Relationship between Genotypes Sult1a2 and Cyp2d6 and Tamoxifen Metabolism in Breast Cancer Patients
et al. (2013) Relationship between Genotypes Sult1a2 and Cyp2d6 and
Tamoxifen Metabolism in Breast Cancer Patients. PLoS ONE 8(7): e70183. doi:10.1371/journal.pone.0070183
Relationship between Genotypes Sult1a2 and Cyp2d6 and Tamoxifen Metabolism in Breast Cancer Patients
Ana Ferna ndez-Santander 0
Mara Gaibar 0
Apolonia Novillo 0
Alicia Romero-Lorca 0
Margarita Rubio 0
Luis Miguel Chicharro 0
Armando Tejerina 0
Fernando Bandre s 0
Rui Medeiros, IPO, Inst Port Oncology, Portugal
0 1 Department of Basic Biomedical Sciences, Faculty of Biomedical Sciences, Ca tedra Florencio Tejerina-Universidad Europea de Madrid, Universidad Europea de Madrid , Madrid , Spain , 2 Department of Applied Medical Specialities, Psychology and Education, Faculty of Biomedical Sciences, Universidad Europea de Madrid , Madrid , Spain , 3 Department of Morphological Sciences, Faculty of Health Sciences, Universidad Europea de Madrid , Madrid , Spain , 4 School of Advanced Studies, Ca tedra Florencio Tejerina-Universidad Europea de Madrid , Fundacio n Tejerina, Madrid , Spain
Tamoxifen is a pro-drug widely used in breast cancer patients to prevent tumor recurrence. Prior work has revealed a role of cytochrome and sulfotransferase enzymes in tamoxifen metabolism. In this descriptive study, correlations were examined between concentrations of tamoxifen metabolites and genotypes for CYP2D6, CYP3A4, CYP3A5, SULT1A1, SULT1A2 and SULT1E1 in 135 patients with estrogen receptor-positive breast cancer. Patients were genotyped using the RocheAmpliChipH CYP450 Test, and Real-Time and conventional PCR-RFLP. Plasma tamoxifen, 4-hydroxy-tamoxifen, N-desmethyltamoxifen, endoxifen and tamoxifen-N-oxide were isolated and quantified using a high-pressure liquid chromatographytandem mass spectrometry system. Significantly higher endoxifen levels were detected in patients with the wt/wt CYP2D6 compared to the v/v CYP2D6 genotype (p,0.001). No differences were detected in the remaining tamoxifen metabolites among CYP2D6 genotypes. Patients featuring the SULT1A2*2 and SULT1A2*3 alleles showed significantly higher plasma levels of 4-hydroxy-tamoxifen and endoxifen (p = 0.025 and p = 0.006, respectively), as likely substrates of the SULT1A2 enzyme. Our observations indicate that besides the CYP2D6 genotype leading to tamoxifen conversion to potent hydroxylated metabolites in a manner consistent with a gene-dose effect, SULT1A2 also seems to play a role in maintaining optimal levels of both 4-hydroxy-tamoxifen and endoxifen.
Competing Interests: The authors have declared that no competing interests exist.
Tamoxifen (TAM) is widely used to prevent recurrence in
patients with estrogen or progesterone receptor-positive breast
cancer (BC) due to its estrogen receptor blocking effect .
Tamoxifen is described as a pro-drug since two of its metabolites,
4-hydroxy-tamoxifen (4OH-TAM) and
N-desmethyl-4-hydroxytamoxifen (endoxifen), both have an affinity for the estrogen
receptor that markedly exceeds that of TAM itself . Endoxifen
is considered the main active metabolite of TAM, since its estrogen
receptor affinity is 100-fold that of TAM and its serum levels are
10-fold those of 4OH-TAM .
Tamoxifen is metabolized via the cytochrome P450-mediated
pathway to several primary and secondary metabolites that show
variable potency toward the estrogen receptor.
N-desmethyltamoxifen (NDM-TAM), produced by CYP3A4/5-mediated
metabolism, is the major primary metabolite, accounting for 90% of
primary TAM oxidation, whereas 4OH-TAM, mediated by
CYP2D6 activity, is a minor metabolite. Both NDM-TAM
(produced via CYP2D6) and 4OH-TAM (produced via
CYP3A4/5) are secondarily metabolized to endoxifen .
Although it has been established that tamoxifen and its metabolites
undergo phase II conjugation reactions including glucuronidation
and sulfation, few studies have examined the role of phase II
sulfotransferase enzymes (SULTs) in TAM metabolism. Given that
4-OH-TAM and endoxifen are known substrates of SULT1A1 [5
and 6, respectively], different SULT enzyme activity levels could
markedly influence the efficacy of TAM .
