The Influences of Adherence to Tamoxifen and CYP2D6 Pharmacogenetics on Plasma Concentrations of the Active Metabolite (Z)‐Endoxifen in Breast Cancer

Abstract

Tamoxifen efficacy in breast cancer is suspected to depend on adherence and intact drug metabolism. We evaluated the role of adherence behavior and pharmacogenetics on the formation rate of (Z)‐endoxifen. In 192 Brazilian patients we assessed plasma levels of tamoxifen and its metabolites at 3, 6, and 12 months of treatment (LC‐MS/MS), adherence behavior (Morisky medication adherence scale), and CYP2D6 and other pharmacogene polymorphisms (MALDI‐TOF mass spectrometry, real‐time PCR). Adherence explained 47% of the variability of tamoxifen plasma concentrations (P<.001). While CYP2D6 alone explained 26.4%, the combination with adherence explained 40% of (Z)‐endoxifen variability at 12 months (P<.001). The influence of low adherence to not achieving relevant (Z)‐endoxifen levels was highest in patients with non‐compromised CYP2D6 function (RR 3.65, 95% CI: 1.48‐8.99). As a proof‐of‐concept we demonstrated that (Z)‐endoxifen levels are influenced both by patient adherence to tamoxifen and CYP2D6 which is particularly relevant for patients with full CYP2D6 function.

Full text

Citation: Clin Transl Sci (2020) 13, 284–292; doi:10.1111/cts.12707
ARTICLE
The Influences of Adherence to Tamoxifen and CYP2D6
Pharmacogenetics on Plasma Concentrations of the
Active Metabolite (Z)-Endoxifen in Breast Cancer
Jeanine Marie Nardin1,2,3 , Werner Schroth4,5, Thais Abreu Almeida6, Thomas Mürdter4,5, Solane Picolotto7, Evelyn Castillo Lima
Vendramini7, Reiner Hoppe4,5, Jenifer Primon Kogin2, Diandra Miqueleto2, Silvia Dark Robaskievicz de Moraes2,
Matthias Schwab4,8,9,10, Roberto Flavio Pecoits-Filho3, Hiltrud Brauch4,5,9,10,† and José Claudio Casali-da-Rocha3,11,*,†
Tamoxifen efficacy in breast cancer is suspected to depend on adherence and intact drug metabolism. We evaluated the role
of adherence behavior and pharmacogenetics on the formation rate of (Z)-endoxifen. In 192 Brazilian patients, we assessed
plasma levels of tamoxifen and its metabolites at 3, 6, and 12months of treatment (liquid-chromatography tandem mass
spectrometry), adherence behavior (Morisky, Green, and Levine medication adherence scale), and cytochrome P450 2D6
(CYP2D6) and other pharmacogene polymorphisms (matrix-assisted laser-desorption-ionization time of flight) mass spec-
trometry, real-time polymerase chain reaction). Adherence explained 47% of the variability of tamoxifen plasma concentra-
tions (P<0.001). Although CYP2D6 alone explained 26.4%, the combination with adherence explained 40% of (Z)-endoxifen
variability at 12months (P<0.001). The influence of low adherence to not achieving relevant (Z)-endoxifen levels was highest
in patients with noncompromised CYP2D6 function (relative risk 3.65; 95% confidence interval 1.48–8.99). As a proof-of-
concept, we demonstrated that (Z)-endoxifen levels are influenced both by patient adherence to tamoxifen and CYP2D6,
which is particularly relevant for patients with full CYP2D6 function.
One-third of patients with early breast cancer with estrogen
receptor (ER)-positive tumors treated with the selective es-
trogen receptor modulator tamoxifen either relapse or die
from the disease in the following decade.1,2 Improvement
of tamoxifen efficacy requires a better knowledge of
factors determining outcome of which the medication-
taking behavior (i.e., adherence), is a strong suspect.3,4
Discontinuation and nonadherence to tamoxifen are fre-
quent and result in increased mortality.5–9 A large study
using automated pharmacy records showed that 31% of
patients discontinued therapy, and among those who con-
tinued, only 70% adhered at 4.5years with only 50% being
1Clinical Research Department, Erasto Gaertner Hospital, Curitiba, Brazil; 2School of Health Science, UniBrasil, Curitiba, Brazil; 3Pontifical Catholic University of
Parana, Curitiba, Brazil; 4Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany; 5Universit y of Tuebingen, Tuebingen, Germany;
6Clinical Oncology Department,Erasto Gaertner Hospital, Curitiba, Brazil; 7Pharmacy Department,Erasto Gaertner Hospital, Curitiba, Brazil; 8Departments of Clinical
Pharmacology, Pharmacy and Biochemistry,University of Tuebingen, Tuebingen, Germany; 9German Cancer Consortium (DK TK),German Cancer Research Center
(DKF Z), Heidelberg, Germany; 10iFIT Cluster of Excellence,University of Tuebingen, Tuebingen, Germany; 11Department of Oncogenetics,Erasto Gaertner Hospital,
Curitiba, Brazil. *Correspondence: José Claudio Casali-da-Rocha (casalidarocha@gmail.com)
Received: August 19, 2019; accepted: August 20, 2019. doi:10.1111/c ts.12 707
†These authors contributed equally to this work.
Study Highlights
WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?
✔ Poor patient adherence to tamoxifen is associated with
reduced clinical efficacy. cytochrome P450 2D6 (CYP2D6)
is a key factor of tamoxifen metabolism, however, genetic
variants only partially explain the variability of plasma
concentrations of the active metabolite (Z)-endoxifen.
