The Association of G2677T Polymorphism in MDR1 Gene with

Neutropenia Incidence in Breast Cancer Patients Treated by

Doxorubicin based Chemotherapy

Siti Syarifah

, Dita Hasni

Tri Widyawati

and Dwi Rita Anggraini

Department of Pharmacology and Therapeutic, Faculty of Medicine, Universitas Sumatera Utara,

Medan, 20155, Indonesia

Department of Pharmmacology, Faculty of Medicine, Universitas Baiturrahmah, Padang, Indonesia

Department of Anatomy, Faculty of Medicine, Universitas Sumatera Utara, Medan, 20155, Indonesia

Keywords: Breast Cancer, Doxorubicin, MDR1, Polymorphism.

Abstract: MDR1 gene is a gene that encoded P-glycoprotein (P-gp), an active efflux pump for a variety of carcinogens

and cytostatics. It has been suggested that MDR1 polymorphisms G2677T contribute to the variability of

therapeutic outcome and side effects. The present study was conducted to investigate the relation of G2677T

polymorphisms in MDR1 gene with neutropenia incidence in breast cancer patients treated with doxorubicin

based chemotherapy. As many as 147 Indonesian women’ isolated DNA samples were amplified using the

PCR method. The analysis process of G2677T polymorphism were done by using PCR-RFLP method. The

frequencies of MDR1 G2677T genotype for homozygous GG, heterozygous GT and variant TT was 50

(34.01%), 78 (53.1%), and 19(12.9%) respectively. No association were found between MDR1 G2677T

polymorphisms with degree of neutropenia (p > 0.05). However, there was no significant deviation of allele

and genotype frequency from Hardy-Weinberg Equilibrium

1 INTRODUCTION

Breast cancer is the second most frequent cancer

experienced by women in both well developed and

developing countries with the number of newly

diagnosed cases being 1.7 million women in the

world.(World Cancer Research, 2015). The

administration of chemotherapy as one of the

important things in the management of breast cancer

patients increased life expectancy but also various

side effects (Vulsteke et al, 2013). One of the most

dangerous side effects of chemotherapy is bone

marrow suppression that can be measured through

absolute neutrophil count (ANC). The condition of

neutropenia may cause patients at risk for infection

and result in delayed chemotherapy (Fung et al,

2014).

Current studies have showed, the development of

medical technologies such as gene sequencing, DNA

analysis and popularization of the idea of

"personalized medicine" have contributed significant

advances on how genetic patterns can be used to

predict the efficacy and safety of chemotherapy in

breast cancer (Milojkovic et al, 2011). The presence

of genetic polymorphisms in the MDR1 G2677T

(rs2032582) gene in exon 21 which encoded P-

glycoprotein (P-gp) associated with the increased of

neutropenia incidence due to chemotherapy. P-gp is a

transporter protein that acts as an active effluent

pump for various toxins including carcinogens and

medicines such as antineoplastic drugs like

doxorubicin and taxan. Interestingly, P-gp is mainly

expressed in bone marrow and peripheral leukocytes,

the presence of P-gp in bone marrow and peripheral

leukocytes certainly has a protective effect of cells

against drug accumulation into cells (Taheri et al,

2010). Several studies showed the relation of MDR1

polymorphism with hematological toxicities, but the

results was inconsistent and there was no many data

about MDR1 polymorphism in Indonesia (Cascorbi et

al, 2011).

Therefore, we evaluated the relationship of G2677

polymorphism with degree of neutropenia both

individually breast cancer patients who treated by

doxorubicin based chemotherapy. The results of this

study are expected to provide information related to

428

Syarifah, S., Hasni, D., Widyawati, T. and Anggraini, D.

The Association of G2677T Polymorphism in MDR1 Gene with Neutropenia Incidence in Breast Cancer Patients Treated by Doxorubicin based Chemotherapy.

DOI: 10.5220/0010044204280431

In Proceedings of the 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and Technology (ICEST 2018), pages 428-431

ISBN: 978-989-758-496-1

the role of pharmacogenomics in response to

treatment.

2 MATERIAL AND METHODS

147 Indonesian women who met the inclusion criteria

were included in the study. The isolated DNA

samples were then amplified using the PCR method

[Syarifah et al, 2016]. DNA Amplification of Gen

MDR1 G2677T using GoTaq® Green Master Mix

(Promega) of 12.5 μl, F5’- TGC AGG CTA TAG

GTT CCA GG – 3’ and R5’- TTT AGT TTG ACT

CAC CTT CCC G – 3’ to amplify 224bp fragment.

respectively 1 μl, nuclease free water as much as 7.5

μl and 3 μl DNA with final volume is 25 μl. The

amplification process consisted of an initiation

denaturation stage of 5 min at 94°C, followed by a 30-

second denaturation step at 58°C at 35 cycles,

annealing stage for 30 seconds at 72

, extension stage

for 45 seconds at 72°C and elongation at 72°C for 10

minutes. [Sailaja et al, 2010].

The analysis process of G2677 polymorphism is

done by using PCR-RFLP method. [16] For SNP

MDR1 G2677T analysis, 5 μl of PCR product will be

rested with restriction enzyme Ban I (Promega) of 1

unit, incubated at 37°C for 1 hour. The distilled

fragment was then separated by electrophoresis on

4% agarose gel for 60 min at 90 mV and analyzed

after staining with Ethidium Bromide under UV light.