CYP-regulated drug metabolism is prone to genetic variability,
which can lead to normal, low or null activity levels of a given
enzyme. The main enzyme responsible for 4OH-TAM and
endoxifen formation, CYP2D6 , is highly polymorphic. Over
80 different alleles resulting in reduced or impaired CYP2D6
activity have been reported . Subjects carrying two null
CYP2D6 alleles are classed as poor drug metabolizers (PMs), with
510% of Caucasians classified as PMs . The CYP2D6*4 allele,
followed by CYP2D6*5, CYP2D6*3 and CYP2D6*6, is the main
null allele that gives rise to the PM phenotype in Europeans (12
22%) . Around eighty single nucleotide polymorphisms (SNPs)
of CYP3A4/5 have been reported to the Human P450 Allele
Nomenclature Committee . The CYP3A5*3 SNP at intron 3
causes alternative protein splicing and truncation . This
mutant allele is considered the main defective CYP3A5 allele and
frequencies of this allele as high as 95% have been described in
some European populations [12,13]. This variant plays an
important role in interindividual and interethnic differences in the
metabolic profiles of many drugs . Two inactive genetic
variants of CYP3A4, CYP3A4*3 and CYP3A4*17, have also been
described in Caucasian populations [15,16]. The CYP3A4*3 allele,
which has a T1473C change that produces a Met445Thr
substitution in exon 12, induces structural differences in the
enzyme modifying its activity. The CYP3A4*17 polymorphism
with a T.C mutation in exon 7 is a putative defective allele that
leads to .99% reduction in catalytic activity . Breast cancer
patients under treatment with TAM who feature the CYP3A4*1B
allele and a single A to G change in the promoter may be at an
increased risk of developing endometrial cancer, as described by
Chu et al. . Recently, a lower production of NDM-TAM in
CYP3A5*3/*3 microsomes compared to the wild-type genotype
has been described by Mugundu et al. . However, so far no
variant CYP3A4 allele has been linked to a modified TAM
metabolism. Some authors have reported considerable
interindividual variation in plasma levels of TAM metabolites that could
affect the response to treatment [19, for instance]. Hence, genetic
variability in the genes coding for the enzymes CYP2D6, CYP3A4
and CYP3A5 could explain such variations in metabolite
Sulfotransferase enzymes are a family of phase II liver enzymes
involved in the detoxification of a variety of xenobiotic and
endogenous compounds. These enzymes catalyze the transfer of a
sulfonyl group to nucleophilic groups increasing their solubility
and facilitating their excretion. SULT1A1 is the most highly
expressed SULT in the liver and some studies have shown that the
high-activity SULT1A1*1 allele is linked to a better overall survival
rate in BC patients receiving TAM . Among all known SULTs,
SULT1E1 shows the highest affinity for estrogens indicating its
activity at physiologically significant estrogen concentrations .
Moreover, SULT1E1 is highly expressed in normal human
mammary epithelial cells  and may play an important role
in estrogen-driven BC development. In effect, genetic
polymorphisms in SULT1E1 have been associated with both an increased
risk of BC and disease-free survival in Asian women . Further,
a study examining the role of the sulfotransferase gene, SULT1A2,
has revealed its contribution to the TAM-resistant phenotype in
the presence of certain combinations of CYP2C9 and SULT1A2
allelic variants .
Given the complexity of TAM metabolism and the inconsistent
results provided in the literature, this descriptive study was
designed to examine relationships between TAM metabolite
concentrations and genotypes for CYP2D6, CYP3A4, CYP3A5,
SULT1A1, SULT1A2 and SULT1E1 in 135 patients with estrogen
receptor-positive breast cancer. Besides our findings related to the
CYP2D6 genotype, this paper presents the first data on the effects
of SULT1A2 genotypes on TAM metabolite levels.
Patients and Methods
The study protocol was approved by the Review Board of the
Hospital de Getafe (Madrid, Spain). Written informed consent to
participate in the study was obtained from all participants.
One hundred and thirty five Caucasian patients of Spanish
descent recently diagnosed with BC were recruited from the center
Fundacion Tejerina-Centro de Patologa de la Mama (Madrid).
