The role of pharmacogenomics in personalising the use of tamoxifen in breast cancer

Abstract

Tamoxifen is a well-established endocrine therapy used in the treatment of hormonereceptor positive breast cancer. In recent years, its role in adjuvant and advanced breast cancer settings has been superseded by aromatase inhibitors. The prodrug tamoxifen is metabolised in the liver into several primary and secondary metabolites via cytochrome P450 enzymes. The main active forms are endoxifen and 4-hydroxytamoxifen, with CYP2D6 being a key enzyme in the production of both. There is evidence that tamoxifen-associated hot flushes are an indicator of clinical efficacy of tamoxifen. Co-prescription of CYP2D6 inhibitors, including selective serotonin re-uptake inhibitors (SSRIs) commonly used to treat hot flushes, may decrease the clinical efficacy of tamoxifen and should be used with caution. Moreover, there is evidence that patients with poor CYP2D6-mediated tamoxifen metaboliser phenotype ave worse clinical outcomes, and fewer hot flushes, than those with other phenotypes. Further research is necessary to clarify whether extensive metabolisers have the best clinical outcomes on tamoxifen. Preliminary data suggest that in individuals of certain CYP2D6 phenotype, amoxifen may have equivalent efficacy, and be considered as an alternative to aromatase inhibitors if there were concerns over the side effect profile and cost of the latter. Thus the role of tamoxifen may be redefined in a new era of personalised breast cancer treatment.

Full text

In: Tamoxifen: Properties, Biological Activity and Health Benefits ISBN
Editor: Editors Name, pp. © 2008 Nova Science Publishers, Inc.
Chapter
The role of pharmacogenomics in personalising the use of tamoxifen
in breast cancer
SJ Johnston1, LC Chen2, and KL Cheung1*
1Division of Breast Surgery and 2School of Pharmacy, University of Nottingham, UK
Abstract
Tamoxifen is a well-established endocrine therapy used in the treatment of hormone-
receptor positive breast cancer. In recent years, its role in adjuvant and advanced breast
cancer settings has been superseded by aromatase inhibitors. The prodrug tamoxifen is
metabolised in the liver into several primary and secondary metabolites via cytochrome
P450 enzymes. The main active forms are endoxifen and 4-hydroxytamoxifen, with
CYP2D6 being a key enzyme in the production of both. There is evidence that tamoxifen-
associated hot flushes are an indicator of clinical efficacy of tamoxifen. Co-prescription
of CYP2D6 inhibitors, including selective serotonin re-uptake inhibitors (SSRIs)
commonly used to treat hot flushes, may decrease the clinical efficacy of tamoxifen and
should be used with caution. Moreover, there is evidence that patients with poor
CYP2D6-mediated tamoxifen metaboliser phenotype have worse clinical outcomes, and
fewer hot flushes, than those with other phenotypes. Further research is necessary to
clarify whether extensive metabolisers have the best clinical outcomes on tamoxifen.
Preliminary data suggest that in individuals of certain CYP2D6 phenotype, tamoxifen
may have equivalent efficacy, and be considered as an alternative to aromatase inhibitors
if there were concerns over the side effect profile and cost of the latter. Thus the role of
tamoxifen may be redefined in a new era of personalised breast cancer treatment.
* E-mail address: kl.cheung@nottingham.ac.uk

