al. The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene. Am J Hum Genet. 1989 Dec;45(6):889-904 62. Marez D, Legrand M, Sabbagh N, et al. Polymorphism of the cytochrome P450 CYP2D6 gene in a European population: characterization of 48 mutations and 53 alleles, their frequencies and evolution. Pharmacogenetics. 1997 Jun;7(3):193-202 63. Dahl ML, Johansson I, Bertilsson L, et al. Ultrarapid hydroxylation of debrisoquine in a Swedish population. Analysis of the molecular genetic basis. J Pharmacol Exp Ther. 1995 Jul;274(1):516-20 64. Johansson I, Lundqvist E, Bertilsson L, et al. Inherited amplification of an active gene in the cytochrome P450 CYP2D locus as a cause of ultrarapid metabolism of debrisoquine. Proc Natl Acad Sci U S A. 1993 Dec;90(24):11825-9 65. Panserat S, Mura C, Gérard N, et al. DNA haplotype-dependent differences in the amino acid sequence of debrisoquine 4-hydroxylase (CYP2D6): evidence for two major allozymes in extensive metabolisers. Hum Genet. 1994 Oct;94(4):401-6 66. Raimundo S, Fischer J, Eichelbaum M, et al. Elucidation of the genetic basis of the common 'intermediate metabolizer' phenotype for drug oxidation by CYP2D6. Pharmacogenetics. 2000 Oct;10(7):577-81 67. Sakuyama K, Sasaki T, Ujiie S, et al. Functional characterization of 17 CYP2D6 allelic variants (CYP2D6.2, 10, 14A-B, 18, 27, 36, 39, 47-51, 53-55, and 57). Drug Metab Dispos. 2008 Dec;36(12):2460-7 68. Gaedigk A, Bhathena A, Ndjountché L, et al. Identification and characterization of novel sequence variations in the cytochrome P4502D6 (CYP2D6) gene in African Americans. Pharmacogenomics J. 2005;5(3):173-82 69. Gaedigk A, Ndjountché L, Leeder JS, Bradford LD. Limited association of the 2988G>A single nucleotide polymorphism with CYP2D6*41 in black subjects. Clin Pharmacol Ther. 2005 Mar;77(3):228-30 70. Shimada T, Tsumura F, Yamazaki H, et al. Characterization of (+/-)-bufuralol hydroxylation activities in liver microsomes of Japanese and Caucasian subjects genotyped for CYP2D6. Pharmacogenetics. 2001 Mar;11(2):143-56 71. Kagimoto M, Heim M, Kagimoto K, et al. Multiple mutations of the human cytochrome P450IID6 gene (CYP2D6) in poor metabolizers of debrisoquine. Study of the functional significance of individual mutations by expression of chimeric genes. J Biol Chem. 1990 Oct ;265(28):17209-14 72. Yokota H, Tamura S, Furuya H, et al. Evidence for a new variant CYP2D6 allele CYP2D6J in a Japanese population associated with lower in vivo rates of sparteine metabolism. Pharmacogenetics. 1993 Oct;3(5):256-63