Premenopausal and postmenopausal women were enrolled when started
on TAM as standard adjuvant therapy after undergoing primary
surgery, radiation and adjuvant chemotherapy. Patients were
excluded if they had started TAM therapy simultaneously with
either adjuvant chemotherapy or adjuvant radiation therapy (or
both) or if they were undergoing other adjuvant endocrine
therapies. Patients who were pregnant or breast-feeding were also
excluded from the study. Enrolled patients were allowed to take
vitamin E, selective serotonin reuptake inhibitors (SSRIs) or herbal
remedies. Blood samples were collected within 3 to 60 months of
initiating TAM treatment.
Venous blood was collected from all 135 subjects before taking
their daily 20- mg dose of TAM. Ten milliliters of heparin plasma
were separated by centrifugation and immediately stored at
220uC until analysis. In addition, one milliliter of an EDTA blood
sample was taken for subsequent DNA extraction and genotyping.
DNA was isolated from peripheral leukocytes using a QIAamp
DNA Blood Mini KitH (Qiagen, Madrid, Spain) according to the
manufacturers instructions. The DNA concentration was
determined and adjusted to 220 ng/ mL.
The Roche-AmpliChipH CYP450 Test (Roche, Spain) was used
to identify 33 CYP2D6 alleles (including duplications and deletions)
in the plasma samples. The AmpliChip CYP450 Test microarray
serves to examine both sense and antisense strands of an amplified
target DNA sample. The test was powered by Affymetrix
microarray technology. In most patients, the presence of common
mutations was confirmed using a different genotyping method: a
long PCR product was used as a template to type alleles in
separate multiplex allele-specific PCRs, based on SBE with
fluorescent labeled ddNTPs (ABI Prism SNaPshot Multiplex Kit,
Applied Biosystems, USA).
The CYP3A4*3 variant allele was determined by PCR, using the
forward primer 59-TGG ACC CAG AAA CTG CAT ATG C-3
and reverse primer 59- GAT CAC AGA TGG GCC TAA TTG-3
under the PCR conditions described by van Schack et al. .
The nucleotides underlined are mismatches with the normal
CYP3A4 sequence that create a NsiI restriction site in the wild-type
CYP3A4 PCR product. The CYP3A4*17 variant allele was also
identified by PCR, using the forward primer
59-CTGGACATGTGGGTTTCCTGT-39 and reverse primer 59-
AGCAGTTATTTTTAAGAGAGAAAGATAAAT-39 followed by
digestion with the BpmI restriction enzyme as described by Lee
et al. . The CYP3A4*1B polymorphism, a single A to G
transition in the CYP3A4 promoter, was detected using the ABI
Prism 7700 Sequence Detection System and the standard SDS
allele discrimination protocol. To this end, a 104- bp PCR
product was amplified using the forward and reverse primers
59-GTGGAGCCATTGGCATAAAAT-3, respectively, and detected using the
fluorescently-labeled probes 59-6-carboxy-fluorescein
HEX-AATCG CCTCTCTCcTGCCCTTGTCT-BHQ13 for
the A and G alleles, respectively, according to the method of
Spurdle et al. . Results were confirmed by RFLP using the
forward 59-GGACAGCCATAGAGACAAGGGGA- 39 and
reverse 5-CACTCACTGACCTCCTTTGAGTTCA-39 primers
followed by digestion with the MboII enzyme . CYP3A5*3
was detected by the PCR-RFLP procedure described by van
Schaik et al.  using the Sspl restriction enzyme and the forward
and reverse primers 59-CATCAGTTAGTAGACAGATGA-39
and 59-GGTCCAAACAGGGAAGAAATA-39, respectively.
For the SULT1A1 gene, alleles *1 and *2 were identified by
PCR-RFLP using the HaeII enzyme and the forward and reverse
primers 59-GGTTGAGGAGTTGGCTCTGC-39 and
59-ATGAACTCCTGGGGGACGGT-39 respectively under the PCR
conditions described in Coughtrie et al. . For the SULT1A2
gene, two pairs of primers and two different restriction enzymes
were used to genotype alleles *1 (wt), *2 and *3, as described by
Arslan et al. . The PCR product amplified with the primers
R59CTGAGGTGAGCATGACCTCG-39 was digested with the
BstEII enzyme and the product amplified with
R59-GCCTCTGCAAAGTACTTGATGCG-39 was digested with BstUI. To genotype the SULT1E1
gene, samples were amplified with the primers F
59CTCCTTCTCTGGCATTCAGG-39 and R
59-CAACCTGTTTAGTTGATCCTGTG-39 and digested with the DdeI enzyme, as
described by Adjei et al. .
Any mutations detected were confirmed by repeating the
procedure or using a different technique whenever possible.