SJ Johnston, LC Chen and KL Cheung
Introduction
With the introduction of tamoxifen in 19731 it became possible to personalise breast cancer
treatment according to oestrogen receptor (ER) status, as determined by an immunocytological
or histochemical technique. Selection of individual patients according to ER status for 5 years
treatment with tamoxifen following surgery for early breast cancer is made in the light of data
from worldwide randomised trials. Meta-analysis of these data indicates a reduction of around
50% in recurrence and 40% in breast cancer mortality in patients with ER positive tumours, but
no effect in patients with ER negative tumours2. For several decades, tamoxifen has been the first
line endocrine agent for adjuvant treatment and for post-menopausal women with hormone
receptor-positive advanced breast cancer. Tamoxifen is an effective and well-tolerated endocrine
treatment in these settings, but has side effects which are potentially serious, e.g. endometrial
proliferation and deep vein thrombosis, and more common side effects such as hot flushes, which
may impact adversely on quality of life and affect compliance to treatment. Moreover, between
30% and 50% of those taking adjuvant tamoxifen will eventually relapse or die of breast cancer,
which may in part be explained by tumour-dependent ER activation3. In recent years, tamoxifen
has been superseded by the third-generation aromatase inhibitors (AIs), anastrozole4,5,6,
letrozole7,8 and exemestane9, with evidence demonstrating a significant efficacy advantage for
AIs over tamoxifen. However, the magnitude of this benefit is not large: for example, long-term
follow-up of the Arimidex (anastrozole), Tamoxifen, Alone or in Combination (ATAC) trial10
demonstrated an absolute difference in disease-free survival of 4% at 100 months in favour of
anastrozole. The wide inter-individual variation in clinical effectiveness of tamoxifen may be
due to intrinsic host factors including phenotypic variation of tamoxifen metabolism into its
active components. This chapter outlines key developments over the last decade that have
increased knowledge of the metabolism of tamoxifen via cytochrome P450 enzymes and
explored links between pharmacogenetic factors and response to tamoxifen. The implications for
the role of pharmocogenetics in personalising the use of tamoxifen therapy in breast cancer are
then discussed, including the potential to enable clinicians to predict the benefits and risks of
tamoxifen therapy through testing for individual genes, and hence tailor the choice of hormone
treatment to the individual patient.

The role of pharmacogenomics in personalising the use of tamoxifen in breast cancer 3
Tamoxifen metabolism
Irrespective of selection for hormone receptor positivity, there remains wide inter-individual
variation in clinical response to tamoxifen, both in terms of efficacy and in relation to side
effects, which may be caused by variation in the metabolism of the prodrug, tamoxifen, into its
active components.
Tamoxifen is metabolised extensively in the liver into several primary and secondary metabolites
(Figure 1) via cytochrome P450 (CYP) enzymes. Studies on tamoxifen metabolites have
established that 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) are the
main active therapeutic components, being of similar potency, with at least 100-fold greater
affinity to the ER, and 30- to 100-fold greater potency in the suppression of oestrogen-dependent
proliferation of breast cancer cells compared with the prodrug1.
The relative roles of endoxifen and 4-hydroxytamoxifen serum concentrations in determining the
response to tamoxifen therapy are unknown; however, the steady state concentration of
endoxifen in the plasma of patients receiving chronic tamoxifen therapy is five to ten times
higher than that of 4-hydroxytamoxifen 11 suggesting a major role for endoxifen. In-vitro
characterisation of tamoxifen sequential metabolism by CYP isoforms12 demonstrated that
CYP2D6 is the major enzyme involved in the formation of both endoxifen and 4-
hydroxytamoxifen, with a more dominant, rate limiting role in endoxifen formation via 4-
hydroxylation of the primary tamoxifen metabolite, N-desmethyltamoxifen.
Figure 1: Metabolic pathway of tamoxifen. The width of the arrow represents the relative
contribution of each pathway. [Adapted from Goetz et al, J. Clin. Oncol. 2005;23(36)9312-8]
Endoxifen
N-desmethyltamoxifen
CYP2D6
4-hydroxytamoxifen
Tamoxifen
CYP3A
CYP2C9
CYP2C9
CYP2C19
CYP2D6
CYP3A4