WHAT QUESTION DID THIS STUDY ADDRESS?
✔ We studied the influence of patient adherence behav-
ior and CYP2D6 phenotype (and other pharmacogenes)
on plasma metabolite concentrations. We investigated
whether (Z)-endoxifen concentrations depended on either
factor alone or in combination.
WHAT DOES THIS STUDY ADD TO OUR KNOW-
LEDGE?
✔ Adherence and CYP2D6 status are independent de-
terminants of tamoxifen and (Z)-endoxifen plasma levels.
Their combined influence is particularly relevant for pa-
tients with full CYP2D6 function.
HOW MIGHT THIS CHANGE CLINICAL PHARMA-
COLOGY OR TRANSLATIONAL SCIENCE?
✔ The dual monitoring of tamoxifen (surrogate for adher-
ence) and (Z)-endoxifen (surrogate for clinical response)
plasma levels could be a strategy to improve tamoxifen
outcome in the future.

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Nardin et al.
fully adherent.10 Nonadherence gradually evolves over time
from 10% in the first year to > 50% in the fifth year6 , 8,10 ,11
being most pronounced in young women being below
40years of age and women older than 75years.10 Ten-year
survival rates significantly differed between patients who
continued treatment (82%) compared with patients who did
not adhere (78%).11 Recently, the BIG I-98 trial reported a
considerably reduced disease-free survival (hazard ratio
1.45; 95% confidence interval (CI) 1.09–1.93) for patients
who ceased protocol-assigned endocrine treatment.12
To assess drug adherence, commonly used prescrip-
tion-refill patterns infer that the medication is taken exactly
as prescribed, yet, partial adherence or actual use of the
medication cannot be controlled.13 In contrast, an objective
surrogate of tamoxifen adherence is the monitoring of drug
concentrations, including the parent drug and its active me-
tabolite (Z)-endoxifen. (Z)-endoxifen can be easily measured
in the patients’ plasma, however, individual concentrations
depend on pharmacogene polymorphisms, particularly
those of the liver enzyme cytochrome P450 2D6 (CYP2D6).14
Polymorphisms are responsible for different phenotypes,
which aside from individuals with normal CYP2D6 func-
tion (extensive metabolizer (EM)), comprise individuals with
absent (poor metabolizer (PM)), reduced (intermediate me-
tabolizer (IM)), and increased (ultrarapid metabolizer (UM))
CYP2D6 activity. Among Europeans, the frequency of PM
and IM individuals is 9% and 40%, respectively. In Brazil,
the observed PM frequency is 2.5% with an increased 4%
frequency in patients with ER-positive breast cancer.15,16
For patients receiving tamoxifen therapy, CYP2D6 ge-
notyping allows the prediction of (Z)-endoxifen plasma
concentrations.17–19 A putative threshold of 5.9ng/mL has
been proposed in the Women’s Healthy Eating and Living
(WHEL) study suggesting that a minimal concentration
threshold is required above which (Z)-endoxifen is more ef-
fective against the recurrence of breast cancer and below
which patients are at higher risk for recurrence,20 likely due
to incomplete inhibition of ER-dependent growth signal-
ing.21 Although several outcome studies demonstrated the
importance of CYP2D6 genotyping for the prediction of the
risk-to-relapse in adjuvant and metastatic settings,22–25
other studies did not confirm this association,26–29 the rea-
son why current clinical guidelines do not support the use
of CYP2D6 genotypes for predicting tamoxifen response.30
Therefore, it is important to identify confounders that may
mask the tamoxifen CYP2D6 association, with tamoxi-
fen adherence being a prime candidate.13 The combined
analysis of adherence behavior and tamoxifen metabolism
may shed new light on this important issue, particularly
because first evidence has been reported of an increased
effect of CYP2D6 genotype on patient outcome when
adjusted for adherence to tamoxifen therapy.31 Here, we
present a prospective study of mainly patients with early
breast cancer from Brazil, in which interview-informed
tamoxifen adherence together with CYP2D6 metabolizer
status have been measured in order to evaluate their com-
bined contribution to the lowering of the patients’ plasma
(Z)-endoxifen concentrations to potentially subtherapeutic
levels. The study demonstrates how patients with breast
cancer of underserved patient populations can contribute
valuable pharmacokinetic and pharmacogenetic informa-
tion in the field of breast cancer biomarker research.
MATERIALS AND METHODS
Patients and study design
Patients were consecutively recruited at the Erasto
Gaertner Hospital, Curitiba, Southern Brazil, a national ref-
erence center for oncology treatment. Between April 2014
and June 2017, 192 patients with ER-positive breast cancer
treated with tamoxifen were included to investigate the rel-
evance of drug adherence on the plasma levels of active
tamoxifen metabolites. According to recommendations for
tumor marker studies (REMARK),32 inclusion criteria were
defined as women aged 18years or older who were diag-
nosed with any stage, histologic, or molecular subtype of
ER-positive breast cancer, and who started daily treatment
with 20 mg tamoxifen for an intended 5 years of therapy.
Exclusion criteria were age beyond 82years and patients
unable to complete the study schedule and questionnaire.
Patients were followed during the first year of treatment
at months 3, 6, and 12 for the assessment of adherence
to tamoxifen intake based on interview, measurement of
plasma levels of tamoxifen and its metabolites, and as-
sessment of CYP2D6 metabolizer status (and other relevant
drug metabolizing enzymes (DMEs)) based on genotypes.
