Prevalence of Epstein–Barr virus in North-East Indian breast cancer patients' blood

Kamalika Bhandari Deka; Pallavi Sarma; Anupam Sarma; Gaurav Das; Subhradip Karmakar; Goura Kishore Rath; Avdhesh Kumar Rai

doi:10.4103/aort.aort_24_21

ORIGINAL ARTICLE

Year : 2021 | Volume : 1 | Issue : 2 | Page : 111-115

Prevalence of Epstein–Barr virus in North-East Indian breast cancer patients' blood

Kamalika Bhandari Deka¹, Pallavi Sarma¹, Anupam Sarma², Gaurav Das³, Subhradip Karmakar⁴, Goura Kishore Rath⁵, Avdhesh Kumar Rai¹
¹ DBT Center for Molecular Biology and Cancer Research Dr. Bhubaneswar Borooah Cancer Institute, Guwahati, Assam, India
² Department of Pathology, Dr. Bhubaneswar Borooah Cancer Institute, Guwahati, Assam, India
³ Department of Surgical Oncology, Dr. Bhubaneswar Borooah Cancer Institute, Guwahati, Assam, India
⁴ Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
⁵ Department of Radiation Oncology, National Cancer Institute, National Cancer Institute, AIIMS, Jhajjar Campus, Haryana, India

Date of Submission	20-Oct-2021
Date of Decision	09-Nov-2021
Date of Acceptance	09-Nov-2021
Date of Web Publication	22-Dec-2021

Correspondence Address:
Dr. Avdhesh Kumar Rai
DBT Center for Molecular Biology and Cancer Research, Dr. Bhubaneswar Borooah Cancer Institute, Guwahati-16, Assam
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/aort.aort_24_21

Abstract

BACKGROUND: Breast cancer (BC) has been the most frequently diagnosed cancer among women and it has multiple risk factors. Epstein–Barr virus (EBV) has been shown to be associated with BC as one of the risk factors. Our aim was to detect EBV DNA in blood samples of BC patients of North-East India.
MATERIALS AND METHODS: A total of 53 eligible BC patients' samples were analyzed by Nested polymerase chain reaction for EBV Antigen-1 (EBNA1) gene. All statistical analysis was carried out using the software of statistical package for social sciences (SPSS) version 21.
RESULTS: 20.8% (11 of 53) BC patients' blood samples have EBNA1 gene DNA. The median age of BC patients was 42 and 43 years for EBV-positive and EBV-negative groups. 96.22% EBV-positive BC patients were diagnosed with high-grade malignancy. 54.54% of EBV-positive BC patients were diagnosed with T3 and T4 stage cancer. EBV-positive patients have not shown statistically significant association with ER positive (P-0.877, odds ratio [OR]: 1.111), PR positive (P-0.256, OR-2.16), Her-2/neu positive (P-0.93, OR-1.017), Ki-67 positive (P-0.487, OR: 1.8), and AR positive (p-1.000, OR-0.836).
CONCLUSION: We conclude from our study that EBNA1 gene was found in the blood samples of subset of BC patients of North-East India. Further studies with BC tissue and adjacent normal tissue samples of BC patients will be required to provide evidence about EBV's role with BC of North-East India.

Keywords: Breast cancer, Epstein–Barr virus antigen-1, Epstein–Barr virus, nested polymerase chain reaction, North-East India

How to cite this article:
Deka KB, Sarma P, Sarma A, Das G, Karmakar S, Rath GK, Rai AK. Prevalence of Epstein–Barr virus in North-East Indian breast cancer patients' blood. Ann Oncol Res Ther 2021;1:111-5

How to cite this URL:
Deka KB, Sarma P, Sarma A, Das G, Karmakar S, Rath GK, Rai AK. Prevalence of Epstein–Barr virus in North-East Indian breast cancer patients' blood. Ann Oncol Res Ther [serial online] 2021 [cited 2022 Aug 8];1:111-5. Available from: http://www.aort.com/text.asp?2021/1/2/111/333307