SJ Johnston, LC Chen and KL Cheung
CYP2D6 activity and effect on plasma endoxifen levels
Factors affecting CYP2D6 activity and thus tamoxifen metabolism in individuals on tamoxifen
may include genetic polymorphism of the enzyme, and concomitant drug interactions via
competitive inhibition (acting as substrate to the enzyme) and direct inhibition of enzyme
function.
The influence of CYP2D6 enzyme on plasma endoxifen levels during tamoxifen treatment was
demonstrated in three prospective clinical trials. In the first of these, Stearns et al13 measured the
plasma concentration of tamoxifen and its metabolites (N-desmethyl-tamoxifen, 4-
hydroxytamoxifen and endoxifen) in the plasma of 12 women of known CYP2D6 genotype with
breast cancer who were taking adjuvant tamoxifen before and after 4 weeks of coadministered
paroxetine. The trial found that the mean plasma concentration of endoxifen decreased by 56%
(p=0.02), whilst none of the other metabolites underwent statistically significant changes in
concentration. Analysis by CYP2D6 genotype indicated that baseline plasma endoxifen
concentration in those with variant genotype were lower, and that the reduction in endoxifen
concentration was evident primarily in women with wild-type compared to variant genotype
(64% versus 24%, p=0.03). Indeed, there was no statistical reduction in endoxifen concentration
for those with variant genotype.
Although this was a small preliminary study, the results raised the possibility that
pharmacogenetic variation in CYP2D6 activity may influence therapeutic outcomes from
tamoxifen treatment, with potential implications for CYP2D6 variant allele carriers, having
lower plasma endoxifen, and those with wild-type genotype, whose plasma endoxifen
concentrations may be reduced by concomitant enzyme inhibiting drugs.
Further evidence of the importance of CYP2D6 genotype and concomitant use of SSRIs was
subsequently presented in a larger prospective clinical trial14 of 80 patients with newly diagnosed
breast cancer beginning adjuvant tamoxifen therapy. Of these patients, 24 were taking CYP2D6
inhibiting drugs (SSRIs paroxetine (n=10), sertraline (n=4), citalopram (n=4), fluoxetine (n=2)
and the SNRI venlafaxine (n=3)). Patients were genotyped for common CYP2D6 and other
tamoxifen-metabolising enzyme (CYP2C9, CYP3A5 and sulfotransferase 1A1) alleles. The
primary outcome measure was plasma concentration of tamoxifen and its metabolites, after one
and four months of tamoxifen therapy. The study found that those with a CYP2D6 homozygous
wild-type genotype had statistically higher levels of plasma endoxifen after four months of
tamoxifen therapy compared to those with homozygous variant or heterozygous genotypes, and
that for homozygous wild-type genotype, plasma endoxifen concentration was reduced
substantially when co-administered with paroxetine. Of the other CYP2D6 inhibiting drugs,
venlafaxine was the weakest inhibitor and the effect of sertraline was intermediate between
paroxetine and venlafaxine, which is consistent with in vitro data (although data for the two
patients taking fluoxetine was not reported).

The role of pharmacogenomics in personalising the use of tamoxifen in breast cancer 5
The largest of these prospective trials was conducted by Borges et al15, in which 158 breast
cancer patients taking tamoxifen were genotyped for 33 CYP2D6 alleles and had medication
history taken. The outcome measure was plasma concentration of tamoxifen and its metabolites
at the fourth month of tamoxifen treatment. The study used a mixture model approach to define
phenotypic groups according to endoxifen/N-desmethyltamoxifen plasma concentration ratio,
identifying three distinct genotype groups in the distribution of this ratio (Table 1).
Phenotypic group
Mean ratio
endoxifen/NDM
p-value
No functional CYP2D6 allele
0.04
<0.001
1 active CYP2D6 allele
0.08
2 or more active CYP2D6 allele
0.15
Table 1: Mixture model in Borges et al, (Clin. Pharmacol. Ther. 2006;80(1):61-74). “NDM” =
N-desmethyltamoxifen
The study also identified two phenotypic groups by plasma endoxifen concentration, and showed
that those with CYP2D6 extensive metaboliser phenotype taking potent CYP2D6 inhibitors had
significantly lower mean endoxifen plasma concentration than those who were not taking
CYP2D6 inhibitors (23.5 +/- 9.5 nmol/L versus 84.1 +/- 39.4 nmol/L, p<0.001).
Together, these three prospective clinical trials provided evidence that steady-state endoxifen
plasma concentrations during tamoxifen treatment were reduced in women that carry certain
CYP2D6 genetic variants, or who carry wild-type alleles and are co-prescribed CYP2D6
inhibitors, while the concentration of tamoxifen or other metabolites remained unaffected by
CYP2D6 metabolic status. Moreover, the plasma concentrations of endoxifen were on average
ten-fold higher than those of 4-hydroxytamoxifen, with a large degree of interpatient variability:
up to one hundred-fold greater endoxifen exposure relative to 4-hydroxytamoxifen in some
patients13,15,16.
Endoxifen and potential indicators of tamoxifen response
CYP2D6 phenotype
In order to determine whether changes in active metabolite concentration resulting from genetic
polymorphisms affect clinical outcomes of women receiving adjuvant tamoxifen, a prospective
adjuvant tamoxifen trial17 was used to explore the association between variation of tamoxifen