Samples were discarded if there was disagreement between the
methods or repetitions.
Quantifying Tamoxifen and its Metabolites in Plasma
Reagents and chemicals. Tamoxifen, 4-hydroxy-tamoxifen,
N-desmethyl-tamoxifen, N-desmethyl-4-hydroxy-tamoxifen (1:1
E/Z mixture), tamoxifen-N-oxide, tamoxifen-d5,
4-hydroxy-tamoxifen-d5, N-desmethyl-tamoxifen-d5, and
N-desmethyl-4-hydroxy-tamoxifen-d5 (1:1 E/Z mixture) were purchased from
Toronto Research Chemicals (North York, Ontario, Canada).
Acetonitrile, methanol, distilled water and formic acid were
obtained from Fluka Analytical (Sigma-Aldrich, Spain). All
chemicals used were of analytical grade. Small (1 mL) volumes
of drug-free human serum were pooled and used for validation
HPLC. TAM and its metabolites were separated and
quantified by high-pressure liquid chromatography-tandem mass
spectrometry using an Agilent HPLC 1200 system. HPLC
experiments were performed using a binary pump G1312A, a
G1316A column oven, G1379B degasser and an automatic
injector H-ALS G1367B. Mobile phase A and phase B consisted
of 0.1% formic acid in water and acetonitrile, respectively. Mobile
phases A and B were pumped through a ZORBAX Eclipse
XDBC18 column (150 mm62.1 mm I.D., 3.5 mm, Agilent USA) at a
flow rate of 0.2 mL/min using the gradient shown in Table 1.
Separation was conducted at 30uC. 15- mL aliquots were injected
and the autosampler needle was rinsed in acetonitrile/water
solution (1:1). The total run time was 20 min. During the first 4.0
and last 2.0 min, the eluate was removed using a divert valve to
avoid endogenous compounds entering the mass spectrometer.
As a detector, we used an A 6410 Triple Quadrupole mass
spectrometer (Agilent Technologies, USA) equipped with a heated
aMobile phase A: 0.1% formic acid in water.
bMobile phase B: acetonitrile.
Flow rate (mL/min)
Mobile phase Aa (%)
Mobile phase Bb (%)
electrospray ionization source (Thermo Fisher Scientific,
Waltham, MA, USA) operating in positive ion mode. For
quantification, multiple reaction monitoring (MRM) chromatograms were
acquired using Mass Hunter software version B.01.04 (Agilent
Technologies, USA). Positive ions were created at atmospheric
pressure. Quadrupoles operated at unit resolution (0.7 Da). The
HESI/MS/MS operating parameters and mass transitions are
provided in Table 2.
Calibration Standards, Quality Controls and Internal
Two separate stock solutions of all analytes (1 mg/mL) and
internal standards (1 mg/mL) were prepared by dissolving
accurately-weighed approximate 1 mg amounts in 1 mL of
methanol. One stock solution was used to prepare calibration
standards and the other stock solution to prepare quality control
standards. A mixture of internal standard stock solutions was
prepared and this mixture was diluted in acetonitrile to obtain a
working solution for sample pretreatment. This internal standard
working solution contained: tamoxifen-d5,
4-hydroxy-tamoxifend5, N-desmethyl-tamoxifen-d5 and
N-desmethyl-4-hydroxy-tamoxifen-d5 (1:1 E/Z mixture).
A 300- mL volume of 1% formic acid in acetonitrile containing
internal standards was added to a 100- mL serum aliquot. After
vortexing and centrifugation, the clear supernatant was transferred
to a HybridSPE column (Supelco) and the eluents stored at 28uC
until analysis. Samples were analyzed in triplicate.
Eleven non-zero calibration standards were prepared in
duplicate for each run and analyzed in three independent runs.
Calibration curves (area ratio obtained with the internal standard
versus nominal concentration) were fitted by least-squares linear
regression using the reciprocal of the squared concentration (1/
62) as a weighting factor. The intra- and inter-assay accuracy and
precision of the method were determined by assaying three
replicates of each of the quality control samples at the lower limit
of quantification (LLQ), at a low, a medium and a high
concentration level in three separate runs. The concentration of
each quality control sample was calculated using the calibration
standards that were analyzed in duplicate in the same run.