Introduction

Breast cancer (BC) is the most common malignancy, with 11.7% of newly diagnosed cases worldwide.^[1] BC is the most common cancer developed in women in India. According to the projected incidence of cancer statistics in India, in 2020, 1 in 29 females will be at risk for developing BC.^[2] Although several hormonal and reproductive risk factors have been identified and well established for BC development, studies have also suggested numerous viruses' contribution to the development of BC.^[3] The role of Epstein–Barr virus (EBV), mouse mammary tumor virus, bovine leukemia virus, and human papillomavirus (HPV) has been proposed in BC development.^[4],[5] EBV, a ubiquitous herpes virus, DNA (>50%) was detected in BC by multiple studies.^[6],[7],[8] Some studies reported EBV association in BC with aggressive behavior and lymph node metastasis.^[9],[10]

The association between EBV and BC has been projected based on high incidence of male BC in Mediterranean countries, EBV-associated lymphomas in breast and medullary carcinoma of the breast presented with morphological similarities with nasopharyngeal carcinoma.^[11] Chu et al.^[12] used multiple analytical methods for EBV detection and found 40% EBV-positive tumors by at least one method, but only 13% of tumors were EBV-positive when more than one method was used. EBV Antigen-1 (EBNA-1) is present in viral episome and is expressed in all known forms of viral latency. EBNA1 is essential for the replication, transcription, and maintenance of the virus genome and is the only viral protein expressed in all EBV-positive tumors.^[13] However, Deshpande et al. reported EBV negativity in BC tumor samples tested by in situ hybridization (ISH) and immunohistochemistry (IHC).^[14]

There is a lack of evidence about EBV DNA status in BC patients of North-East India.

Our present study aimed to detect EBV DNA in blood samples of BC patients of North-East India.

Materials and Methods

Patient selection

A total of 60 BC patients visiting the Outpatient Department of Department of Surgical Oncology at Dr. B. Borooah Cancer Institute during November 2020-August 2021 were enrolled with informed written consent. Following the inclusion criteria, 53BC patients were eligible for the study and 7 were excluded. All the enrolled BC patients were female with confirmed histopathology of BC. The age of enrolled BC patients ranged between 27 and 69 years. Enrolled BC patient demographic and clinical details were recorded in a structured questionnaire pro forma. The Institutional Ethics Committee of BBCI has approved the study (BBCI-TMC/Misc-01/MEC/178/2020).

Blood specimen collection and processing

Five-milliliter peripheral venous blood was collected in K2-EDTA vials from enrolled BC patients. The blood samples of the BC patients were processed using buffy coat enrichment procedure. Whole blood was centrifuged (Eppendorf, Germany) at 3500 rpm for 10 min at room temperature, and buffy coat layer (200 μl) was gently aspirated and transferred in a new sterile 1.5 ml microcentrifuge tube. Red blood cell (RBC) lysis buffer (×1) was added to the buffy coat cell suspension (5:1ratio) and gently vortexed. Tubes were incubated at −20°C for 10 min. Incubated tubes were centrifuged at 2500 rpm for 10 min at room temperature to obtain the white blood cell (WBC) pellet. The RBC Lysis step was repeated if RBCs were present after centrifugation from the previous step. PBS buffer wash was done for WBC pellet by centrifugation at 2500 rpm for 10 min. WBC pellets were stored in RNAlater solution at −20°C (Qiagen, Germany) until further processing for genomic DNA isolation.

Genomic DNA extraction

Genomic DNA (gDNA) was extracted from WBC of BC patients' samples. Two hundred microliter of NET buffer (×1) (Amresco, USA) and 20 μl of 10% SDS (Amresco, USA) were added to WBC and mixed thoroughly by pipetting. 10 μl of 20 mg/ml Proteinase K (Qiagen, Germany) was added to the cell suspension and kept in water bath for overnight at 56°C. Next day, additional 10 μl of Proteinase K was added and the tube was kept at 56°C in water bath for another 2 h. After the incubation period, the solution was centrifuged at 10,000 rpm for 10 min at, 4°C, and the supernatant was collected in a new sterile microcentrifuge tube. To the supernatant an equal volume of chloroform (1:1 ratio) was added and vortexed vigorously. The solution was centrifuged at 10,000 rpm for 10 min at 4°C. The upper aqueous layer (UAL) was collected carefully in a new 1.5 ml microcentrifuge tube. 1M NaCl (20 μl per 100 μl of UAL) solution and 2–2.5 volume (1M NaCl solution + UAL) of 96%–100% chilled ethanol was added. Tubes were gently mixed by inversion and incubated at -20°C for 30 min. After incubation, the solution was centrifuged at 10,000 rpm for 5 min. The precipitated DNA pellet was washed with 500 μl of 70% ethanol by centrifugation at 10,000 rpm, 5 min. This step repeated twice. Alcohol was carefully removed, and DNA pellet was air-dried for 15–20 min. DNA pellet was dissolved in 50–100 μl of 1X TE buffer and stored overnight at 4°C. Genomic DNA was assessed for quantity at 260, 280 nm wavelength using Biophotometer Plus nanospectrophotometer (Eppendorf, Germany). Ratio of 260/280 for DNA was 1.7–1.8. The quality of the genomic DNA was verified by 0.8% agarose gel electrophoresis (Bio-Rad, USA).