Ethical approval was obtained from the Brazilian National
Commission of Ethical Research. All patients provided writ-
ten informed consent. Study size calculation (99.9% power)
revealed a minimum of 42 patients required to detect an
association of (Z)-endoxifen variability with CYP2D6 poly-
morphism, based on the prevalence of IM and PM patients
of 40% in Brazil33 and an expected effect size of R2=0.4.17
Assessment of adherence to tamoxifen therapy
Tamoxifen adherence was assessed using the Morisky,
Green, and Levine Medication Adherence Scale ques-
tionnaire, a structured four-item self-reported adherence
measure validated for a wide range of diseases, including
cancer.34 This questionnaire has been successfully used in
patients with low literacy and its feasibility was previously
confirmed in our hospital for inpatients and outpatients
with cancer.35 Four trained pharmacists per formed the
questionnaire-based interviews at 3, 6, and 12 months
after starting tamoxifen therapy and included the following
questions: (i) Do you ever forget to take your medicines?; (ii)
Are you careless at times about taking your medicines?; (iii)
When you feel better do you sometimes stop taking your
medicine?; and (iv) Sometimes, if you feel worse when you
take the medicine, do you stop taking it? For each patient
and visit “yes” (1 point) or “no” (0 point) answers were doc-
umented and the four-item scores summed up to define
three adherence levels: high (0), medium (1–2), and low ad-
herence (3–4). Information on concomitant medication and
self-reported adverse events during tamoxifen treatment
were recorded during all visits.
Genotyping and quantification of tamoxifen and its
active metabolites from plasma
Genomic DNA obtained from peripheral blood mononu-
clear cells was genotyped for CYP2D6 polymorphisms as

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Table 1 Adherence behavior to tamoxifen treatment (20mg daily) of Brazilian patients with breast cancer in relation to their demographic and clinical characteristics
Characteristic
Overall
distribution
(n=192)
Adher ence a t
3months (n=163) OR
P value
Adher ence a t
6months (n=173) OR
P value
Adher ence a t
12month s (n=170) OR
P value
High,
n (%)
Medium/
low, n (%) 95% CI
High, n
(%)
Medium/
low, n (%) 95% CI
High,
n (%)
Medium/
low, n (%) 95% CI
Ethnicity,a n (%)
White 139 (7 2.4) 89 ( 74. 8) 30 (25.2) 0.804 (0.37–1.73) 0.689 98 (79.7) 25 (20.3) 2.019 (0.97–4.20) 0.078 78 (62.9) 46 ( 37.1) 0.9 04 (0.45–1.84) 0.859
Black 11 ( 5 .7 ) 31 (70.5) 13 (29.5) 33 (66.0) 17 ( 34. 0) 30 (65. 2) 16 (3 4.8 )
Pardo 37 (19.3)
Asian/Indian 5 (2.6)
Educational level,b n (%)
None or incomplete basic 68 (35.4) 46 ( 76.7) 14 (23.3) 0.777 (0.37–1.62) 0.582 4 8 (77. 4) 14 (2 2.6) 1.157 (0 .56 –2 .41) 0.853 4 0 ( 67.8 ) 19 (32. 2) 1.331 (0.68–2.59) 0.503
Basic 50 (26.0) 74 ( 71 .8 ) 29 (28.2) 83 ( 74. 8 ) 28 (25.2) 68 ( 61.3 ) 43 (38 .7)
Upper secondary 53 ( 27.6 )
University (Bachelor/
Master/Doctoral)
21 (10.9)
Age at diagnosis, median
(range), yea rs
51.5 ( 24 –82) 54 (33–82) 47 (28–7 6)  <0.001 54 (24– 82) 49 (32–77) 0.007 56 ( 24– 82) 48 (28 –79)  <0.001
<65 145 (7 5. 5) 87 (69.0) 3 9 ( 31.0 ) 0.270 (0.09– 0.82) 0.018 94 (71.8) 37 (28.2) 2.913 (1.03–7.98) 0.038 71 (5 5.9) 56 (4 4 .1) 4.86 (1.92–12.34) <0.001
65 47 (24.5) 33 (89.2) 4 (10. 8) 37 ( 8 8.1 ) 5 ( 11. 9) 37 (86.0) 6 (14 .0 )
Menopausal status, n (%)
Premenopausal 127 ( 66 .1) 74 (6 6 .7 ) 37 (3 3.3) 0.261 (0.10–0.67) 0.004 81 (70.4) 34 (29.6) 2 .6 23 ( 1.13 –6 .12 ) 0.02 5 60 (53.6) 52 (46.4) 4.160 (1.92–9.04) <0.001
Postmenopausal 65 (33.9) 46 (88.5) 6 (11 .5 ) 50 (86.2) 8 (13. 8) 48 (82.8) 10 ( 17. 2)
ECOG performance status, n (%)
0–1 1 87 ( 97.4 ) 116 ( 73. 0) 43 ( 27. 0) 1.371 (1.25 –1.51) 0.5 74 126 ( 75.0) 42 (25.0) 1. 33 3 (1. 22 –1.46) 0.337 103 (62. 4) 62 (37.6) 1.60 2 (1. 42–1.80) 0.1 6 0
2–4 5 (2.6) 4 (100.0) 0 (0.0) 5 (100. 0) 0 (0.0) 5 (100.0) 0 (0.0)
Clinical stage at diagnosis,c n (%)