Differences between nominal and measured concentrations were
used to calculate accuracy. Accuracy should be within 85115%
and precision should not exceed 15% of the CV. Carry-over was
determined by injecting a processed control human serum sample
after an upper limit of quantification sample. Areas of peaks in the
blank processed sample should be within 20% of the peak area of
aMultiple reaction monitoring.
bLower limit of quantification.
the LLQ sample. Four individual batches of control human serum
were used to assess the specificity and selectivity of the method. To
determine whether endogenous constituents interfere with the
assay, a double blank and a sample spiked at the LLQ were
processed from these batches.
15.79% (SULT1A2*3) and 15.04% (SULT1E1*2) (Table 4). All the
CYP and SULT genes frequencies examined exhibited good
agreement with Hardy-Weinberg equilibrium. The data for some
patients (,3%) were discarded due to conflicting genotyping
Genotype frequencies, allele frequencies and Hardy-Weinberg
equilibria were determined using the Genepop software package (v
4.1). Descriptive statistics were calculated using standard methods.
Metabolic ratios were calculated as the concentration of substrate/
concentration of metabolite. The non-normal distribution of data
was confirmed by the Kolmogorov-Smirnov test. Levels of TAM,
metabolites and ratios between genotype groups were compared
using Wilcoxon-Mann-Whitney or Kruskal-Wallis tests. All
statistical tests (two-sided) were performed using SPSS software
(version 18.0 SPSS, Chicago, IL). Significance was set at a
The cohort examined was comprised of 135 Spanish patients
with BC from different geographic regions. Mean age was
52.33 years (SD = 9.90, range 30 to 81 years). Tumor types were:
51.7% infiltrating ductal carcinoma, 29.9% ductal carcinoma in
situ, 11.5% infiltrating lobular carcinoma and 6.9% both
infiltrating ductal carcinoma and ductal carcinoma in situ. Seven
patients (5.1%) were receiving concomitant CYP2D6 inhibitors
(fluoxetine, paroxetine or citalopram) and five patients (3.6%) were
receiving concomitant CYP2D6 substrates (propanolol and
CYP2D6 genotype and allele frequencies are provided in
Tables 3 and 4, respectively. The heterozygous polymorphism
occurring at the highest frequency was wt/*4 (15.04%, Table 3).
Null and intermediate allele frequencies of CYP2D6 variants were
as follows: 0.75% (*3), 11.65% (*4), 3.38% (*5), 0.38% (*6), 1.5%
(*9), 0.38% (*10), 0.75% (*17), and 3.76% (*41) (Table 3). For the
CYP3A4 gene, low frequencies of the defective alleles CYP3A4*1B,
CYP3A4*3 and CYP3A4*17 were observed (1.23%, 0.83% and
3.72%, respectively, Table 4). Conversely, a high incidence
(97.78%) was detected of the null CYP3A5*3 allele (Table 4).
Among the SULT genes, SULT1A2 was the most polymorphic,
showing up to 49.62% heterozygosity. Mutation frequencies in
SULT genes were 30.08% (SULT1A1*2), 30.83% (SULT1A2*2),
Plasma Concentrations of Tamoxifen and its Metabolites
The data for some patients were eliminated because of
conflicting results or technical problems. The mean TAM
concentration of the samples analyzed (n = 125) was
202.32694.55 ng/mL (median = 176.98 ng/mL). The mean
NDM-TAM concentration was over 2 times the TAM
concentration (450.546188.79 ng/mL; median = 418.46 ng/mL). Of
TAMs known clinically active hydroxylated metabolites,
endoxifen showed the highest mean concentration being almost three
times the 4OH-TAM mean concentration (24.75619.37 ng/mL
and median = 19.11 ng/mL vs. 9.0167.13 ng/mL and
median = 6.75 ng/mL). The mean Tamoxifen-N-oxide level was
50.88623.09 ng/mL (median = 48.81 ng/mL).
No significant differences were detected in mean plasma
concentrations of TAM, NDM-TAM, 4OH-TAM and
TAM-Noxide between patients receiving CYP2D6 inhibitors
concomitantly with TAM and those not receiving these inhibitors
(180.10661.25 ng/mL versus 203.63696.19 ng/mL (p = 0.68));
453.71695.47 ng/mL versus 450.386192.56 ng/mL (p = 0.60);
6.8164.69 ng/mL versus 9.1267.22 ng/mL (p = 0.46);
57.04624.25 ng/mL versus 50.51623.08 ng/mL (p = 0.44),
respectively. However, mean plasma endoxifen concentrations were
significantly lower in patients taking CYP2D6 inhibitors than
those not taking these drugs (15.55617.77 ng/mL versus
25.30619.39 ng/mL (p = 0.03)). These findings reflect the
importance of the CYP2D6 enzyme in the formation of endoxifen.
