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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 2  |  Page : 180-183

Detection of Epstein–Barr virus in prostate tissue from prostatic cancer patients in Iraq


Department of Medical Microbiology, Al-Sadr Medical City Hospital, Kufa, Iraq

Date of Submission08-Apr-2021
Date of Acceptance01-May-2021
Date of Web Publication16-Jun-2021

Correspondence Address:
Anwar Abd Hashim Al-Ramahy
Department of Medical Microbiology, Al-Sadr Medical City Hospital, Kufa
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_57_21

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  Abstract 


Introduction: The prostatic tumor was the third form of cancer in males being more than 55 years of age, after the pulmonary and colon cancer. The prostate infected by many viruses causing repeated or chronic inflammation or enhance carcinogenesis. The aim of this study was to determine the presence and prevalence of Epstein–Barr virus (EBV) in the prostate tissue of patients with prostate cancer. Methods: Prostate tissue samples were collected from paraffin embedding block of 70 patients diagnosed with EBV from archives of AL-Sadder Hospital in AL-Najaf City and some archives of private histopathology laboratories from January 2016 to the December of the same year. We investigated the presence of EBV DNA by using the polymerase chain reaction (PCR). Results: The sample size was 60 patients and 10 as controls. The present study shows that 7/60 prostatic patients have EBV. Five patients of them have a malignancy prostatic tumor and only two with a benign prostatic tumor. On the other hand, there are 53/60 prostatic patients without EBV. Conclusions: EBV gene sequences were identified in benign and prostate cancer specimens. These sequences were located in the nuclei of prostate epithelial cells. The presence of EBV gene sequences in some of the benign and malignant prostate specimens is particularly noteworthy because of recent experimental evidence demonstrating that EBV can increase the proliferation of cultured cervical cells.

Keywords: Benign prostatic hyperplasia, Epstein–Barr virus, polymerase chain reaction, prostate cancer


How to cite this article:
Al-Ramahy AA. Detection of Epstein–Barr virus in prostate tissue from prostatic cancer patients in Iraq. Biomed Biotechnol Res J 2021;5:180-3

How to cite this URL:
Al-Ramahy AA. Detection of Epstein–Barr virus in prostate tissue from prostatic cancer patients in Iraq. Biomed Biotechnol Res J [serial online] 2021 [cited 2021 Jul 23];5:180-3. Available from: https://www.bmbtrj.org/text.asp?2021/5/2/180/318433




  Introduction Top


Prostate cancer is one of the life-threatening disorders and the most common malignancy seen worldwide in men with more than 160 000 new cases each year in the U. S. A.[1] It was also considered the most common cancer in males in 84 countries.[2] Viral infections contribute to approximately 12% of cancers worldwide, with the vast majority occurring in developing countries and areas. Human papillomavirus and Epstein–Barr virus (EBV) are associated with 38% of all virus-associated cancers.[3]

EBV is a double-stranded DNA virus belonging to the Herpes family (an oncogenic gamma-1 herpesvirus), infects more than 90% of the adult population worldwide, and its associated with close to 200,000 malignancies worldwide each year, contributing to several lymphoid malignancies, including T, B, and natural-killer cell lymphomas and epithelial carcinomas such as nasopharyngeal carcinoma.[4],[5],[6],[7],[8],[9],[10],[11]

Despite, EBV is a ubiquitous virus with known oncogenic potential, a possible association with prostate cancer has rarely been investigated. Only three reports in which EBV have been identified in prostate tissues. In Sweden, identified EBV in 31 of 352 (8.8%) benign and malignant prostate tissues.[12] In the U.S.A, identified EBV in 7 of 18 (37%) prostate adenocarcinomas, and 16 of 200 (8%) of normal, benign, and malignant prostate tissues in another study in the U.S.A.[13],[14]

The prevalence of EBV DNA in tumor tissue of patients with prostate cancer has been poorly investigated in Iraq. Therefore, the present study examined the prevalence of high- and low-risk EBV, using a polymerase chain reaction technique in AL-Najaf/Iraq.

Ethical approval

The study protocol was approved by the Ethical Committee University of Kufa Faculty of Science on September 17, 2017, approval number 3723. In addition, verbal approval was obtained from the patients before taking the sample.


  Materials and Methods Top


Patients

Paraffin embedding blocks were collected for 70 patients who were diagnosed with prostate cancer (20 malignant prostates, 40 benign, and 10 control) from archives of AL-Sadder Hospital in AL-Najaf City and some archives of private histopathology laboratories from January 2016 to the December of the same year.