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73. Gaedigk A, Blum M, Gaedigk R, et al. Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism. Am J Hum Genet. 1991 May;48(5):943-50 74. Steen VM, Molven A, Aarskog NK, Gulbrandsen AK. Homologous unequal crossover involving a 2.8 kb direct repeat as a mechanism for the generation of allelic variants of human cytochrome P450 CYP2D6 gene. Hum Mol Genet. 1995 Dec;4(12):2251-7 75. Tyndale R, Aoyama T, Broly F, et al. Identification of a new variant CYP2D6 allele lacking the codon encoding Lys-281: possible association with the poor metabolizer phenotype. Pharmacogenetics. 1991 Oct;1(1):26-32 76. Broly F, Meyer UA. Debrisoquine oxidation polymorphism: phenotypic consequences of a 3-base-pair deletion in exon 5 of the CYP2D6 gene. Pharmacogenetics. 1993 Jun;3(3):123-30 77. Gaedigk A, Hernandez J, García-Solaesa V, et al. Detection and characterization of the CYP2D6*9x2 gene duplication in two Spanish populations: resolution of AmpliChip CYP450 test no-calls. Pharmacogenomics. 2011 Nov;12(11):1617-22 78. Rau T, Diepenbruck S, Diepenbruck I, Eschenhagen T. The 2988G>A polymorphism affects splicing of a CYP2D6 minigene. Clin Pharmacol Ther. 2006 Nov;80(5):555-8 79. Zanger UM, Schwab M. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther. 2013 Apr;138(1):103-41 80. Fang H, Liu X, Ramírez J, et al. Establishment of CYP2D6 reference samples by multiple validated genotyping platforms. Pharmacogenomics J. 2014 Dec;14(6):564-72 81. Wang D, Poi MJ, Sun X, et al. Common CYP2D6 polymorphisms affecting alternative splicing and transcription: long-range haplotypes with two regulatory variants modulate CYP2D6 activity. Hum Mol Genet. 2014 Jan;23(1):268-78 82. Bernard S, Neville KA, Nguyen AT, Flockhart DA. Interethnic differences in genetic polymorphisms of CYP2D6 in the U.S. population: clinical implications. Oncologist. 2006 Feb;11(2):126-35 83. Sistonen J, Sajantila A, Lao O. CYP2D6 worldwide genetic variation shows high frequency of altered activity variants and no continental structure. Pharmacogenet Genomics. 2007 Feb;17(2):93-101 84. Neafsey P, Ginsberg G, Hattis D, Sonawane B. Genetic polymorphism in cytochrome P450 2D6 (CYP2D6): Population distribution of CYP2D6 activity. J Toxicol Environ Health B Crit Rev. 2009;12(5-6):334-61

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26

Figure Caption Figure 1 Tamoxifen metabolism

27

Table 1. CYP2D6 alleles and functional activity Mean frequency CYP2D6 allele

Function

CYP2D6* 1A -*2M; CYP2D6* 32 -*35B and CYP2D6* 39

Normal

CYP2D6* 1XN , CYP2D6* 2XN and CYP2D6* 35X2

CYP2D6* 9*10X2; CYP2D6* 14B, CYP2D6* 17, CYP2D6* 29, CYP2D6* 41, CYP2D6* 49, CYP2D6* 50, CYP2D6* 54, CYP2D6* 55, CYP2D6* 59, CYP2D6* 69

References

Increased

Metabolizer phenotypes

Genotype

EM

UM

References

Caucasia ns

Asians

Africans

Two functional alleles

75–85%

69-91%

50-77%

Three or more functional alleles (gene duplication)

1-2%

0%

5-40%

12,13; 61-78

Reduced

79-88

IM

One functional allele or two reduced function alleles

28

10-15%

6-29%

10-15%

CYP2D6* 3A CYP2D6* 4XN; CYP2D6* 5 CYP2D6* 6A - *8; CYP2D6* 11 - *14A and CYP2D6* 15; CYP2D6* 19 *21B; CYP2D6* 38 - *42; CYP2D6* 92, CYP2D6* 100 and CYP2D6* 101

None

PM

Two nonfunctional alleles

29

5-10%

1,2%

0-19%

Table 2. Summary of described studies on CYP2D6 genotyping and endoxifen concentrations in plasma Primary endMaterials and N. pts Results Ref. point methods

Endoxifen plasma concentrations in PM, IM, EM and UM patients

Endoxifen plasma concentrations in PM, IM, EM and UM patients

Disease-free survival

Tamoxifen pharmacokinetics and CYP2D6 enzimatic activity

111, 24, 42

279

1370, 677

In vitro

The increase of tamoxifen dose in CYP2D6 PM raise the endoxifen concentrations levels similar to EM, even if in some case was 20, 21, insufficient to reach those 25 observed in EMs. No increasing of side effects was reported

