Genetic Polymorphisms of the Efflux Transporter Gene ABCB1 and Their Effects on the Anastrozole Response in Iraqi Breast Cancer Patients

Abstract

Background: The ABCB1 gene (ATP-binding cassette) encodes the ABCB1 transporter. Anastrozole is asubstrate for the ABCB1 efflux transporter. The objective of the study was to detect the genetic polymorphismsof this efflux transporter in Iraqi breast cancer patients and their effects on the anastrozole response.Method: Blood samples were taken from breast cancer patients, and a portion of each sample was used forbiochemical analysis (measuring estradiol and cancer antigen 15.3 levels), while the remaining portion wasused for genetic analysis. Genotyping demonstrates that the mutant genotype being the most frequent typefor both C1236T and C3435T SNPs.Results: The result indicated nonsignificant differences in the serum levels of estradiol or cancer antigen15.3 among different genotype groups of the ABCB1 gene.Conclusion: It is concluded that the ABCB1 gene is highly polymorphic in Iraqi women with breast cancer.This study indicated that ABCB1 gene may not affect the response to anastrozole.

Full text

Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2 4401
Genetic Polymorphisms of the Efux Transporter Gene
ABCB1 and Their Effects on the Anastrozole Response in Iraqi
Breast Cancer Patients
Hiba Salah Mahdi1, Ahmed Salih Sahib2, Hassan Mahmood Mousa Abo Almaali3, Karrar Kadhim Mohsin4
1Bachelor in Pharmacy, Department of Pharmacology and Toxicology, College of Pharmacy, University of
Kerbala, Iraq, 2Professor, Department of Pharmacology and Toxicology, College of Pharmacy, University of
Kerbala, Iraq, 3Assistant Professor, Department of Clinical Laboratory Sciences, College of Pharmacy, University
of Kerbala, Iraq, 4Diploma, Imam Al-Hussein Hematology and Oncology Center, The Holy City of Kerbala, Iraq
Abstract
Background: The ABCB1 gene (ATP-binding cassette) encodes the ABCB1 transporter. Anastrozole is a
substrate for the ABCB1 efux transporter. The objective of the study was to detect the genetic polymorphisms
of this efux transporter in Iraqi breast cancer patients and their effects on the anastrozole response.
Method: Blood samples were taken from breast cancer patients, and a portion of each sample was used for
biochemical analysis (measuring estradiol and cancer antigen 15.3 levels), while the remaining portion was
used for genetic analysis. Genotyping demonstrates that the mutant genotype being the most frequent type
for both C1236T and C3435T SNPs.
Results: The result indicated nonsignicant differences in the serum levels of estradiol or cancer antigen
15.3 among different genotype groups of the ABCB1 gene.
Conclusion: It is concluded that the ABCB1 gene is highly polymorphic in Iraqi women with breast cancer.
This study indicated that ABCB1 gene may not affect the response to anastrozole.
Keywords Anastrozole, ABCB1 gene, estradiol, cancer antigen CA15.3, arthralgia.
Introduction
Breast cancer (BC) is one of the most frequent
cancers worldwide, with an elevated incidence rate in
all countries (1). In Iraq, BC is the rst among the top
ten malignancies affecting society. In 2016, hundreds of
females died from this disease, which is recorded as the
leading cause of cancer-related mortality among Iraqi
women (2). Of the risk factors that have been associated
with BC, such as family history, genetic mutations,
lifestyle and others, estrogen is a major factor in breast
cancer pathogenesis. Factors such as obesity, age at
menarche and menopause, and hormone replacement
therapy have been demonstrated to increase the risk of
BC (3).
Tumor markers are biomarkers that are produced at
higher levels by cancer cells or by the host in response
to cancer. One of these markers is cancer antigen (CA)
15-3, which is used to determine breast cancer prognosis
and to monitor the efcacy of therapy (4).
Because of the role of estrogen in carcinogenesis,
hormonal therapy targeted at decreasing estrogen
production or its action has been applied in various ways
to manage BC (3). Decreasing the quantity of estrogen by
inhibiting its production can be achieved with aromatase
inhibitors (AIs), such as anastrozole (5). Anastrozole is
a nonsteroidal inhibitor that inhibits aromatase enzyme
competitively by binding to the heme group of the
enzyme, thus decreasing estrogen biosynthesis in the
breast and periphery (6).
One study showed that anastrozole is a substrate
for ATP-binding cassette B1 (ABCB1) transporter (7),
an energy-dependent xenobiotic efux pump that is
expressed at the apical membrane in tissues that are
involved in excretion or form blood-tissue barriers.