Prostate cancer was confirmed by a pathologist through the standard criteria. Tumors were staged using standard criteria by the Gleason score. Demographic and medical information including age, habitat, and Gleason score was collected from patients' medical records.

DNA extraction

The step of DNA extraction according to the commercial kit (Geneaid Biotech Ltd.). To extract DNA, up to 25 mg of tissue cut into 10 mm fragments were incubated in 200 μl of GST and 20 μl of proteinase K than at 60°C overnight or until complete fragment digestion. Subsequently, the cell lysis was done by add 200 ml of Gel Extraction Buffer (GSB) Buffer and shake vigorously for 10 s, and the total product was stored at −20°C freezer to use for the detection of EBV.

Detection Epstein–Barr Virus EBNA1

The nucleotide sequences of the forward and reverse primer used for polymerase chain reaction (PCR) are 5-ATC-GTG-GTC-AAG-GAG-GTT-CC-3 and 5-ACT-CAA-TGG-TGT-AAG-ACG-AC-3, respectively.[15] PCR master mix was prepared by using (AccuPower PCR PreMix Kit) included 0.75 μl of each specific primer, 6 μl of PCR water, and 2.5 μl of DNA then placed in standard AccuPower PCR PreMix Kit that containing all other components which needed to PCR reaction such as (Taq DNA polymerase, dNTPs, Tris-HCl pH: 9.0, KCl, MgCl2, stabilizer, and tracking dye).

The amplification was reformed by including the reaction mix for 30 cycles in a thermocycler. Each cycle consisted of denaturation of DNA at 95°C for 30 s, followed by annealing at 56°C for 30 s, and extension at 72°C for 1 min with initial delay for 3 min at 95°C at the beginning of the first cycle and 5 min delay at 72°C at the end of the last cycle.

The reaction products were separated on 1% agarose gels with ethidium bromide at 100 V for 60 min. PCR products 209 bp were visualized using ultraviolet transilluminator, as shown in [Figure 1].
Figure 1: Agarose gel electrophoresis image appear the polymerase chain reaction product analysis of Epstein–Barr virus from in DNA extracted from formalin-fixed, paraffin-embedded (FFPE) prostate tissue section samples, where ladder (2000–100 bp), some positive DNA samples for Epstein–Barr virus at 209bp polymerase chain reaction product size

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Statistical analysis

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 21 (GraphPad Software, San Diego, California, USA). The groups of men patients were analyzed by the Chi-square. Values of P < 0.001 were measured to be statistically significant.


  Results Top


[Table 1] shows the characteristics of patients with prostatic disease, whereas the distribution of prostate cancer according to the presence of EBV using PCR is shown in [Table 2].
Table 1: Characteristics of the patients with prostatic disease

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Table 2: Distribution of prostate cancer according to the presence of Epstein-Barr virus by using polymers chain reaction

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It was observed the age range of patients between 50 and 90 years old. The resident of patients in urban was more than in rural (66.67% of prostate patients in urban). The sample size was 60 patients and 10 as controls. The number of benign patients is 40/60 (66.7%) while the number of malignancies patients is 20/60 (33.3%). There were five types of prostatic patients according to Gleason Grade for differentiating the prostatic cell carcinoma. The total number of malignant prostate was 20 and the distribution is as the following: Grade 1 was 6/20 (30%), Grade 2, 3, and 5 was 4/20 (20%), and Grade 4 was 2/20 (10%). The immunohistochemistry (IHC) test gave positive results of 13/20 of malignancy patients, while only 7/40 of benign patients gave positive results in IHC.

The present study shows that 7/60 prostatic patients have EBV. Five patients of them have a malignancy prostatic tumor and only two with a benign prostatic tumor. On the other hand, there are 53/60 prostatic patients without EBV. Fifteen patients with malignancy and 38 patients with benign prostatic patients. There is a highly significant associated with EBV with malignancy prostatic disease compared with benign(P = 0.001), but it is lowly associated with prostatic cancer (only 11.6% from prostatic cancer patients had EBV).

PCR results showed a highly significant specificity (P < 0.001) to detect the presence of EBV in malignancy and benign prostatic disease.


  Discussion Top


EBV gene sequences were identified in benign and prostate cancer specimens. These sequences were located in the nuclei of prostate epithelial cells. These outcomes are likely to be correct because of the use of several different analytical techniques on a range of specimens. The differences in the prevalence of EBV sequences between the fixed and fresh specimens may reflect the greater sensitivity of the PCR technique used on the fixed specimens.

