Single Nucleotide Polymorphism of MDR1 C1236T Gene and Its

Association with Neutropenia Event in Breast Cancer Patients

Treated b

Chemotera

S. Syarifah

, T. Widyawati

D. Hasni

and D. R. Anggraini

Department of Pharmacology and Therapeutic, Faculty of Medicine, Universitas Sumatera Utara, Medan, 20155,

Indonesia

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

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

Keywords: Neutropenia, Polymorphism, Breast cancer.

Abstract: Neutropenia event was one of the side effect which could be occurred in breast cancer patients receiving

chemotherapy. 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 C1236T contribute to the

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

association of C1236T polymorphisms in MDR1 gene with neutropenia incidence in breast cancer patients

treated with antracycline based chemotherapy. As many as 144 Indonesian women’ isolated DNA samples

were amplified using the PCR method. The analysis process of MDR1 C1236T polymorphism were done by

using PCR-RFLP method. The frequencies of MDR1 C1236T genotype for homozygous CC, heterozygous

CT and variant TT was 13 (9,03%), 93 (64,58%), and 38(26,39%) respectively. There was no association

between MDR1 C1236T polymorphisms with neutropenia event (p > 0.05). However, 69 patients (47.9%)

.suffered for neutropenia event. Limitation: the data of patients were collected only after 3 cycles of

chemotherapy.

1 INTRODUCTION

The incidence of breast cancer increased every year,

especially in developing countries due to the

increased of life expectancy, lifestyle, urbanization,

and the majority of cases detected when it is already

achieved in advanced stage (World Cancer Research,

2015). Chemotherapy as one of the important things

in the management of breast cancer patients increased

life expectancy but also various side effects

(Vulsteke, 2013). Neutropenia event is one of the side

effects which can be harmful to the patients due to

increased of infection’s risk and delayed

chemotherapy (Fung, 2009).

The pharmacogenomic studies have contributed

significant advances on how genetic patterns can be

used to predict the efficacy and safety of

chemotherapy in breast cancer (Franke, 2010). The

presence of genetic polymorphisms in the MDR1

C1236T gene in exon 12 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 (Tazzite, 2016).

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

(Milojkovic, 2016).

Therefore, we evaluated the relationship of

C1236T 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 the role of pharmacogenomics in response to

treatment.

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

Single Nucleotide Polymorphism of MDR1 C1236T Gene and Its Association with Neutropenia Event in Breast Cancer Patients Treated by Chemoterapy.

DOI: 10.5220/0010092808330836

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

833-836

ISBN: 978-989-758-449-7

833

2 MATERIALS AND METHODS

We conducted a cohort study recruited 144 breast

cancer patients receiving anthracycline base

chemotherapy in Adam Malik Hospital.

2.1 Study Area

This study was conducted at Adam Malik Hospital

and Facullty of Medicine, Universitas Sumatera

Utara.

2.2 Samplings

Protocol of this study has been approved by Medical

Ethics Committee Universitas Sumatera Utara

(No. 386/TGL/FK/KEPK FK USU-RSUP

HAM/2018).

2.2.1 Recruiting Methods

Subjects of this study were purposively selected

according to the inclusion criteria. Most of the

subjects who had been diagnosed and treated by

anthracycline base chemotherapy at Adam Malik

Hospital were recruited for this study.

2.2.2 Subjects Characteristics

This study has recruited 144 breast cancer patients.

The characteristic of the patients fulfil the inclusion

criteria: had histologically confirmed breast cancer,

had been planned to receive anthracycline base

regimen of chemotherapy, ages 16– 68 years old, had

normal liver function and kidney function, had a

normal complete blood count (CBC).

