Association of Epstein Barr Virus Infection to Prognosis Factors of

Invasive Breast Cancer in Semarang Kariadi General Hospital

Population

Wasisto Dwi Yudisaputro

, Djoko Handojo

2 b

, Yan Wisnu Prajoko

and Dik Puspasari

Surgical Oncology Trainee, Faculty of Medicine University of Diponegoro / RSUP Dr. Kariadi Semarang

Department of Oncology Surgery, Faculty of Medicine University of Diponegoro / RSUP Dr. Kariadi Semarang

Department of Pathology Anatomy, Faculty of Medicine University of Diponegoro / RSUP Dr. Kariadi

Keywords: Breast Cancer, Prognosis Factors of Breast Cancer, EBV Infection, Molecular Detection of EBV

Abstract: Breast cancer is the most common malignant disease in female. In addition to internal factors, external factors

such as viral infections are thought to have a role in the carcinogenesis process of breast cancer. In the case

of nasopharyngeal cancer, Epstein-Barr Virus (EBV) has been shown to be able to activate several signaling

pathways so that cancer cells become more aggressive and have a worse prognosis. This study aims to

determine the relationship of the detection status of EBV DNA with the prognosis factor of breast cancer. The

research design used in this study was analytic observational with a cross-sectional approach, with 71 paraffin

block samples from the Anatomical Pathology Laboratory of Kariadi General Hospital who obtained from

mastectomy or Breast Conserving Surgery. Samples was examined using PCR to detect EBV DNA in tumor

cells. age of breast cancer cases ranged from 23 to 78 years with median age of 52 years. From 71 samples,

there were 28 positive samples (39.4%) of EBV DNA and 43 samples (60.6%) were negative. Significant

results were obtained (p < 0.05) in the relationship between EBV infection and tumor size (p = 0.002), axillary

lymph node metastasis (p = 0.001), and lymphovascular invasion (p = 0.001). Our research could find a

significant statistical association in the status of axillary lymph nodes, lymphovascular invasion status and

tumor size in breast cancer samples infected by EBV and those not infected by EBV..

1 INTRODUCTION

Breast cancer is the most common malignant disease

in female (Bray et al.,2018). The incidence of breast

cancer in Indonesia in female is the highest (Manuaba

, Burmansyah, and Tjindarbumi, 2010; WHO, 2014)

. In addition to internal risk factors such as genetic

and sex hormones, external factors such as viral

infections also play a role in the process of

carcinogenesis (Alibek et al., 2013; Ahmed et

al.,2019). The relationship of the epstein-barr virus

(EBV) with breast cancer is based on several reasons,

such as the high incidence of male breast cancer,

which is reported in Mediterranean countries,

endemic areas for EBV, the occurrence of several

https://orcid.org/0000-0002-9791-1200

https://orcid.org/0000-0003-1694-2054

https://orcid.org/0000-0002-1126-4939

https://orcid.org/0000-0002-8043-564X

EBV-related lymphomas in the breast, and

morphological similarities between breast medullary

cancers and nasopharyngeal cancer (KNF) (Zekri et

al., 2012). EBV is a family of herpesviruses and has

two variations (type 1 and type 2) (Joshi and

Buehring, 2012;. Hjalgrim, Friborg, and Melbye,

2007). In the human body, EBV will settle in B

lymphocytes, and from B lymphocytes EBV will

produce several viral proteins (LMP, EBNA, and

EBER) that can affect cell growth signals from its

host (Chang, Moore, and Weiss, 2017; Kim, Kim,

Park, 2013; Kanda, 2018;He et al., 2003). Some

authors believe that EBV might play a role in the

oncogenesis of breast cancer not as a major etiology

but as a cofactor in the development of breast cancer

and can influence the aggressive nature of breast

116

Yudisaputro, W., Handojo, D., Prajoko, Y. and Puspasari, D.

Association of Epstein Barr Virus Infection to Prognosis Factors of Invasive Breast Cancer in Semarang Kariadi General Hospital Population.

DOI: 10.5220/0010488701160120

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 116-120

ISBN: 978-989-758-499-2

cancer (Louise et al.,1 995;Murray et al.,2003;

Arbach et al.,2006; Mazouni et al., 2011)

Treatment modalities for breast cancer include

surgery, chemotherapy, hormonal therapy,

immunotherapy, and external radiation (Panigoro and

Purwanto, 2012). The success of the therapy given

will be related to predictive factors and prognostic

factors (Ramli, 2015; Cianfrocca and Goldstein,

2004) Several established prognostic factors such as

hormone receptors, Her2 expression, tumor size,

axillary lymph node metastases and lymphovascular

invasion can affect the aggressive nature of breast

cancer so that it will affect the survival rate and

disease-free interval (Carter et al., 1989; Fisher et al.,

1988)

With the development of molecular biology, EBV

infection in breast cancer can be determined by using

PCR examination by detecting the presence of EBV

DNA in cancer cells22,23. The purpose of this study

is to find out whether there is a relationship between

the detection status of EBV DNA and the prognosis

factor for breast cancer. The results of this study not

only can find out the prevalence of EBV infection in

breast cancer cases in Kariadi Hospital, but also can

be the basis for further research on the role of EBV in

the carcinogenesis of breast cancer.