However, no significant differences in mean plasma endoxifen
concentrations were observed in patients under treatment with
other CYP2D6 substrates (i.e., likely tamoxifen competitors)
compared to those those not taking CYP2D6 substrates.
Correlating CYP and SULT Genotypes with Plasma
In Table 5 we provide the means, standard deviations, medians
and ranges of TAM and its metabolite concentrations for the
CYP2D6, CYP3A4 and CYP3A5 genotypes. Due to CYP2D6 gene
variability, genotypes were classified according to pairs of alleles as
different combinations of: wt including all extensive metabolizer
alleles (CYP2D6*1, CYP2D6*2 and CYP2D6*35), wtxN
including all ultraextensive metabolizer alleles (CYP2D6*1xN and
CYP2D6*2xN), P including the null alleles (CYP2D6*3,
CYP3A4 n = 121
CYP3A5 n = 135
SULT1A1 n = 133
SULT1A2 n = 133
SULT1E1 n = 133
(+)wt allele correspond to normal enzyme activity. In the case of CYP2D6 wt
includes *1, *2 and *35 alleles.
CYP2D6*4, CYP2D6*5, CYP2D6*6, CYP2D6*7, CYP2D6*8) and
I including intermediate metabolizer alleles (CYP2D6*9,
CYP2D6*10, CYP2D6*17, CYP2D6*41). In this manner, 7
different CYP2D6 genotypes were identified in the sample
analyzed: wt/wt, wtxN/P, wt/P, wt/I, I/I, I/P and P/P
(+)wt allele correspond to normal enzyme activity. In the case of CYP2D6 wt
includes *1, *2 and *35 alleles.
(Table 5). Endoxifen was the only metabolite that varied
significantly in the concentration among the different genotypes
for CYP2D6 (p = 0.026, Table 5). Product/substrate ratios were
estimated for the two active hydroxylated TAM metabolites.
Significant differences were observed among CYP2D6 genotypes in
plasma concentration ratios of endoxifen/NDM-TAM (p,0.001)
but not of 4OH-TAM/TAM. No significant differences in TAM
metabolite levels were observed among the genotypes for CYP3A4
and CYP3A5 (Table 5). For the SULT1A1, SULT1A2 and
SULT1E1 genotypes, Table 6 provides the means, standard
deviations, medians and ranges of TAM and its metabolite
concentrations. This table indicates no significant differences in
metabolite concentrations for the SULT1A1 and SULT1E1
genotypes. In contrast, endoxifen levels differed significantly
among the SULT1A2 genotypes (p = 0.027, Table 6) and
4OHTAM levels showed a similar trend, albeit not significant
(p = 0.056, Table 6), whereby higher concentrations of active
metabolites (SULT1A2 substrates) were observed in patients
carrying null alleles (*2, *3).
CYP3A4 n = 121
CYP3A5 n = 135
SULT1A1 n = 133
SULT1A2 n = 133
SULT1E1 n = 133
As may be observed in Table 5, having optimal plasma
concentrations of endoxifen seems dependent on carrying CYP2D6
wt alleles and genotypes were accordingly classified into the three
groups: wt/wt, patients with 2 or more copies of any functional
allele; wt/v, patients carrying one functional allele and one variant
-intermediate or null- allele; v/v, patients featuring intermediate or
null alleles. Among these CYP2D6 genotype subgroups, significant
differences were only observed for the endoxifen metabolite
(p = 0.001, Figure 1). Hence, when endoxifen concentrations were
pairwise compared with these CYP2D6 genotype groups,
significantly lower values were detected in v/v than in wt/wt or wt/
wt+wt/v (p,0.001 and p = 0.002, respectively). For the
comparisons wt/wt vs wt/v and wt/v vs v/v, endoxifen concentrations
were always lower in the groups with a smaller number of wt
alleles though significance was not reached (p = 0.076 and
p = 0.080, respectively). However, significant differences were
observed when endoxifen/NDM-TAM ratios were compared
among the same groups (p = 0.006 and p = 0.015, respectively).
For the SULT1A2 gene, patients were similarly stratified for
comparisons, considering the significantly higher endoxifen levels
observed in carriers of null alleles (Table 6). Thus, among the
SULT1A2 genotype subgroups (see Figure 1), significantly lower
levels of 4OH-TAM and endoxifen were conferred by the wt/wt
SULT1A2 genotype (p = 0.025 and p = 0.006, respectively,
Figure 1). Pairwise comparisons among these SULT1A2 subgroups
revealed significantly lower endoxifen levels in wt/wt compared to
wt/v and v/v patients (p = 0.007 and p = 0.006, respectively).