Epstein–Barr virus DNA detection by polymerase chain reaction

Genomic DNA was used as a template for the EBNA1 gene amplification. DNA sequence detection by Nested polymerase chain reaction (PCR). The total reaction volume was 20 μl using 2x Emerald MasterMix (Takara, Japan), 200nM EBNA1 gene primer sequences^[15] [Specific Primer Details Listed in [Table 1]]. PCR conditions for first EBNA1A were as follows: 95°C for 5 min followed by 36 cycles of 94°C-30 s; 56°C-1 min; 72°C-1 min and final extension at 72°C for 5 min. PCR product size was 609 bp. EBNA1A PCR products were the template for second EBNA1B gene amplification, and its PCR conditions were as follows: 95°C-6 min followed by 35 cycles of 95°C-20 s; 55°C-1 min; 72°C-1 min and final extension at 72°C for 7 min. PCR product size was 308 bp. Genomic DNA of nasopharyngeal cancer patient samples was used as positive control. PCR reaction without template DNA was used as negative control. 8 μl of PCR product were analyzed by 2% agarose gel electrophoresis.

Table 1: List of primers used to amplify the Epstein–Barr virus antigen 1 Gene sequence

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Statistical analysis

All statistical analysis was carried out using the software of Statistical Package for Social Sciences (SPSS) version 21 (PASW, IBM Corp., USA, 2012, IBM Corporation, Armonk, New York). Numerical data (age) were presented as mean ± standard deviation with standard error. Chi-square test was used to compare EBV-positive and EBV-negative BC patients' demographic and clinical characteristics. P < 0.05 was considered statistically significant. Bivariate logistic regression was used to calculate Odd Ratio (OR) at 95% confidence interval.

Results

A total of 53 eligible BC patients' samples were analyzed by Nested PCR for EBNA1A and EBNA1B [Figure 1]. 11 of 53 (20.8%) BC samples were positive for the EBNA1 gene. Clinical details of BC patients include their age, TNM stage, histopathology, and IHC status of ER, PR, AR, Her-2/neu, and Ki-67 [Table 2] and [Table 3].

Figure 1: Agarose gel electrophoresis of polymerase chain reaction amplification for the Epstein-Barr virus Antigen gene in BC patients. Lane 1 and Lane 5 shows amplification of 308 bp Epstein-Barr virus Antigen 1B gene fragment of Epstein-Barr virus from BC patient's sample land negative control includes distilled water. Lane positive control includes the Nasopharyngeal Carcinoma (NPC) sample. Lane L represents a 100bp ladder

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Table 2: Demographic and clinical characteristics of breast cancer patients

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Table 3: Histopathological details of the breast cancer patients

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The median age of the patients was 42, 43 years (range 27–69 years) for EBV-positive and EBV-negative [Table 2]. Among EBV negative BC patients, 53.57% (23/42) (P-0.99; OR-0.991) were diagnosed with tumor stages T3 and T4 whereas among EBV-positive BC patients, 54.54% (6 of 11 patients) were diagnosed with tumor stages T3 and T4. About 9% (1 out of 11) of EBV-positive patients had been diagnosed with metastasized BC. 72.7% of EBV-positive patients had nodal involvement [Table 2].

Seventy-six percent of the BC patients had infiltrating ductal carcinoma (IDC) (p-0.352; OR-0.367). 90% of the EBV-positive samples have IDC. 3.77% of BC patients have low-grade malignancy and 96.22% have high grade malignancy. EBV-positive patients have not shown statistically significant association with ER positive (P-0.877, OR: 1.111), PR positive (p-0.256, OR-2.16), Her-2/neu positive (P-0.93, OR-1.017), Ki-67 positive (p-0.487, OR: 1.8), and AR positive (P-1.000, OR-0.836) [Table 3].