2 MATERIAL AND METHODS

2.1 Patients and Tissue

The research design used in this study was analytic

observational with a cross-sectional approach. This

study consisted of a Formalin-Fixed Paraffin-

Embedded (FFPE) of cancer biopsy from 71 cases of

invasive breast cancer stage I to III that had been

performed mastectomy or Breast Conserving

Surgery. The samples were taken from the pathology

anatomy laboratory of Kariadi Hospital from January

2019 to September 2019. Each paraffin tumor block

will be extracted DNA and then DNA amplification

by PCR was done in the pathology anatomy

laboratory of FKKMK UGM. The primers used were

(forward primers

5‘GGCCTCCAAGGAGTAAGAC-C3‘ and reverse

primers: 5‘CCCCTCTTCAAGGGGTC-TAC3') that

were circulating on the market24. The DNA positive

control for EBV comes from KNF where the virus

was detected and the negative control uses nuclease-

free distilled water instead of DNA.

Histopathological data provided axillary lymph node

metastasis, tumor size, receptor hormone status, Her2

overexpression status, degree of malignancy and

lymphovascular invasion and were obtained from

medical record data and anatomical pathology report

of Kariadi Hospital.

2.2 Statistical Analysis

Data is processed using the SPSS 20.0 for Windows.

The Chi square test was used for parametric test, and

Mann Whitney test for non-parametric test to

determine the relationship between the detection

status of EBV DNA with the degree of malignancy,

estrogen receptor status, progesterone receptor status,

Ki 67 status, lymphovascular invasion status, Her 2

status, and axilla lymph node metastasis. The value of

the significance is <0.05.

3 RESULTS

Study found that some relationship between EBV

DNA status and prognosis factors for breast cancer

among the patients (Table 1).

Association of Epstein Barr Virus Infection to Prognosis Factors of Invasive Breast Cancer in Semarang Kariadi General Hospital Population

117

Table 1. Relationship between EBV DNA status and prognosis factors for breast cancer

Characteristics EBV (+) EBV (-) Mean ± SD Median (min – max) P Value

Age 49,97 ± 11,99 52 (23 – 78)

Tumor size

T1 0 0

T2 3 22 P = 0.002

T3 14 9



T4 11 12



Axillary lymph node metastasis

Negative 1 17

1 – 3 0 15 P = 0.001

4 – 10 18 8



>10 9 3



Estrogen Receptor

 

P = 0.605 Negative 10 18

Positive 18 25

Progesterone Receptor

P = 0.843

Negative 15 22

Positive 13 21

Her 2

P = 0,673

Negative 21 26

Positive 11 7

Ki 67

P = 0.750

< 20% 5 9

> 20% 23 24

Malignancy grade

P = 0.808

1 0 4

2 21 24

3 7 15

Lymphovascular invasion

P = 0.001

Negative 4 24

Positive 25 20

4 DISCUSSION

In this study the prevalence of breast cancer samples

which EBV Epstein Barr Virus (EBV) were detected

was 28 samples (39.4%). According to

epidemiological studies carried out by Huo Q et al. on

the basis of PCR examination, the prevalence of EBV

infection worldwide is 29.32% and prevalence at the

Asian level is 35.25% (Huo, Zhang, and Yang, 2012)

The prevalence of EBV DNA detection status in

this study has a higher rate than the prevalence

worldwide, or in Asia. The higher incidence can be

caused by demographic differences and the

characteristics of the research sample (Hjalgrim,

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(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

118

Friborg, and Melbye, 2007)

This study proves the link between the EBV

infection and the incidence of breast cancer, but only

a small proportion of positive EBV cases develop into

breast cancer. This shows that only EBV alone is not

enough as an etiological factor in carcinogenesis, a

number of biological and environmental cofactors are

also needed for the occurrence of breast cancer

(Alshammari, 2017).

The results of our study have significant statistical

association of EBV infection with tumor size. This

result is in accordance with some studies

conductedpreviously. Arbach in his research found

that EBV infection in breast cancer cells can increase

the ability of these tumor cell mutations such as

invasion, angiogenesis, and metastasis. As in the case

of nasopharyngeal cancer, the LMP-1 oncoprotein

produced by EBV induces cyclooxygenase 2 (COX-

2) which then induces vascular endhotelial growth

factor that plays a role in the process of angiogenesis

through NF-κB signaling (Arbach et al., 2015).

LMP-1 is directly related to oncogenesis, because

it is able to activate several cellular signaling

pathways such as Nuclear Factor-κB, c-Jun NH2-

terminal kinase (JNK), p38 kinase,

phosphatidylinositol 3-kinase (PI3K), and several

other possible pathways. thereby resulting in

continuous proliferation and inhibits apoptosis (Sun

et al.,2015)

One of the oncoproteins found in Epstein-Barr

virus is EBNA-1. The oncogenic role of EBNA-1

protein has been reported by some previous

researchers. EBNA-1 can reduce p53 levels by

triggering ubiquitin specific protease USP7 so that

p53 becomes unstable. In the end the breast gland

epithelium is infected with EBV so that the cells

become proliferated continuously and anti-apoptotic

(Frappier, 2012)

The results of our study have significant statistical

association of EBV infection with The metastatic

status of axillary lymph nodes and lymphovascular

invasion status. These findings come in agreement

with studies conducted by Fessahaye et al., 2017. Our

study shows that tumor cells contained in the EBV

gene have the potential for metastases to occur, which

suggest aggressive tumor behaviour, and making

them a poor prognostic factor. Arbach et al. who

showed that EBV infection of breast tumor enhances

its mutagenic properties, such as invasion,

angiogenesis, and metastasis.

5 CONCLUSION

The conclusion of this study is, there are significant

statistical association in the status of axillary lymph

nodes, lymphovascular invasion status and tumor size

in breast cancer samples infected by EBV and those

not infected by EBV.

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