The electrophoresis pattern shows one band (272 bp)

for TT variant genotype, 2 bands (198 and 26 bp) for

homozygous GG genotype and two band (224 and

198 bp) for heterozygous GT genotype. Data on

neutropenia will be classified according to Common

Terminology and Criteria of Adverse Events

(CTCAE) v.4.0

Data analysis will use IBM SPSS ver.23.0,

Comparison of degree of neutropenia with MDR1

G2677T polymorphism will be calculated by using

Kruskal Wallis test. P < 0.05 was statistically

significant. The frequency distribution of alleles and

genotypes will use Hardy-Weinberg Equilibrium.

3 RESULT AND DISCUSSIONS

3.1 Characteristic of Subjects

Characteristics of subjects can be seen in Table 1.

Frequencies of GG, GT and TT genotypes were 50

(34.01%), 78 (53.1%), and 19(12.9%) respectively.

The electrophoresis pattern of MDR1 G2677T

polymorphism can be seen in Figure 1. We found five

ethnics include Bataknese, Malay,

Javanese,Acehnese and others (Tionghoa and India).

Distribution of MDR1 G2677T was varied among

these ethnics. Bataknese had the highest frequency

for three type of G2677T polymorphism which are

homozygous GG genotype (wildtype) with 25 (17%),

heterozygous variant (GT) with 44 (29.9%) and

homozygous variant (TT) with 7(4.7%)

Table 1. Characteristic of Subjects

Variables

N(%)

Group of age

<40

13(8.5)

40-50

63(41.2)

51-60

53(34.6)

>60

18(11.8)

Ethnic

Bataknese

76(49.7)

Javanese

39(25.5)

Acehnese

16(10.5)

Malay

12(7.8)

Others

4(2.6)

BMI Classification

Underweight

2(1.3)

Normal

60(39.2)

Overweight

63(41.2)

Obese

22(14.4)

Stages of breast cancer

II A

15(9.8)

II B

26(17.0)

III A

51(33.3)

III B

41(26.8)

14(9.2)

Histopathology of breast cancer

Infiltrative ductal carsinoma

14(9.2)

Invasive ductal carsinoma

133 (90.8)

Histopatology grading

Unknown

14 (9.5)

Grade I

41(27.9)

Grade II

70(47.6)

Grade III

22(15.0)

The Association of G2677T Polymorphism in MDR1 Gene with Neutropenia Incidence in Breast Cancer Patients Treated by Doxorubicin

based Chemotherapy

429

Figure 1: Electrophoresis pattern of MDR1 G2677T by

PCR-RFLP: GG genotype (B-E), TT genotype (I), GT

genotype (F,H,K), 25 bp DNA ladder marker (1),

Undigested PCR product (A).

3.2 Frequency of Allele and Genotype

of MDR1 G2677T Polymorphism

Frequency of allele and genotype can be seen in Table

2 below.

Table 2: Frequency of allele and genotype of MDR1

G2677T polymorphism.

Polymorphism

Genotype

n (%)

Allele

(%)

Hardy-

Weinberg

G2677T

(34.01)

78 (53.1)

0,56

19 (12.9)

The G allele frequencies tend to be higher than T

allele in G2677T. Based on the distribution of

polymorphism, the frequency of alleles and genotype

in this study more closely related to Asian

populations than Caucasians. In this study, p> 0,05

shows that there is no significant genotype and allele

frequency deviation based on Hardy-Weinberg

Equilibrium.

3.3 The Association of MDR1 G2677T

Polymorphism with Neutropenia

Degree of Neutropenia

Total

Polymorphis

Norma

Degree

1-2

Degree 3-4

16.

12.

5.4

0.09

30.

18.

4.1

53.

4.1

6.8

12.

Total

37.

11.

147

100

Kruskal-

wallis Test

In this study, as shown in Table 3, 55 people (37.4%)

had mild neutropenia (1-2 degrees) and 17 people

(11.5) had severe neutropenia (grade 3-4). G2677T

polymorphisms had no significant association with

neutropenia (p> 0.05). The results of this study are in

line with a study by Cizmarikova et al (2009) which

shows no significant association between MDR1

polymorphism and bone marrow suppression events.

Studies conducted by Chang et al (2008) in 121

cancer patients who received paclitaxel

chemotherapy also showed that there was no

association between MDR1 polymorphism with the

incidence of neutropenia of grade 3 and 4. The results

of this study contradict the studies conducted by Tran

et al (2010) and Sissung et al (2006) which indicate

that the homozygous variant of TT has a relationship

to the incidence of severe neutropenia.

The presence of a TT variant is known to cause

lower P-gp expression. The absence of any

association between MDR1 G2677T with the

occurrence of neutropenia may be due to other

influencing factors such as the presence of other gene

polymorphisms and the influence of MDR1 C3435T,

C1236T polymorphism, it is known that the common

haplotype in the MDR1 gene were C3435T, C1236T

and G2677T (Syarifah, 2016)

4 CONCLUSIONS

In this study, all forms of GG, GT and TT

polymorphisms in G2677T were found. There was no

significant association between MDR1 G2677T

polymorphisms with neutropenia grading. Advanced

research is needed with larger sample quantities to

1 A B C D E F G H I J K

ICEST 2018 - 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and

Technology

430

confirm and compare the results obtained with respect

to gene polymorphisms related to treatment response.

Conflict of Interest Statement

The authors declare that there are no conflicts of

interest.

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