Discussion

The mean age of the BC patients was 42.8 years, which is described as the young subset of BC patients.^[16] Young BC patients present a highly heterogeneous, potentially aggressive, and complex biological feature carcinoma.^[17] Similarly, Metwally et al. have found that among Egyptian BC women EBV, HPV, and HMTV was significantly associated with younger age.^[18] Although a statistically significant association between EBV positive and EBV negative group was indifferent to the lymph node involvement (P > 0.05). However, among EBV-positive BC patients, most were found to have axillary lymph node involvement. A study conducted at Chung Shan Medical University Hospital, Taiwan, also reported that lymph node positivity was not significantly different between the EBV positive and negative BC patients. However, it was observed that various viruses, including EBV, infecting the BC cells is significantly related to overall survival.^[19] As most EBV-positive BC patients were diagnosed with high-grade tumors, it may suggest a possible association between EBV and high-grade tumor. Four out of 59 breast tissue samples from the Iranian female BC patients have EBNA1 DNA sequence. All of the EBV-positive patients were diagnosed with high-grade malignancy.^[20] A significant association (P < 0.05) of the EBV genome with high histological grade tumors suggests EBV may play a role in development of aggressive tumors.^[9]

In our study, EBV-positive patients show no significant association with ER/PR expression status. Similarly, an EBV associative study on Pakistani BC patients also found no effect with ER/PR status among EBV-positive samples.^[4]

We observed Ki-67 positivity in most of the EBV-positive BC patients. In a retrospective study on BC patients, a high index proliferation-related activity (Ki-67 >50%) was observed in 53.5% EBV positive cases (15/28).^[21]

Among the first studies to demonstrate association of BC and EBV, using PCR in the DNA isolated from lymphocytes of BC patients, found 21%(19 out of 91 BC cases) of samples were positive for EBV.^[6] A study from India has shown 55% positivity in BC samples using IHC for EBNA1 in tissue samples.^[3] A possible causal association of EBV in BC was also observed in another study from India. 30.1% BC samples were positive for Epstein–Barr virus encoded RNA-ISH (EBER-ISH) test.^[22] 39.4% of BC patients stained slides of ductal and lobular tumor type displayed the presence of EBV.^[23] In a study done on the BC tissue samples of Syrian women using the tissue microarray technique, 51.58% patient tissues have shown expression of EBV.^[24] However, a higher percentage of EBV positivity may be attributed to the sensitivity of the technique used and the type of samples used for analysis.

However, no definite consensus has been established regarding EBV association with BC.^[7],[25] Studies from India have also reported the lack of expression of EBV among BC samples. One study in BC patients was conducted to establish an association of EBV by studying the EBNA1, LMP1, and LMP2A (latent membrane proteins) by using ISH for detecting nonpolyadenylated EBERs, along with using protein localization technique of IHC. The study found that none of the samples expressed the EBV viral gene products.^[14] The presence of contradictory reports on the association of EBV and BC, indicate the importance of viral etiology of EBV study to elucidate its role in BC progression and therapeutic response.

Limitations of our study are small sample size, use of one method of EBV detection. In our study, we found EBV DNA in a subset of BC patients of North-East India. Even though we used only blood samples to observe the EBNA1 gene in BC patients, we included the clinical and histopathological parameters to study the association of EBV with clinical characteristics of patients.

Conclusion

We conclude from our study that EBNA1 gene was found in the blood samples of subset of BC patients of North-East India. Further studies with BC tissue and adjacent normal tissue samples of BC patients will be required to provide evidence about EBV's role with BC of North-East India.

Acknowledgements

This study was supported by the National Centre for Disease Informatics and Research (NCDIR), Govt. of India, Bengaluru, India (Grant no.: NCDIR/CaRes/NER/6/2020/34). Authors express their gratitude towards Dr. Abhijeet Talukdar and Dr. Deep Jyoti Kalita from the Department of Surgical Oncology, Dr. B. Borooah Cancer Institute, Guwahati, India, for their immense support throughout the patient enrolment period and also Mr. Manoj Kalita, Population Based Cancer Registry, Dr. B. Borooah Cancer Institute, Guwhati, India for assistance in statistical analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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