CYP2D6 genotyping; LC-MS/MS

Upon receiving tamoxifen standard dose, most of breast cancer patients in Poland not achieve a therapeutic level of endoxifen, independently from the genotype

CYP2D6 genotyping; LC-MS/MS

80% of tamoxifen treated PMs patients achieve the minimal threshold at which endoxifen is effective

CYP2D6 genotyping and phenotyping; HPLC PCR and DHPLC

CYP2D6*4 have an increased disease-free survival

Cloning and sitedirected mutagenesis; expression of CYP2D6 variant proteins in COS-7 cells; Western blotting

8 CYP2D6 variants showed <50% of CYP2D6 activity (CYP2D6*2; *9; *26; *28; *32; *43; *45; *70)

30

23

22; 29

24

Distant relapse free-survival

Relapse-free time

Risk of recurrence

587

Recurrencefree survival

CYP2D6 genotyping; LCMS/MS

99

CYP2D6 genotyping and phenotyping; HPLC

226

PCR and Restriction fragment length polymorphism

Time to cancer recurrence

190

Disease-free survival

A reduced endoxifen concentration/formation and a decreased CYP2D6 activity predict shorter DRFS

Despite a trend for longer relapse-free time in UM patients, none of the analysed CYP2D6 variants had significant impact on it

Patients carriers of the CYP2D6*4 and SULT1A1*1 genotype have improved survival

Multivariate analysis demonstrated that patients with a decreased metabolism displayed a CYPD6 genotyping shorter TTBR, RFS, DFA and phenotyping and a reduced OS in comparison with patients with an extensive CYP2D6 metabolism

26

27

28

30

Overall survival

Risk of death

Disease-free survival Overall survival

85

53

The risk of breast cancer mortality is increased in patients carrying the CYPD6 genotyping CYP2D6*4 allele and phenotyping compared to wild type patients

CYP2D6*4 patients have Immunohistochemi decreased disease-free cal analysis; survival and overall CYPD6 genotyping survival respect to and phenotyping CYP2D6*1

31

31

32

32

Table 3. CYP2D6 inhibitors

Strong CYP2D6 inhibitors

Moderate CYP2D6 inhibitors

Weak CYP2D6 inhibitors

Fluoxetine (SSRI)

Sertraline (SSRI)

Buprenorphine (opioid)

Paroxetine (SSRI)

Duloxetine (SSRI)

Amiodarone (antiarrhyth

Bupropione (NDRI)

Terbinafine (antifungal)

Quinidine (antiarrhythmic) Cinacalcet (calcimimetic) Ritonavir (anti-HIV)

33

34

Table 4. Summary of described studies on drug-drug interactions Materials and Primary end-point N. pts Results methods Efficacy and toxicity of venlafaxine for treatment of hot flashes in survivors of breast cancer

Efficacy of fluoxetine for the treatment of hot flashes in breast cancer survivors

Endoxifen plasma concentrations

Ref

191

Randomized controlled trial: placebo vs venlafaxine (37.5 mg / 75 mg/ 150 mg)

Venlafaxine 75 mg/daily was effective against hot flashes

37

81

A double-blinded, randomized, twoperiod (4 weeks per period), cross-over trial: placebo vs fluoxetine (20 mg/die)

Treatment with fluoxetine resulted in modest improvement against hot flashes

38

12, 158, 80, 10

CYP2D6 genotyping; HPLC; UPLC-MS/MS; prospective trial: coadministration of tamoxifen and paroxetine

35

Breast cancer patients treated with tamoxifen in combination with paroxetine displayed a decreased in endoxifen concentrations 39-42 Treatment with escitalopram, a weak CYP2D6 inhibitor, determinated a clinically relevant rises in endoxifen concentrantions