Further, similar results were obtained for the same pairwise
comparisons for the 4OH-TAM metabolite (p = 0.022 and
p = 0.012).
Prevalences of the commonly observed CYP2D6 genotypes and
alleles observed in our study are in good agreement with those
reported for other European populations [29, for instance] with
CYP2D6*4 being the most frequently detected null allele (12%,
Table 4). Several studies examining worldwide genetic variation in
the CYP2D6 gene have revealed that this allele occurs most
commonly in Caucasian populations (12%21%) . The
prevalence of CYP3A4 and CYP3A5 variants in different
populations varies considerably [31,32]. The scarce prevalence of
defective CYP3A4 alleles in the subjects examined here is consistent
with the data reported for other European populations of
Caucasian origin . In contrast, CYP3A5*3 polymorphism
appears at a high incidence (97.78%, Table 4) in European
populations, such as 94.35% in Greek , 94% in British  or
91.7% in Dutch  subjects. Similarly, SULT genes are
polymorphic and show variable allele prevalences among ethnic
groups . The rates of the defective alleles SULT1A1*2,
SULT1A2*3 and SULT1E1*2 determined in the present study
are also in agreement with figures provided for other Caucasians
populations [34,27,28, respectively].
The results of our study revealed plasma endoxifen
concentrations of 24.75619.37 ng/mL (mean) and 19.11 ng/mL (median)
in 125 women under treatment with TAM, which is slightly lower
than the levels detected by Borges et al.  and higher than those
reported in other studies [36,37]. Such variations are likely
attributable to differences in sample handling, storage and
measurement methods . When endoxifen plasma
concentrations were compared in patients taking or not taking selective
serotonin reuptake inhibitors (CYP2D6 inhibitors), the difference
was significant. Our patients who were taking both TAM and
SSRIs were wt/wt except two who were wt/P for CYP2D6. These
data are consistent with an effect of the CYP2D6 genotype on
endoxifen plasma concentrations, as described by other authors
[39,37], and suggest that pharmacogenetic variation in CYP2D6
activity may affect therapeutic outcomes of TAM treatment.
However, larger trials are needed to determine the clinical
implications of low circulating endoxifen concentrations.
When endoxifen plasma levels were compared according to the
presence of two wt, one wt or no wt alleles, significant differences
were detected in mean endoxifen concentrations between wt/wt
CYP2D6 and v/v CYP2D6 patients (p,0.001). Other authors have
also reported lower endoxifen concentrations in patients with the
v/v CYP2D6 genotype than those with the wt/wt genotype,
regardless of the alleles tested [39,40,41,42]. In our cohort, similar
endoxifen levels were noted in patients showing the wt/wt or wt/v
CYP2D6 genotype, in accordance with the findings of other studies
. Although endoxifen concentrations differed between wt/v
and v/v CYP2D6 patients, significance was not reached
(p = 0.080). Other authors have associated reduced CYP2D6
activity with a poor treatment outcome in terms of a higher risk of
recurrence and shorter time of recurrence-free survival .
CYP3A4 and CYP3A5 contribute to the biotransformation of
TAM into its primary metabolites: NDM-TAM and 4OH-TAM.
We detected no differences in TAM metabolite levels for the
variant alleles CYP3A4*1B, CYP3A4*3, CYP3A4*17 and CYP3A5*3
(Table 5). Although the possible relationship between CYP3A5*3
and TAM metabolism or clinical outcome of TAM therapy has
been addressed, no significant link has been so far detected .
We should highlight the absence of the wt/wt CYP3A5 genotype in
our cohort of women with BC. This polymorphism should be
assessed in future studies including a larger number of patients.
According to the results obtained by Mugundu et al. ,
microsomes reveal a marked NDM-TAM reduction in wt/*3
and *3/*3 CYP3A5 relative to wt/wt CYP3A5. In contrast, CYP3A4
polymorphisms do not seem to be relevant in TAM metabolism,
although some authors propose that certain combinations of
CYP3A4 and CYP2C9 allelic variants may contribute to a TAM
resistance phenotype .
While pharmacogenetic studies of SULTs have lagged behind
studies examining other enzyme families, it is becoming
increasingly clear that phase II drug metabolism plays an important role
in the response shown by an individual to therapeutic agents.