4402 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2
Several studies have conrmed the notable impact of
drugs transport via ABCB1 on the pharmacokinetics of
these drugs in humans (8). Additionally, the ABCB1 gene
has been identied to have multiple single nucleotide
polymorphisms (SNPs) that effect on drug disposition
and clinical outcomes (9). Of these mutations, the most
well studied are two synonymous transitions (C1236T
and C3435T) located in exons 12 and 26, respectively
(10).
Patients and Methods
Study samples
This cross-sectional observational study included
Iraqi females with breast cancer taking 1 mg of
anastrozole by tablet daily. This study was carried out
at the Oncology Center in Kerbala at Imam AL-Hussein
Medical City.
The protocol of the study was approved by the
Ethical Committee of Pharmacy College at Kerbala
University, and each participant was given a written
informed consent form for their participation.
The study was conducted on 100 females aged 34-
76 years, who had estrogen receptor and/or progesterone
receptor (ER, PR)-positive breast cancer.
The exclusion criteria for this study involved:
taking other adjuvant endocrine therapies, taking
anastrozole therapy concomitantly with either adjuvant
chemotherapy or adjuvant radiotherapy (or both),
females with a history of gastrointestinal surgery or
disorders, women who took CYP3A4/5 or UGT1A4
inducers or inhibitors or any drugs affecting ABCB1
transporter, and females who were pregnant or lactating.
Clinical data collection
The clinical data were taken from the medical
records of the patients and from the women themselves
and included weight, height, age, workplace, academic
achievement, marital status, breast feeding, pre- or
postmenopause, family history of breast cancer, date of
breast cancer and duration, site, grade and stage of breast
cancer, presence of liver or any other diseases, and time
and duration on anastrozole.
Sample collection and analysis
A 5 ml peripheral blood sample was taken from
each female, 3ml of the blood was placed in a gel tube
and used to measure the biochemical parameters, and 2
ml was placed in an EDTA tube for the genetic assay.
Biochemical parameters
Estradiol level (E2)
E2 levels in the serum of breast cancer patients were
determined by using a CL-series chemiluminescence
immunoassay (CLIA) analyzer (Mindray/China). This
assay is a competitive binding immunoenzymatic assay
(11). The method was performed according to a kit.
Normal values:
Postmenopausal (<25-84 pg/ml)
Follicular phase (20-138 pg/ml)
Ovulation phase (100-440 pg/ml)
Luteal phase (31-317 pg/ml)
Tumor marker CA15.3
Levels of the serum tumor marker CA15.3 in breast
cancer patients were determined by using a Minividas
(Biomerieux/France) instrument, which utilizes the
enzyme linked uorescent assay (ELFA) technique (12,
13). The method was performed according to a kit.
Normal value: 0-30 U/ml.
Genotyping
Genomic DNA was extracted from blood samples
using the G-DEX™IIb kit (iNtRON/Korea).
ABCB1 gene polymorphism genotyping
C1236T and C3435T were detected using
amplication refractory mutation system PCR (ARMS
PCR). Primers (synthesized by Macrogen/Korea) were
used for C1236T and C3435T identication, and the
detection of alleles is shown in Table (1).

Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2 4403
Table (1) Primer sequences for determining the genotypes of C1236T and C3435T based on product size
(14).
SNPs Primer sequences Product size
1236P1 5ʼ AAT GTT CAC TTC AGT TAC CCA TCT CG 3ʼ 508
1236P2 5ʼ AAT GAT TTC CCG TAG AAA CCT TAC 3ʼ
1236C 5ʼ TGG TAG ATC TTG AAG CGC 3ʼ 305
1236T 5ʼ TGC ACC TTC AGG TTC TGA 3ʼ 238
3435P1 5ʼ TGC TGG TCC TGA AGT TGA TCT GTG AAC 3ʼ
300
3435P2 5ʼ GGC CAG AGA GGC TGC CAC AT 3ʼ
3435C 5ʼ GTG TCA CAG GAA GAG TTC 3ʼ 126
3435T 5ʼ TCC TTT GCT GCC CTC TCA 3ʼ 209
The PCR mixture was prepared in an AccuPower®
PCR PreMix (Bioneer/Korea) tube by adding 1 µl of
each primer at 10 pmol/µl, and 5 µl of DNA, and the
volume was brought to 20 µl with distilled water. PCR
was carried out with the following program: initial
denaturation for 3 minutes (min) at 94ºC, followed by
35 cycles of denaturation at 94ºC for 30 seconds (s),
annealing at 60ºC and 58 ºC for C1236T and C3435T
respectively for 30 s, and extension at 72ºC for 55
s. Final extension was performed at 72ºC for 5 min.
Amplied segments were separated by electrophoresis
on a 1.5% agarose gel, stained with ethidium bromide
and observed under ultraviolet (UV) light.
Statistical Analysis
For statistical analysis, SPSS software for Windows
(version 15.0 USA) was used. Single-factor ANOVA
was performed to examine the differences in the mean of
parameters tested within genotype groups. Odds ratios
and the corresponding 95% CIs were used to examine
associations. For all tests, p-values of <0.05 were
considered statistically signicant.
Results
Study Population
The general characteristics of the participants:
the mean age of women who participated in this study
was 57.51 (range: 34-76); the percentages of women
who were married and unmarried were 94% and 6%,
respectively; the menopausal status of the patients
was 95% postmenopausal and 5% premenopausal; the
proportion of women who depended on lactation to feed
their babies was 64%, while 19% did not lactate and 17%
undertook mixed feeding; the percentages of females
who had and did not have a family history of breast
cancer were 12% and 88%, respectively; some patients
presented with breast cancer on the right side (48%),
others on the left side (49%) and only 3% presented with
breast cancer on both sides; the percentage of patients
who were both ER- and PR-positive was 94%, while
only 6% of patients were ER- or PR-positive; and the
proportion of women who suffered from arthralgia (as a
side effect of anastrozole) was 89%, while 11% did not
have arthralgia.
Frequency and distribution of ABCB1 gene
polymorphisms
The percentage of ABCB1 genotypes detected in
100 Iraqi breast cancer patients are shown in Table (2).
For C1236T, the frequencies of patients who carried CC,
CT, and TT were 28%, 18%, and 54%, respectively. For
C3435T, 9% patients had the wild genotype (CC), 76%
had the mutant genotype (TT), and only 15% patients
carried the heterozygote genotype (CT). The most
frequent genotype for both SNPs was TT.