2.2.3 Data Collection

The data of subjects’s characteristic and neutropenia

were collected from medical records for three cycles

of chemotherapy. The data of MDR1 C1236T

polymorphism were collected using PCR-RFLP

method. DNA Amplification of MDR1 C1236T using

GoTaq® Green Master Mix (Promega) of 12.5 μl,

primer of exon forward 12 MDR1 5-

'GCCACAGTCTGCCCACTC-3 'and reverse exon

12 MDR1 5-'CCCATCGAAAAGAAATTAAG-3',

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, followed by a 30-second

denaturation step, annealing stage, extension stage

and elongation stage for 10 minutes (Syarifah, 2016)

3 RESULTS

The results consist of characteristic of subjects,

frequency of allele and genotype of MDR1 C1236T

polymorphism, the association of MDR1 C1236T

polymorphism with neutropenia.

3.1 Characteristic of Subjects

This study include 144 subjects. We found five

ethnics include Bataknese, Malay,

Javanese,Acehnese and others (Tionghoa and India).

The majority of the subjects were Bataknese (52,8%),

the group of age were 40-50 years old (39,6%), had

job as housewives (63,9%). Most of the subjects were

detected at late stage (61,8%) and 69 patients suffered

for neutropenia after 3 cycles of chemotherapy

(47,9%)[Table 1].

Table 1: Characteristic of subjects

Variables N (%)

Group of age

<40 14(8.5)

40-50 57(39.6)

51-60 54(37.5)

>60 18(11.8)

Ethnic

Bataknese 76(52.8)

Javanese 40(27.8)

Acehnese 14(9.7)

Malay 11(7.6)

Others 3(8.4)

Job

Housewives 92(63.9)

Public Employee 35(24.3)

Private Employee 10(6.9)

Farmer 7 (4.9)

Stages of breast cancer

II 41(28.5)

III 89(61.8)

IV 14(9.7)

Degree of neutropenia

Normal 75(52.1)

Degree 1-2 52(36.1)

Degree 3-4 17(11.8)

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

834

3.2 Frequency of Allele and Genotype of

MDR1 C1236T Polymorphism

Frequency of allele and genotype can be seen below

[Table 2]

Table 2: Frequency of allele and genotype of MDR1

C1236T polymorphism

Polymo

rphism

Gen

otyp

n (%)

Allel

)

Hardy-

Weinber

g p

C1236T CC

(9.02)

(64.58

)

C 62 0,61

(26.38

)

T 38

3.3 The Association of MDR1 C1236T

Polymorphism with Neutropenia

The association of MDR1 C1236T polymorphism

with neutropenia can be seen below [Table 3]

C1236

Degree of Neutropenia

Total

Normal

Degree

1-2

Degree

3-4

n % n % n % n %

CC 7 4.8 5

13 34

32.

53.

14.

12.

Tot

100

P: 0.096 (Kruskal- wallis Test)

4 DISCUSSION

The C allele frequencies tend to be higher than T

allele in C1236T. 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.In this study, as shown in Table 3, 52

subjects (36%) had mild neutropenia (1-2 degrees)

and 17 people (12%) had severe neutropenia (grade

3-4). C1236T polymorphisms had no significant

association with neutropenia (p> 0.05). The results of

this study are in line with a study which shows no

significant association between MDR1

polymorphism and bone marrow suppression events

(Cizmarikova, 2010). Studies conducted) 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 (Chang,

2010). The results of this study contradict the studies

which indicate that the homozygous variant of TT has

a relationship to the incidence of severe neutropenia

(Taheri, 2010) (Sissung, 2006). The presence of a TT

variant is known to cause lower P-gp expression. The

absence of any association between MDR1 C1236T

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)

5 CONCLUSION

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

confirm and compare the results obtained with respect

to gene polymorphisms related to treatment response.

ACKNOWLEDGEMENT

The authors gratefully acknowledge to the

Universitas Sumatera Utara for supporting this study.

The research was supported by the research grant

TALENTA USU of The Year 2018 Contract Number

2590 / UN5.1.R / PPM / 2018, date 16 March 2018.

Single Nucleotide Polymorphism of MDR1 C1236T Gene and Its Association with Neutropenia Event in Breast Cancer Patients Treated by

Chemoterapy

835

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