0 (in situ)2 (1.0 ) 9 3 ( 72.1) 3 6 ( 27.9) 0.670 (0.27–1.67) 0.513 104 (75.4) 34 (24.6) 1.103 (0.46–2.66) 1.000 85 (61.6) 53 (38.4) 1.59 3 (0.6 9– 3.70) 0 .314
IA/IB 56 (29. 2)
IIA/IIB 9 2 (4 7.9)
IIIA/IIIB/IIIC 3 3 (1 7. 2) 27 (79.4) 7 (20.6) 27 ( 77.1) 8 (22.9) 23 ( 71.9) 9 ( 28 .1)
IV 9 (4.7)
Concomitant medications,d n (%)
<3 medications 122 (63.6) 92 (71.3) 37 (28.7) 1.877 (0.72–4.91) 0. 274 1 07 (7 7.0 ) 32 (23.0) 1.393 (0.60–3.22) 0.504 92 (62.2) 5 6 (3 7.8) 0.616 (0.23–1.67) 0.477
3 or more medication 70 (36.5) 28 (82.4) 6 (1 7.6 ) 24 (70. 6) 10 (29.4) 16 (72.7 ) 6 (27. 3)
Self-reported adverse events
Yes 15 5 (8 0.7 ) 60 (69.8) 26 (3 0.2) 0.654 (0.32–1.33) 0.287 66 ( 70.2) 28 (29.8) 1.970 (0.95–4.08) 0.076 6 3 (55.8) 50 (44 .2) 2.976 (1.42– 6.22) 0.004
No 37 (19.3) 60 (77.9) 17 ( 2 2.1 ) 65 (82.3) 14 ( 17.7 ) 45 ( 78.9) 12 (21.1)
CI, confi dence inte rval; ECOG, Easter n Cooperative Oncology Gro up; OR, odds ratio.
P: signifi cance val ue for Fisher’s exact test for qualitative variables and Mann–Whitney test for quantitative variable (age).
aBlack, Pardo, and Asi an/Ind ian were grouped to be tested against white. In B razil, Pardo is an ethnic/skin color category use d by the Brazilian Institute of Geography and Statistics (IBGE) in the Brazilian
census es. The ter m “pardo” is c ommonly used to refer to Brazilians of mixed e thnic anc estrie s. Pardo Brazilian s represent a wide ra nge of skin c olors an d backgrounds. They are typically a mixture of white
Brazilian, Afro-Brazilian, and Native Brazilian. Indian refers to Native Brazilian.bBasic, upper secondary and universit y levels were grouped to b e tested against no instruction.c0 (in sit u), IA/IB a nd IIA/IIB were
grouped to be tested ag ainst IIIA/III B/IIIC and IV.dConcomita nt medication in this analysis was assessed to evalu ate the impact of polypharmacy i n adheren ce behavi or. The main pre scribe d pharma cologic
classe s were antihyperte nsive drugs (29.2%, 29.4%, and 26.3% for 3, 6, and 12months, respectively) and nonopioid analges ics and nonsteroidal anti-inflammatory drugs (13.2%, 16.0%, and 22.0% for 3, 6,
and 12months, res pectively). Strong cytochrome P450 2D6 (CYP2D 6) inhibitors were ne ar 1.0% of all conco mitant me dicatio ns for all time points (0.5%, 0.9%, and 1.2% for 3, 6, and 12months, r espectively).

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previously described.24 A CYP2D6 enzyme activity score
(AS) was assigned to genotypes (diplotypes) based on al-
lele scores of 0 (PM), 0.5 (IM), 1 (EM), and 2 (UM)36 ,37 and
CYP2D6 phenotypes were deduced from AS: PM (0), IM
(0.5 to 1.0), EM (1.5 to 2.0), and UM (3.0). Other DME gene
polymorphisms included CYP2C9*2 and *3, CYP2C19*2
and *17, and CYP3A4*22 and CYP3A5*3 (Supplemental
Material).
Heparinized plasma samples were obtained at months 3,
6, and 12 after the start of tamoxifen therapy. Plasma levels
of tamoxifen and the inactive major metabolite DM-Tam as
well as active metabolites (Z)-endoxifen and (Z)-4-OH-Tam)
were measured by liquid chromatography tandem mass
spectrometry as previously described.17
Statistical analysis
DME genotype frequencies were tested for Hardy–Weinberg
Equilibrium. Parametric and nonparametric tests were applied
to determine whether tamoxifen and its metabolite concentra-
tions as well as metabolic ratio’s (MRs) differ between DME
genotypes and adherence behavior. Multiple linear regression
modeling was applied to evaluate the contribution of fac-
tors to the variability of plasma concentrations of tamoxifen,
(Z)-endoxifen, DM-Tam, and (Z)-4-OH-Tam, as well as the re-
spective MRs. Relative risk (RR) and 95% CIs were calculated
at the 12-month time point to evaluate the risk of patients not
achieving a previously proposed clinical threshold of 5.9ng/
mL (15.8nM) (Z)-endoxifen.20 All P values were two-sided,
and values < 0.05 were considered statistically significant
(details provided in Supplemental Material).