4404 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2
Table (2): The percentage of C1236T and C3435T detected in Iraqi breast cancer patients.
SNPs Genotypes Percentage
C1236T
CC (wild type) 28%
CT (heterozygote) 18%
TT (mutant type) 54%
C3435T
CC (wild type) 9%
CT (heterozygote) 15%
TT (mutant type) 76%
SNPs: single nucleotide polymorphisms, C: cytosine, T: thiamine.
Effect of ABCB1 gene polymorphisms on estradiol levels (E2)
Table (3) shows the mean and standard deviation (SD) of estradiol levels in the detected genotypes for C1236T
and C3435T SNPs. For C1236T, patients with CC had a lower mean level of E2 (19.482 ± 15.06 pg/ml), while
those with CT had the highest mean level of E2 (22.656 ± 8.02 pg/ml), and the mean level in those carrying the TT
genotype was 19.716 ± 9.717 pg/ml.
In C3435T, the mean and SD values of E2 in patients who had the CC, CT, and TT genotypes were 15.275 ±
6.817 pg/ml, 23.023 ± 18.803 pg/ml, and 20.2 ± 9.45 pg/ml, respectively. There were nonsignicant differences in
the mean levels of E2 and genotype groups for both SNPs.
Table (3) Mean and standard deviation of E2 levels in the detected ABCB1 (C1236T and C3435T) genotypes.
SNPs Genotypes
E2 level
Mean ± SD
Pg/ml
P value
C1236T
CC 19.482 ± 15.06 a
0.585CT 22.656 ± 8.02 a
TT 19.716 ± 9.717 a
C3435T
CC 15.275 ± 6.817 a
0.26CT 23.023 ± 18.803 a
TT 20.2 ± 9.45 a
P value derived from ANOVA test, p< 0.05 is signicant, p> 0.05 is nonsignicant. Same letters indicate nonsignicant
differences. SD: standard deviation.
Effect of ABCB1 gene polymorphisms on cancer
antigen CA15.3 levels
Table (4) shows the mean and standard deviation
values of CA15.3 levels in different genotype groups
for C1236T and C3435T. For C1236T, the mean and
SD of CA15.3 in patients with the wild genotype was
15.616 ± 6.26 pg/ml which is the lowest mean among
the different genotypes. The highest mean of CA15.3
appeared in the heterozygous genotype (23.235 ± 14.73
pg/ml). The mean and SD of CA15.3 in patients with

Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2 4405
the mutant genotype was 20.507 ± 16.446 pg/ml. There
were signicant differences in the mean levels of CA15.3
between the wild type and heterozygote genotypes,
while there were nonsignicant differences between the
wild type and mutant or between the heterozygote and
mutant genotypes.
For C3435T, there were nonsignicant differences
between the mean CA15.3 and genotype groups. The
mean and SD values of CA15.3 for CC, CT, and TT
were 14.438 ± 5.43 pg/ml, 14.882 ± 6.42 pg/ml, and
21.417 ± 15.93 pg/ml, respectively.
Table (4) Mean and standard deviation of CA15.3 levels for the detected genotypes of ABCB1 gene
polymorphisms.
SNPs Genotypes CA15.3 level
Mean ± SD P value
C1236T
CC 15.616 ± 6.26a
0.163CT 23.235 ± 14.73b
TT 20.507 ± 16.446ab
C3435T
CC 14.438 ± 5.43a
0.09CT 14.882 ± 6.42a
TT 21.417 ± 15.93a
SNPs: single nucleotide polymorphisms, CA15.3: Cancer Antigen 15.3, SD: standard deviation. P value derived from
ANOVA test, p< 0.05 is signicant, p> 0.05 is nonsignicant. The same letters indicate nonsignicant differences,
and different letters indicate signicant differences.
Association of ABCB1 gene polymorphisms and the elevation of CA15.3 levels
The association between the genetic polymorphisms in ABCB1 and the elevation in serum levels of CA15.3 was
determined by using odds ratios (Table (5)). For both SNPs, there was a nonsignicant association (p> 0.05) between
ABCB1 gene polymorphisms and the elevation in CA15.3.
Table (5) Association of ABCB1 gene polymorphisms (C1236T and C3435T) with the elevation of CA15.3
levels.
SNPs Odds ratio (CI-95) P value
C1236T 1.56 (0.427-5.721) 0.499
C3435T 1.477 (0.296-7.364) 0.633
CI-95: condence interval 95%, p< 0.05 is signicant.
Association of ABCB1 gene polymorphisms with the occurrence of arthralgia
Table (6) shows the association between ABCB1
gene polymorphisms and the onset of arthralgia.
For C1236T and C3435T, there were nonsignicant
associations with the development of arthralgia (p>
0.05).
Table (6) Association of ABCB1 gene