Sulfotransferases catalyze the formation of sulfated compounds of
4-hydroxytamoxifen and endoxifen . The sulfation of a
compound is considered to render it inactive, as sulfated molecules
are poor ligands for the estrogen receptor . However, prior
studies have provided contradictory information. For instance,
some authors observe no relation between SULT1A1 genotypes
and BC survival  while other authors have detected a strong
link between survival and the common SULT1A1*1 allele,
contrary to the expected outcome if a greater activity of the
enzyme does in fact lead to rapid removal of the drug from target
tissues . In our study, no association between serum levels of
TAM and its metabolites and SULT1A1 genotypes was observed
(Table 6), as noted by others . Despite some authors having
described that some TAM metabolites, for instance 4OH-TAM,
are more rapidly sulfated by SULT1E1 than by SULT1A1 ,
our results for the SULT1E1 genotype were very similar to those
recorded for SULT1A1, with TAM metabolite concentrations not
varying significantly between wild type and null SULT1E1
genotypes (Table 6).
SULT1A2 appears to be the most efficient human enzyme at
sulfating several aromatic compounds . Our results provide
evidence that carriers of null SULT1A2 alleles have significantly
higher plasma levels of 4OH-TAM and endoxifen, the two
hydroxylated substrates of the enzyme (p = 0.025 and p = 0.006,
respectively, Figure 1). Although the findings of some studies have
suggested a role of other SULT enzymes in TAM metabolism
[22,46], no previous study has addressed the relationship between
SULT1A2 and plasma concentrations of TAM metabolites. Our
results point to a possible benefit of carriers of alleles leading to
lower enzyme activity levels (SULT1A2*2 and SULT1A2*3) in
maintaining optimal levels of 4OH-TAM and endoxifen. Thus,
significant higher 4OH-TAM levels were recorded here in wt/v
and v/v than in wt/wt SULT1A2 patients (p = 0.007 and
p = 0.006, respectively). Similar results were obtained for
endoxifen levels (p = 0.022 and p = 0.012, respectively). Consequently,
only one defective SULT1A2 allele seems to be sufficient to slow
down the conversion of the two hydroxylated substrates into
sulfonated substrates. When CYP2D6 and SULT1A2 were
considered together, it was observed that some allelic variant
combinations of both genes seem to markedly affect an individuals
response to TAM therapy, as noted by other authors .
Accordingly, the wt/wt CYP2D6 genotype gave rise to endoxifen
levels of over two-fold those observed in patients with the v/v
CYP2D6 genotype and the wt/v and v/v SULT1A2 genotypes
rendered significantly higher levels of both endoxifen and
4OHTAM (Figure 1). One of the limitations of our study was the
sample size, although the number of patients analyzed is similar to
those included in other studies [43,45,47]. However, the small
numbers of some classes of genotypes compared might have led to
a low statistical power in some of the tests. It could therefore be
that some differences would have emerged if we had data for a
larger patient cohort. This issue will be no doubt resolved in future
In conclusion, our findings indicate that besides the CYP2D6
genotype inducing the conversion of TAM to potent hydroxylated
metabolites in a manner consistent with a gene-dose effect, the
SULT1A2 genotype also seems to play an important role in
maintaining optimal levels of both 4OH-TAM and endoxifen.
Consequently, patients who are wt/wt for CYP2D6 and also
feature the SULT1A2*2 or SULT1A2*3 alleles could be the best
candidates for a good response to TAM therapy in terms of
eliciting adequate plasma endoxifen and 4OH-TAM levels.
Indeed, CYP2D6 and SULT1A2 genotype distributions may partly
explain the wide interindividual variations detected in the
pharmacokinetics of TAM. Given that several other drugs and
enzymes may also affect TAM metabolism, we recommend
therapeutic drug monitoring in TAM trials designed to assess
(+)For the CYP2D6 genotypes:
wt includes all extensive metabolizer alleles (CYP2D6*1, CYP2D6*2, CYP2D6*35).
wtxN includes all ultraextensive metabolizer alleles (CYP2D6*1xN, CYP2D6*2xN).
P includes all null alleles (CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*6, CYP2D6*7, CYP2D6*8).
I includes all intermediate metabolizer alleles (CYP2D6*9, CYP2D6*10, CYP2D6*17, CYP2D6*41).
the effects of both CYP2D6 and SULT1A2 genotypes on treatment
The authors thank Teresa Bela and Pilar Conde for their help with the
blood sample collection process.
Conceived and designed the experiments: FB AF-S. Performed the
experiments: AR AN MG AT LMC. Analyzed the data: MR. Contributed
reagents/materials/analysis tools: AR AN MG AT LMC FB AF-S. Wrote
the paper: AF-S.
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