RESULTS
Patient adherence to treatment
Demographic and clinical characteristics together with
adherence assessment at specific time points are given
in Tab l e 1 and Tabl e S1. Adherence was assessed for
163 patients at month 3 (85%), 173 patients at month 6
(90%), and 170 patients at month 12 (89%). Median age
at diagnosis was 51.5 years (range 24– 82 years); 127
patients (66%) were premenopausal. At 3 and 6 months,
74–76% of patients showed high adherence rates to
tamoxifen treatment, which dropped to 63% at 12months
(Figure 1a). Low adherence was not observed during
the first 3 months but increased to 10.6% at 12months
(Figure 1a; P<0.05). High adherence at 12months was
more prevalent in patients without reported adverse
events compared with those who reported adverse events
(Figure 1b; P<0.05). With the exception of age at diagno-
sis, menopausal status, and self-reported adverse events
at 12months, patient and tumor characteristics did not
differ between adherence subgroups across time points
(Tab l e 1). Adverse events at 3, 6, and 12months were re-
ported for 53%, 54%, and 66% of patients, respectively
(Tab l e 1), with hot flashes being most frequent (62%, 65%,
and 68%, respectively). Others included edema of the in-
ferior members (17%, 25%, and 20%), fatigue (14%, 25%,
and 30%), and nausea/vomiting (15%, 12%, and 12%) at 3,
6, and 12months, respectively.
Genotype frequencies and enzyme AS
Genotypes met Hardy–Weinberg Equilibrium with the
exception of CYP2D6*29 (Tabl e S2). Although allele fre-
quencies differed significantly between ethnic groups
(P<0.001; Tab le S2), ethnicity was not a prognostic factor
for DME phenotypes or plasma concentration of tamoxifen
and its major metabolites. DME phenotypes/AS predicted
from 17 polymorphic loci tested in the 192 patients are
summarized in Figure S1.
Association between treatment adherence and
plasma concentrations of tamoxifen and its
metabolites
At 3months of treatment, patients with good adherence
had 26% higher tamoxifen concentrations than patients
with medium adherence (318±97nM vs. 236±115nM;
P<.001; Figure 2a). Multiple linear regression analysis
showed that 16–21% of the variability of tamoxifen lev-
els at months 3 and 6 were explained by adherence to
treatment and age at diagnosis (P<0.001; Ta b l e 2). At
Figure 1 Adherence levels at consecutive time points after starting tamoxifen therapy of 20mg daily. (a) 3months (n=163); 6months
(n=173); and 12months (n =170). (b) 12months, stratified by the occurrence of self-reported adverse events: presence of adverse
events (Yes; N=113); absence of adverse events (No; N=57). Categories high (light grey), medium (dark grey), and low (black) are given
as % for each group; P values refer to contingency Chi-square tests of a difference between visit a or grouping by adverse events b.
(a) (b)

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12months, tamoxifen plasma levels strongly correlated
with adherence (r = 0.70; P < 0.001) with tamoxifen
levels being highest in patients with high adherence
(389±99 nM) compared with those with low or medium
adherence (157 ± 67 nM and 258 ± 61 nM; P<0.001;
Figure 2a). In multivariate analyses, adherence was con-
firmed as the sole determinant that explained 47% of
tamoxifen plasma concentration variability (P<0.001;
Tab l e 2).
Similar associations with adherence were obtained for
tamoxifen metabolite (Z)-endoxifen (Figure 2b). In subgroup
analysis of patients with functional CYP2D6 (EM/EM) treat-
ment adherence was significantly correlated with tamoxifen
metabolites at all three time points (Figure 2c, data shown
for months 3 and 12).
Association between genotypes of pharmacogenes
and plasma concentrations of tamoxifen and its
metabolites
Plasma concentrations of all tamoxifen metabolites were
affected by CYP2D6 phenotype with a strongest effect
for (Z)-endoxifen (Tab l e S3). CYP2D6 AS demonstrated
gene-dose effects on both (Z)-endoxifen and MR of
N-desmethyltamoxifen (DM-Tam)/(Z)-endoxifen confirming
the importance of CYP2D6 for the bioactivation of tamoxifen
to (Z)-endoxifen via DM-Tam (Figure S2; P<0.0 01). CYP2D6
deficient PM patients (PM/PM) had 4.5 to 5.5 times lower (Z)-
endoxifen concentrations than EM patients with functional
CYP2D6 (EM/EM). The m ean concentrations of PM c ompared
with EM patients at 3, 6, and 12months were 6.5± 2.7nM
vs. 29.6 ± 12.9 nM; 7.2 ± 3.3 nM vs. 30.3 ± 14.5nM; and
Figure 2 Influence of treatment adherence on tamoxifen and (Z)-endoxifen plasma concentration at 3months (n=156 patients) and
12months (n = 139 patients). Plasma concentrations are presented as boxplots with boxes representing medians, 25% and 75%
percentiles, and whiskers defined by the 5th and 95th percentiles and extreme values outside the whiskers. (a) TAM: Tamoxifen (parent
drug). ( b) (Z)-endoxifen. (c) (Z)-endoxifen for the subgroup of cytochrome P450 2D6 (CYP2D6). Extensive metabolizer (EM/EM) patients
at 3months (n=67) and 12months (n=55). P values refer to test for different plasma concentrations between adherence categories.
Data at 6months not shown.