SJ Johnston, LC Chen and KL Cheung
metabolising genes and disease outcomes and toxicity in the form of hot flushes18. The study
determined the genotype of CYP2D6 (based on aforementioned preliminary study findings) and
CYP3A5 (selected because it has been shown to be the principal enzyme catalysing the
elimination of tamoxifen) from 223 patients in the adjuvant tamoxifen trial. Primary outcome
measures were relapse-free (RF) time, disease-free survival (DFS) and overall survival (OS), and
a secondary outcome measure was incidence of hot flushes. At 12 years of follow-up, for
CYP3A5 there was no difference in RF-time, DFS or OS between genotypes, whilst for
CYP2D6, unadjusted data indicated an advantage in RF-time and DFS, but not OS, for those
with CYP2D6*4*4 (homozygous variant) genotype compared to patients with one or no variant
alleles (Figure 2). However, Cox proportional hazards modelling found a significant association
between both nodal status and tumour size with RF-time, DFS and OS. Once adjustment had
been made for these associations, there was no statistically significant difference in hazard ratios
– so it may have been more forthright for the article to display the Kaplan-Meier curves for the
adjusted rather than unadjusted data.
Figure 2: Kaplan-Meier estimates (approximate representation) of (I) relapse-free time; (II)
disease-free survival for unadjusted† data, Wt (wild-type)/*4 or Wt/Wt vs. *4/*4 (adjusted data
hazard ratios not significant: p=0.176 and 0.089 respectively). [Adapted from Goetz et al,
2005;23(36):9312-8]
Similarly for the secondary outcome measure, the incidence of hot flushes did not vary by
CYP3A5 genotype, but there was a non-significant lower incidence of hot flushes in the
homozygous variant CYP2D6 group compared to those with wild-type alleles (Table 2).
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12
Percentage
Years after random assignment
(I)
One or no variant alleles
Homozygous variant
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12
Percentage
Years after random assignment
(II)
p=0.030†
p=0.020†

The role of pharmacogenomics in personalising the use of tamoxifen in breast cancer 7
CYP2D6 genotype
Total
patients
Patients with moderate or severe hot flushes
Number
%
Homozygous variant
13
0
0
Heterozygous wild-type
40
9
23
Homozygous wild-type
137
27
20
Table 2: Incidence of moderate or severe hot flushes by CYP2D6 genotype
Co-prescription of SSRIs
Goetz et al19 then undertook a retrospective review of the medical records of the patients in the
North Central Cancer Treatment Group adjuvant tamoxifen trial17, from which they defined
metaboliser status based on a combination of CYP2D6 genotype and inhibition via co-prescribed
medications (classified as moderate or potent inhibitors). From this information, they classified
patients into extensive metabolisers (homozygous wild-type genotype not co-prescribed a
CYP2D6 inhibitor) or decreased metabolisers, further classified into intermediate or poor
metabolisers based on the presence of one or two CYP2D6 variant (*4) alleles or the co-
prescription of a moderate or potent CYP2D6 inhibitor. Outcome measures were breast cancer
recurrence and death.
By defining the patients in this manner, the study found that those with decreased metabolism
(n=65) had significantly shorter time to recurrence (HR=1.91, p=0.034) and worse relapse-free
survival (HR=1.74, p=0.017) relative to patients with extensive metabolism (n=115), and that
compared to extensive metabolisers, poor metabolisers had the most significant risk of breast
cancer relapse (HR 3.12, p=0.007).
Thus the study concludes that CYP2D6 metabolism as measured by genetic variation and
enzyme inhibition is an independent predictor of breast cancer outcome in post-menopausal
women receiving tamoxifen for early breast cancer – although the “measurement” of CYP2D6
metabolism in this study is based on several layers of classification of retrospective data, with the
effect of moderate and potent enzyme inhibitors (co-prescribed in 13 patients) being somewhat
indeterminate.