Besides, these observations confirm the identification in three studies of EBV in prostate cancers.[12],[13],[14] The presence of EBV gene sequences in the specimens is particularly noteworthy because of recent experimental evidence demonstrating that EBV can increase the proliferation of cultured cervical cells.[16] However, these experimental studies were based on squamous cervical cells and any viral interactions may differ in glandular prostate cancer cells.

It is of interest that in this current study, it has long been recognized that EBV is ubiquitous, and only rarely is this virus associated with oncogenesis. The reason for EBV to exert its oncogenic influences in a particular patient is unknown but is probably associated with co-factors. In addition, these observations confirm the assessment of a previous study that the prostate is a habitat for multiple viral and other infections, some of which have oncogenic potential.[17]

Declaration of patient consent

The author certifies that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Litwin MS, Tan HJ. The diagnosis and treatment of prostate cancer: A review. JAMA 2017;317:2532-42.  Back to cited text no. 1
    
2.
International Agency for Research on Cancer, Geneva, Switzerland: Distributed by WHO Press, World Health Organization, 2014. (e book 2014) 1-169.  Back to cited text no. 2
    
3.
Shi Y, Peng SL, Yang LF, Chen X, Tao YG, Cao Y. Co-infection of Epstein-Barr virus and human papillomavirus in human tumorigenesis. Chin J Cancer 2016;35:16.  Back to cited text no. 3
    
4.
Sarwari NM, Khoury JD, Hernandez CM. Chronic Epstein Barr virus infection leading to classical Hodgkin lymphoma. BMC Hematol 2016;16:19.  Back to cited text no. 4
    
5.
Plottel CS, Blaser MJ. Microbiome and malignancy. Cell Host Microbe 2011;10:324-35.  Back to cited text no. 5
    
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Lieberman PM. Virology. Epstein-Barr virus turns 50. Science 2014;343:1323-5.  Back to cited text no. 6
    
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Brooks L, Yao QY, Rickinson AB, Young LS. Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: Coexpression of EBNA1, LMP1, and LMP2 transcripts. J Virol 1992;66:2689-97.  Back to cited text no. 7
    
8.
Dolcetti R, Dal Col J, Martorelli D, Carbone A, Klein E. Interplay among viral antigens, cellular pathways and tumor microenvironment in the pathogenesis of EBV-driven lymphomas. Semin Cancer Biol 2013;23:441-56.  Back to cited text no. 8
    
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Kutok JL, Wang F. Spectrum of Epstein-Barr virus-associated diseases. Annu Rev Pathol 2006;1:375-404.  Back to cited text no. 9
    
10.
Hjalgrim H, Askling J, Rostgaard K, Hamilton-Dutoit S, Frisch M, Zhang JS, et al. Characteristics of Hodgkin's lymphoma after infectious mononucleosis. New England J Med 2003;349:1324-32.  Back to cited text no. 10
    
11.
Cohen JI. Epstein–Barr virus vaccines. Clin Transl Immunol 2015;4:e32.  Back to cited text no. 11
    
12.
Bergh J, Marklund I, Gustavsson C, Wiklund F, Grönberg H, Allard A, et al. No link between viral findings in the prostate and subsequent cancer development. Br J Cancer 2007;96:137-9.  Back to cited text no. 12
    
13.
Grinstein S, Preciado MV, Gattuso P, Chabay PA, Warren WH, De Matteo E, et al. Demonstration of Epstein-Barr virus in carcinomas of various sites. Cancer Res 2002;62:4876-8.  Back to cited text no. 13
    
14.
Sfanos KS, Sauvageot J, Fedor HL, Dick JD, De Marzo AM, Isaacs WB. A molecular analysis of prokaryotic and viral DNA sequences in prostate tissue from patients with prostate cancer indicates the presence of multiple and diverse microorganisms. Prostate 2008;68:306-20.  Back to cited text no. 14
    
15.
Baer R, Bankier AT, Biggin MD, Deininger PL, Farrell PJ, Gibson TJ, et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 1984;310:207-11.  Back to cited text no. 15
    
16.
Fisher R, Melton S, Ferguson A, Oddo H, Velasco-Gonzales C, Hagensee M. The role of Epstein-Barr virus in cervical cancer: Exploring the immunological mechanisms for production of anti-Estein-Barr virus IgA in high risk women with dysplasia (HUM6P. 250). J Immunol 2015;194 Suppl 1:190-9.  Back to cited text no. 16
    
17.
Zambrano A, Kalantari M, Simoneau A, Jensen JL, Villarreal LP. Detection of human polyomaviruses and papillomaviruses in prostatic tissue reveals the prostate as a habitat for multiple viral infections. Prostate 2002;53:263-76.  Back to cited text no. 17
    


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