TAM 3 months (nM)
0
200
400
600
Adherence 3 Months
High Medium Low
Adherence 12 Months
High Medium
TAM 12 months (nM)
0
200
400
600
(Z)-Endoxifen 3 months (nM)
0
20
40
60
80
100
(Z)-Endoxifen 12 months (nM)
0
20
40
60
80
100
Low
Adherence 12 Months
High Medium
Adherence 3 Months
High Medium
(Z)-Endoxifen 3 months (nM)
0
20
40
60
80
100
(Z)-Endoxifen 12 months (nM)
0
20
40
60
80
100
Low
Adherence 12 Months (EM/EM)
High Medium
Adherence 3 Months (EM/EM)
High Medium
P<0.001 P<0.001
P= 0.012 P<0.001
P= 0.016 P= 0.006
(a)
(b)
(c)

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5.4 ± 1.1nM vs. 30.3 ± 14.5 nM. In multivariate analyses,
CYP2D6 phenotype was a significant predictor of tamoxifen
metabolites and MRs across all time points (Tab l e 2).
Due to the local prescription practice there were few
patients with concurrent comedication of strong CYP2D6
inhibitors. Although three of the four patients taking a
strong CYP2D6 inhibitor (fluoxetine) showed low levels of
(Z)-endoxifen (6–15 nM), CYP2D6 inhibitor use was not
significantly associated with tamoxifen metabolite concen-
trations. Among other covariates, age at diagnosis was
positively associated with increased tamoxifen and DM-TAM
concentrations, whereas DMEs other than CYP2D6 had little
influence (Table S3).
Combined analysis of factors influencing
(Z)-endoxifen concentrations
Adherence and CYP2D6 phenotype were jointly associated
with (Z)-endoxifen concentrations across all time points
(Tab l e 2). The additive effect of adherence and CYP2D6
phenotype was most evident at 12 months. Although
CYP2D6 as a single factor explained 26.4% of (Z)-endoxifen
variability (P<0.001), the explained variability increased to
40% when adherence was included in multivariate analysis
(P < 0.001; Ta b l e 2). We next evaluated whether adher-
ence or CYP2D6 polymorphisms had a stronger effect on
(Z)-endoxifen concentrations based on standardized
beta coefficients (Ta ble 3). Across all time points, severe
Table 2 Summary of multiple linear regression models for tamoxifen and selected metabolites
Metabolite/time point r2F P valueaParameters in the model
Tam 3months 0.212 19.75 0 <0.001 Medium adherence; age at diagnosis
Tam 6months 0 .16 1 9.520 <0.001 Low and medium adherence; age at diagnosis
Tam 12months 0.471 39.130 <0.001 Low and medium adherence
DM-Tam 3months 0.412 8.995 <0.001 CYP2D6 PM/IM, IM/IM, EM/PM, EM/IM; medium adherenc e; age at
diagnosis; Asian/Indian ethnicity
DM-Tam 6months 0. 217 4.384 <0.001 CYP2D6 PM/PM, EM/PM; low and medium adherence; age at diagnosis
DM-Tam 12months 0.427 10. 451 <0.0 01 CYP2D6 PM/ IM, EM/PM, EM /IM; low and medium adh erence; age at
diagnosis
Z-Endo 3months 0.345 7. 37 0 <0.001 CYP2D6 PM/PM, PM/IM, EM /PM; medium adheren ce; Asian /Indian
ethnicity
Z-Endo 6months 0.322 8.421 <0.0 01 CYP2D6 PM/PM, PM/IM, EM/PM, IM/IM; medium adherence
Z-Endo 12months 0.403 10.8 77 <0.001 CYP2D6 PM/PM, PM /IM, EM/PM, IM/IM; low a nd medium a dherence
Metabolic ratio
DM-Tam/Z-Endo 3month s 0.556 30.002 <0.001 CYP2D6 PM/IM, IM/IM, EM/PM, EM/IM
DM-Tam/Z-Endo 6month s 0.510 2 0 .19 5 <0.001 CYP2D6 PM /IM, IM/IM, EM/PM, EM /IM
DM-Tam/Z-Endo 12months 0.575 15.5 40 <0.0 01 CYP2D6 PM/IM, EM/PM, EM/IM; black ethnicity
Cytochrome P450 2D6 (CYP2D6)diplotypes PM/PM, two null allel es; DM-Tam, N-desmethylta moxifen; EM/ IM, one nor mal and on e reduce d activit y allele;
EM/PM, one normal and one null ac tivity allele; EM/UM, patien t with gene duplicati ons of alleles with normal acti vity; F, F-test ( ANOVA) for the m odel; IM/
IM, two reduced activity alleles; PM/IM, one null activity and one reduced activity all ele; r2, model’s coeffic ient of dete rmination; Tam, tamoxifen; Z-Endo,
(Z)-endoxifen.
aSignifi cance value of F-test (analysis of variance) fo r the propo sed mode l.