SJ Johnston, LC Chen and KL Cheung
Hot flushes
The evidence of increased risk of disease relapse and fewer hot flushes with those with decreased
CYP2D6 mediated metabolism, along with poorer outcome with co-administration of SSRIs in
patients receiving adjuvant tamoxifen therapy, was presented as an “a priori” hypothesis20 at the
Annual Scientific Meeting of the American Society of Clinical Oncology in 2007: that hot
flushes themselves are an indicator of tamoxifen efficacy, and that the absence of hot flushes
may be an indication of poor prognosis.
The evidence for this was taken from the Women’s Healthy Eating and Living (WHEL) study21,
a large randomised trial of the effect of a major increase in vegetable consumption on future
breast cancer events among breast cancer survivors. Between 1995 and 2000, the WHEL study
recruited 3088 women aged 18-70 years, diagnosed (within 2-48 months) with stage I (T1c) to
III breast cancer, and randomly assigned subjects to a WHEL healthy cancer prevention diet or
control arm (National Cancer Institute based diet). Baseline information included dietary intake,
menopause status and anti-oestrogen use, and the study recorded information about cancer
pathology and treatment, along with patient questionnaire data on symptoms.
Retrospective analysis was then performed on the control arm (n=1551) to explore links between
tamoxifen use, hot flushes and breast cancer events. Unadjusted cancer event proportions for hot
flushes were compared to those without. The average time after study entry was 7.3 years, and
there were 864 patients on tamoxifen in the analysis. At 7.3 years of follow-up, the study found
that women with hot flushes had fewer breast cancer specific events (locoregional or
contralateral recurrence, or metastatic disease) than those without hot flushes (12.9% vs. 21%,
p=0.01), and that recurrence-free survival was better for those who reported hot flushes (figure
3).

The role of pharmacogenomics in personalising the use of tamoxifen in breast cancer 9
Figure 3: Kaplan-Meier recurrence-free survival (approximate representation) as a function of
reported hot flashes in patients on tamoxifen (n=864) [Adapted from ASCO Proceedings,
Chicago, 1-5 June 2007]
The study found that hot flushes were more predictive of breast cancer specific outcomes than
age, hormone receptor status or stage I vs. II disease. Thus a relationship between hot flushes and
efficacy of adjuvant tamoxifen was suggested.
In summary, the evidence from trials looking at clinical outcomes according to variously defined
metaboliser phenotypic status has identified CYP2D6 genotype, individually and in conjunction
with SSRI co-prescription, as a potential predictive factor of tamoxifen response. The proposed
mechanism, as suggested by prior clinical trials, is CYP2D6-mediated metabolism of the prodrug
tamoxifen to endoxifen. Furthermore, there is evidence that hot flushes themselves might
indicate an efficacious, endoxifen-mediated response to tamoxifen therapy.
Association between CYP2D6 genetic variation and tamoxifen response
A further study attempted to define the predictive value of genetic variants of CYP enzymes
including CYP2D6 for tamoxifen treatment outcome22 . The study was a non-randomised,
retrospective analysis of 486 ER-positive postmenopausal breast cancer patients, with 206
patients taking adjuvant tamoxifen compared to a control population of 280 patients not taking
tamoxifen, using archival material. Patients receiving adjuvant tamoxifen and concomitant
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8
Percentage
Years after random assignment
Recurrence-free survival
Hot flushes
No hot flushes
p=0.003