4406 Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2
polymorphisms (C1236T and C3435T) with the occurrence of arthralgia.
SNPs Odds ratio (CI-95) P value
C1234T 0.401 (0.099-1.611) 0.1979
C3435T 1.214 (0.295-4.994) 0.787
CI-95: condence interval. p< 0.05 is signicant.
Discussion
An in vitro study introduced by Miyajima et al.
indicated that anastrozole is a substrate for ABCB1
transporters (7). Because ABCB1 is a highly polymorphic
transporter, variability in this gene has been associated
with changes in drug response, disposition, and toxicity
(9). Therefore, to obtain an effective therapeutic response
and lower the side effects of anastrozole, it is important
to study the effects of genetic polymorphisms of ABCB1
on the therapeutic response (by measuring serum E2
and CA15.3 levels) in Iraqi women with breast cancer
treated with anastrozole.
The association between the development of breast
cancer and the continued increase in serum estrogen levels
has been studied by several researchers. These studies
suggest that estrogen is a mammary gland carcinogen (15).
Therefore, the use of aromatase inhibitors is a common
strategy to treat women with hormone receptor-positive
breast cancer (16). Although anastrozole was used as
an aromatase inhibitor, this study found a detectable
quantity of E2 in the serum of breast cancer patients that
was still within normal limits (Table 3), which suggests
that there may be other pathways for estrogen synthesis.
These results were in agreement with a study introduced
by Abd-Allateef et al. (2016), who found a detectable
concentration of estrogen in females with breast cancer
treated with anastrozole despite complete inhibition
of the aromatase enzyme (17). In this study, both
SNPs (C1236T and C3435T) exhibited nonsignicant
differences in the mean E2 among genotype groups, with
those who carried the heterozygote genotype CT (in both
SNPs) having the highest mean E2 level, although it was
still within normal limits. A study conducted on females
with breast cancer found that patients who carried TT
genotypes had signicantly lower ABCB1 expression
(18). Accordingly, the capacity of ABCB1 transporters
may be lowered, so patients with TT may respond better
to treatment than those with wild genotypes, although
this was not indicated in our results(20).
Serum tumor markers are soluble molecules present
in the blood that are discharged from tumor cells, and
they are usually used to determine the response to
anticancer drugs or determine prognosis because their
concentrations may indicate the presence of hidden
metastasis or reect the extent of the tumor mass
(19,21). In the present study, there were nonsignicant
associations between serum CA15.3 and the genotype
groups for both SNPs, except for the CT genotype of
C1236T, which showed a signicant difference in the
mean CA15.3 levels relative to the CC genotype (Table
4). (19)
This study revealed that there were nonsignicant
associations between different genotype groups of
the ABCB1 gene and the elevation of CA15.3 (Table
5), which may be because these SNPs (C1236T and
C3435T) are synonymous and do not alter amino acids
or affect activity (10).
Arthralgia, one of the common adverse effects of
anastrozole, usually affects quality of life and may lead
to the patient discontinuing treatment (22). In the present
study, most women suffered from arthralgia, but there
was nonsignicant association between the occurrence of
this side effect and ABCB1 gene polymorphisms (Table
6). This supports the ndings of a study by Gervasini et
al. (2017), who did not report a statistically signicant
association between C1236T and the occurrence of
arthralgia, although the same study found that C3435T
was inversely associated with arthralgia development
(23).

Indian Journal of Forensic Medicine & Toxicology, April-June 2021, Vol. 15, No. 2 4407
Conclusions
It can be concluded that the ABCB1 gene is highly
polymorphic, with mutant genotype TT being the most
frequent for both SNPs in Iraqi breast cancer patients.
This study revealed that ABCB1 gene polymorphisms
may not impact the anastrozole response.
Funding: This study is funded by individual
nancing.
Ethics approval: This study was approved by the
ethical committee of the College of Pharmacy at the
University of Kerbala.
Conict of Interest - The authors declare no conict
of interest.
Acknowledgment: The researchers would like
to acknowledge all women with breast cancer who
participated in this study and any individual who helped
us to complete this study.
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