Table 3 Evaluation of contributing variables based on linear regression coefficients for the prediction of (Z)-endoxifen plasma variability
3Months 6Mont hs 12Month s
Variable
St beta
coef.aP valuebVariable
St beta
coef.aP valuebVariable
St beta
coef.aP valueb
CYP2D6 PM/IM −0.353 <0.001 CYP 2D6 PM /IM − 0.378 <0.001 CYP2D6 PM /PM −0 .3 74 <0.001
CYP2D6 PM/PM −0. 312 <0.001 CYP2D6 PM/PM −0.307 <0.001 CYP2D 6 PM/I M −0.342 <0.001
CYP2D6 EM/PM −0.249 0.001 Adherence-medium −0. 266 <0.001 Adherence -low −0.3 37 <0.001
Adherence-medium − 0 .18 9 0.008 CY P2D6 E M/PM −0.20 3 0.010 CY P2D6 E M/PM −0.208 0.007
CYP2D6 IM/IM − 0 .119 0 .103 CYP2D6 IM/IM − 0 .17 3 0.016 Adherence-medium −0.206 0.004
CYP2D6 EM/IM − 0.14 6 0.055 CY P2D6 E M/IM −0 .13 4 0.083 CYP2D6 IM/IM −0.136 0.050
Black −0.092 0.206 Adherence-low − 0 .110 0 .114 CYP2D6 EM/IM −0.137 0.069
Brown/mixed −0.052 0.456 CYP2D6 EM/UM −0 .010 0.8 87 CYP2D6 EM/UM 0 .14 0 0.0 51
Asian/Indian 0 .14 4 0.042      
CYP2D6 EM/UM 0.10 2 0 .15 6      
Black, Brown/Mixed, Asian /Indian refers to the non-European ethnicities included in the study c ohort; cytochr ome P450 2D6 (CYP2D6)dipl otypes EM/IM,
one normal and one re duced ac tivity a llele; EM/PM, one norm al and one null activity allele; EM/UM, patient with gene duplications of a lleles wi th normal
activit y; IM/IM, two reduc ed activity alleles; PM/IM, o ne null activity and one reduc ed activi ty allel e; PM/PM, two null allele s; St, stan dard.
aVariables are listed according to their relevance in the model according to standardized beta coefficients.bOnly vari ables with P<0.05 (t-test) are relevant
in the mode l.

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Tamoxifen Adherence, (Z)-endoxifen, and CYP2D6
Nardin et al.
CYP2D6 impairment (AS≤0.5; PM/PM and PM/IM) was the
strongest determinant of (Z)-endoxifen variability. The sec-
ond strongest effect resulted from low or medium treatment
adherence at 6 and 12months with an effect size equal or
greater to that resulting from a CYP2D6 AS > 0.5 (IM and
EM; Tab l e 3). As a putative surrogate predictor of clinical
response, we used plasma concentrations of (Z) endoxi-
fen and evaluated the relevance of adherence vs. CYP2D6
phenotype based on the RR of not achieving the threshold
plasma concentration of 5.9ng/mL (Z)-endoxifen (Figure 3).
The risk to not achieve the threshold was highest in patients
with CYP2D6 PM phenotype (RR 4.1; 95% CI 3.04–5.50),
followed by patients with either CYP2D6 IM phenotype (RR
2.54; 95% CI 1.41–4.60) or low treatment adherence (RR
2.44; 95% CI 1.40–4.25). In subgroup analyses in patients
with noncompromised CYP2D6 function (EM phenotype),
low adherence showed to be a strong risk factor of not
achieving clinical threshold concentrations (RR 3.65; 95%
CI 1.48–8.99). This association was less pronounced in
patients with reduced CYP2D6 activity (IM phenotype; RR
2.19; 95% CI 1.21–3.97).
DISCUSSION
We provide first evidence that the ability to achieve clini-
cally relevant (Z)-endoxifen plasma concentrations during
breast cancer tamoxifen treatment is cooperatively influ-
enced by treatment adherence behavior, CYP2D6 genomic
background, and pharmacokinetic capacity. Conjointly, this
could improve the prediction of active tamoxifen metabo-
lite levels (Z-endoxifen) and possibly clinical efficacy,17,2 0, 3 8
a high priority goal in personalized endocrine treatment.
Previous attempts to predict the variability of (Z)-endoxifen
levels relied on pharmacokinetic and pharmacogenomic
knowledge,17,38,39 which has been implemented into thera-
peutic recommendations based on CYP2D6 genotyping.14
Although a recent drug monitoring study showed that tamox-
ifen metabolites may be predictive of side effects, such as
nausea and vaginal dryness,40 clinical tamoxifen outcome
studies as of yet provided controversial results possibly
due to the influence of unidentified confounders.22–24,26–28
Because it is well known that women stop taking their med-
ication before completing the standard 5-year regimen, we
investigated patients’ adherence behavior during the first
year of tamoxifen treatment in relation to their metabolic ca-
pacity for achieving relevant (Z)-endoxifen concentrations.
Our observed 12-month adherence rate is considerably
lower than the 1-year tamoxifen adherence reported by
Dezentje et al.41 (93%) based on prescription refill data. The
latter assumed that prescription-refilling patterns corre-
spond to patient medication-taking behavior and that the
medication is taken exactly as prescribed independent of
patients’ beliefs or concerns about treatment. A 90% 1-year
adherence rate was also repor ted by Makubate et al.42 in
a retrospective cohort of endocrine-treated breast cancers
that declined to 51% in the fifth year, and that demon-
strated an association of low adherence (<80%) with poor
survival. Our prospective study used the Morisky, Green,
and Levine Medication Adherence Scale questionnaire in
a pharmacist-guided interview to assess adherence rates,
a tool previously used in an oncology setting in Brazil35,4 3
and which is considered reliable based on the validation
of self-report questionnaires in relation to medication-mon-
itoring devices.44 Notably, Morisky’s selected adherence
scale is an easy to perform and fast questionnaire suit-
able for our prospective setting with 35% of patients with
low-literacy.13,4 5 The questionnaire provided us with the
benefit to retrieve valuable tamoxifen adherence behavior
and pharmacokinetic information from a health disparity
cancer population that otherwise would not be accessible.