SJ Johnston, LC Chen and KL Cheung
chemotherapy or patients with an unclear ER status (n=135) were excluded from the analysis.
Information on co-prescription of SSRIs was incomplete and also excluded from analysis.
Outcome measures were relapse-free time (time from surgery to locoregional recurrence or
contralateral disease), event-free survival (time to relapse or death from any cause) and overall
survival.
At a median follow-up of 71 months, carriers of CYP2D6*4, *5, *10 and *41 alleles, with
phenotypes defined as poor and intermediate metabolisers, had significantly more breast cancer
recurrences, shorter relapse-free times, and worse event-free survival than carriers of functional
alleles (Figure 4). No such association was found for the control population.
Figure 4: Kaplan-Meier probabilities (approximate representation) of relapse-free time (RFT) of
breast cancer patients on adjuvant tamoxifen for CYP2D6-metaboliser phenotypes. [Adapted
from Scroth et al J. Clin. Oncol. 2007;25(33):5187-93]
TAM=patients on tamoxifen, NoTAM= patients not on tamoxifen, EM=extensive metabolisers,
hetEM=heterozygous EM, IM=intermediate metabolisers, PM=poor metabolisers
The authors concluded by suggesting that genetic testing for CYP2D6 status could be used to
refine the choice and / or sequencing of hormonal therapy in prospective clinical trials, and
identified a potential role for pharmacogenetics, with upfront genetic testing of CYP2D6
genotype, to predicting response to tamoxifen.
40
50
60
70
80
90
100
0 20 40 60 80 100 120
%
Relapse-free time
A. TAM (N=206)
EM
PM or IM
hetEM
40
50
60
70
80
90
100
0 20 40 60 80 100 120
%
Relapse-free time
B. NoTAM (N=280)
p=0.04
p=0.37

The role of pharmacogenomics in personalising the use of tamoxifen in breast cancer 11
Implications
Research into pharmacogenetics of tamoxifen therapy has identified CYP2D6 as a key enzyme
in the metabolism of tamoxifen, with a critical role in the formation of endoxifen. Along with 4-
hydroxytamoxifen, endoxifen is a major active metabolite of the prodrug. In women treated with
tamoxifen, plasma endoxifen levels are on average five to ten times higher than those of 4-
hydroxytamoxifen, with wide inter-individual variation, suggesting a dominant active role for
endoxifen determined via genetic variation in CYP2D6. By defining CYP2D6 phenotypes such
as extensive and poor tamoxifen metabolisers based on genotype and the co-prescription of
known CYP2D6 inhibitors such as SSRIs that are commonly used to treat hot flushes, evidence
is presented of an association between pharmacogenetic factors and clinical response to
tamoxifen. This implies that CYP2D6 genotype could be used in upfront genetic testing to help
clinicians weigh up the potential benefits versus the risks of tamoxifen therapy for the individual
patient.
Research has also asked questions about the practice of prescribing SSRIs to treat tamoxifen-
associated hot flushes, and found evidence that SSRIs, which are both competitive and direct
inhibitors of CYP2D6, contribute to decreased tamoxifen metaboliser status with an associated
diminished clinical response to tamoxifen. Alongside evidence that hot flushes are associated
with fewer breast cancer events, the implication is that hot flushes indicate an efficacious clinical
response to tamoxifen, via CYP2D6-mediated metabolism into endoxifen and SSRIs should
therefore be avoided, or at least used with caution, in patients treated with tamoxifen. Given that
hot flushes may be interpreted as a surrogate of adequate endoxifen levels in the body, patients
could be reassured that with the development of hot flushes, they are likely to be deriving
clinical benefit from tamoxifen.
Whilst aromatase inhibitors have replaced tamoxifen as the first line endocrine therapy in the
adjuvant breast cancer setting for postmenopausal women, the absolute DFS advantage is small;
thus the subgroup of patients who are extensive CYP2D6 metabolisers (e.g. homozygous wild
type alleles) may benefit more from tamoxifen therapy. Whether or not pharmacogenomics could
be used to tailor therapy to the individual would require prospective clinical trials to test whether
individual genes such as CYP2D6 can be used to predict outcomes. In the meantime, genetic
testing could be applied on an individual basis, for example if there are concerns over toxicities
of aromatase inhibitors such as osteoporotic risk, in which case tamoxifen may be preferable and
of similar efficacy for individuals with CYP2D6 homozygous wild-type genotype. With
improved knowledge of global and ethnic variation in CYP2D6 genotype, the application of
pharmacogenomics could be targeted to certain groups in order to make more informed decisions
in tailoring endocrine therapy. Thus a new role for tamoxifen may emerge in a new era of
personalised medicine.

SJ Johnston, LC Chen and KL Cheung
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