The high observed tamoxifen nonadherence rate within the
first year may reflect the socioeconomic condition of this
population and the high percentage of young women (two-
thirds) known to be at increased risk to stop medication.10
We confirmed that CYP2D6 phenotype is a strong determi-
nant of plasma (Z)-endoxifen levels in that increasing CYP2D6
allele activity correlated with increasing plasma metabolite
concentrations. Our data are sound as the prevalence of re-
duced-function and null-function alleles compares with those
reported from Brazil33 and are in line with population admixture
of Europeans, Native Americans, Africans, and Asians, with
our institution serving as a national reference center for oncol-
ogy treatment. (Z)-endoxifen plasma levels were in the range
of those reported by others,15,17 and, as expected, CYP2D6
polymorphism only partially predicted its variability.19 Potential
confounders, such as DME polymorphisms or strong CYP2D6
inhibitors, do not play a significant role as the former showed
no effect and the latter were infrequently used by our patients
given the low prescription rates at our hospital.
Of note, the observed effect of adherence behavior on
active metabolite levels depended on CYP2D6 functionality.
Although PM status was still the most important predictor
of reduced (Z)-endoxifen concentrations, and the risk to not
achieve sufficient concentration due to low-adherence was
moderate across all patients (RR 2.44; 95% CI 1.40–4.25),
low-adherence was a strong predictor in EM patients (RR
3.65; 95% CI 1.48–8.99). The latter clearly indicates an inde-
pendent influence of low-adherence resulting in suboptimal
concentrations of active tamoxifen metabolites. An immedi-
ate consequence from this finding is that EM and IM patients
who are now in their ensuing year(s) of the 5-year treatment
must be encouraged to adhere to tamoxifen, as the expected
benefit will depend on their own authority. Known barriers to
prevent tamoxifen adherence are manifold and include low
recurrence risk perception, side effects, age extremes, medi-
cation cost, ethnicity, educational level, lack of social support,
and suboptimal patient physician communication.3,4,13
Importantly, we confirmed the relevance of age on tamoxifen
adherence that patients at 65years of age or older showed bet-
ter adherence to tamoxifen than younger patients. Moreover,
multivariate modeling identified age as an independent predic-
tor of parent drug levels (i.e., tamoxifen). Previous findings by
others showed that plasma concentrations of tamoxifen and
its metabolites increase with age,46 which, according to our
findings, may be explained at least in part by patient adher-
ence behavior. Given the strong predictive value of adherence
for the variability of tamoxifen plasma concentrations we pro-
pose that drug monitoring is a powerful surrogate to assess
tamoxifen adherence for which we identified a threshold con-
centration of 157nM (±67nM) to stratify patients into low vs.

291
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Tamoxifen Adherence, (Z)-endoxifen, and CYP2D6
Nardin et al.
medium/high tamoxifen adherent. This is particularly relevant
to young patients, as their risk to discontinue tamoxifen is
among the highest4,10 with tamoxifen currently being the sole
standard-of-care treatment in this patient group.
Our study provides a first link between poor patient ad-
herence to tamoxifen and the risk to not achieve relevant
(Z)-endoxifen plasma concentrations. However, the study is
not without limitations. We are aware that the Morisky, Green,
and Levine Medication Adherence Scale scoring system does
not quantitatively capture adherence, yet we consider this
approach appropriate as it allowed us to assess tamoxifen ad-
herence in this patient group with a high proportion of illiteracy.
Moreover, the putative threshold of 5.9ng/mL (Z)-endoxifen20
has not been prospectively validated and controversies on the
CYP2D6—endoxifen relationship for the prediction of tamox-
ifen efficacy have not been finally resolved, which currently
limits the clinical utility of our findings.29,47–52
In conclusion, our proof-of-concept study suggests that
the dual monitoring of tamoxifen plasma levels as surrogate
marker for adherence and (Z)-endoxifen formation as sur-
rogate marker for clinical response could be a strategy to
improve patients’ chances to avoid recurrence and prema-
ture death in the future.
Supporting Information. Supplementary information accompa-
nies this paper on the Clinical and Translational Science website (www.
cts-journal.com).
Figure S1.
Figure S2.
Tab le S1.
Tab le S2.
Table S3.
Supplementary Material.
Funding. This work was conducted during a scholarship supported
by the International Cooperation Program CAPES/STICAMSUD at the
Pontifical Catholic University of Parana, Curitiba, Brazil and financed by
CAPES – Brazilian Federal Agency for Support and Evaluation of Graduate
Education within the Ministry of Education of Brazil. The work was also
funded in part by the Robert Bosch Foundation, Stuttgart, the HORIZON
2020-PHC-2015 grant U-Px 668353, Deutsche Forschungsgemeinschaft
(DFG, German Research Foundation) under Germany’s Excellence
Strategy - EXC 2180 – 390900677, and DFG, SCHR 1323/2-1 and
MU 1727/2-1), Interfaculty Center for Pharmacogenomics and Drug
Research (ICEPHA) University of Tübingen, and The German Cancer
Consortium (DKTK), partner site Tuebingen), Germany.
Conflict of Interest. The authors declared no competing interests
for this work.
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T.M., R.H., M.S., and R.F.P.-F. wrote the manuscript. J.C.C.-d.-R., J.M.N.,
T.A.A., and H.B. designed the research. J.M.N., T.A.A., E.C.L.V., J.P.K., S.P.,
D.M., S.D.R.d.M., R.F.P.-F., T.M., and W.S. performed the research. J.M.N.,
J.C.C.-d.-R., T.A.A., W.S., T.M., R.H., M.S., and H.B. analyzed the data.
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