The oncogenic potential of viruses such as Epstein-Barr virus (EBV), Human Herpesvirus 8 (HHV-8), BK polyomavirus (BKPyV), and JC Polyomavirus (JCPyV) in breast cancer remains under debate. This study aimed to investigate the presence of these viruses in breast tissues using molecular techniques. Formalin-Fixed Paraffin-Embedded (FFPE) breast tissue samples from 108 patients with invasive breast carcinoma and 100 controls (fibrocystic or normal tissue) were analyzed by quantitative PCR. EBV DNA positive samples were further evaluated using EBER in situ hybridization (EBER-ISH). EBV DNA was detected in 4.6% of cancer tissues (5/108) and 1.0% of controls (1/100), with not statistically significant (p = 0.214). All EBV DNA positive malignant cases were over the age of 40 and diagnosed with Invasive Ductal Carcinoma (IDC). No EBER expression was observed in any sample. HHV-8, BKPyV, and JCPyV were not detected in any tissue. Our findings suggest a low prevalence of EBV DNA in breast cancer and no evidence of other DNA viruses. While no causal relationship was established, the presence of EBV exclusively in older patients with invasive ductal carcinoma may merit further investigation with larger cohorts and multimodal analyses.

Highlights

•  Epstein-Barr virus DNA was detected in 4.6% of invasive breast cancer tissues.
•  No Human Herpesvirus 8, BK polyomavirus, or JC polyomavirus DNA was found in any breast tissue samples
• .Epstein-Barr encoded RNA transcripts were not detected in DNA-positive samples using in situ hybridization.
•  Findings suggest limited involvement of Epstein-Barr virus in breast cancer pathogenesis.
•  Combined molecular and histopathological approaches are recommended for future studies.

• Epstein-Barr Virus

• Breast Cancer

• Human Herpesvirus-8

• Polyomaviruses

• Polymerase Chain Reaction

Korkut G, Bakir A, Ar?kök AT (2025) Oncogenic DNA Viruses in Breast Cancer: Molecular Analysis of EBV, HHV-8, BKPyV, and JCPyV. JSM Surg Oncol Res 6(1): 1026.

Breast cancer is the most commonly diagnosed malignant neoplasm among women both globally and in Turkey. Worldwide, the incidence rate is approximately 46.8 per 100,000 women, while in Turkey it is slightly higher, at around 50 per 100,000 women. According to global statistics, breast cancer ranks fourth among all cancer-related deaths and is the second leading cause of cancer mortality among women [1].

Although the exact etiology of breast cancer remains unclear, various genetic, hormonal, environmental, and individual risk factors are known to play a role in its pathogenesis. These include genetic predisposition, hormonal influences, lifestyle choices, obesity, tobacco and alcohol use, as well as anxiety and stress. Approximately 70-85% of breast cancer cases are sporadic, while around 20% are associated with hereditary factors. Therefore, the potential role of environmental risk factors in the development of the disease warrants careful investigation. Among these, infectious agents, a category of environmental factors, are reported to be responsible for approximately 20% of all cancer cases [2].

An increasing number of scientific studies suggest that various oncogenic viruses may be associated with the development of breast cancer. Viruses such as Mouse Mammary Tumor Virus (MMTV), Epstein-Barr Virus (EBV), Bovine Leukemia Virus (BLV), Human Papillomavirus (HPV), Human Herpesvirus 8 (HHV-8), BK polyomavirus (BKPyV), JC Polyomavirus (JCPyV), and Cytomegalovirus (CMV) have all been implicated as potential contributors to breast carcinogenesis [3,4]. These viruses cause An increasing number of scientific studies suggest that various oncogenic viruses may be associated with the development of breast cancer. Viruses such as Mouse Mammary Tumor Virus (MMTV), Epstein-Barr Virus (EBV), Bovine Leukemia Virus (BLV), Human Papillomavirus (HPV), Human Herpesvirus 8 (HHV-8), BK polyomavirus (BKPyV), JC Polyomavirus (JCPyV), and Cytomegalovirus (CMV) have all been implicated as potential contributors to breast carcinogenesis [3,4]. These viruses cause latent infections and genetic instabilities by expressing oncoproteins that disrupt key regulatory mechanisms such as cell cycle checkpoints and apoptosis pathways. They facilitate persistent infection and immune escape by suppressing tumour suppressor pathways (p53, pRB) and overactivating cellular signalling pathways, and contribute to breast cancer by disrupting DNA damage response mechanisms and miRNA expression [5].

This study was conducted to enhance our understanding of the potential viral etiology of breast cancer. It aims to investigate the presence of EBV, HHV-8, BKPyV and JCPyV, as well as their oncoprotein expression, in Formalin-Fixed, Paraffin-Embedded (FFPE) breast tissue samples. Utilizing molecular techniques, the study seeks to elucidate the possible roles of these viruses in the pathogenesis of breast cancer.

Study group and study design

This study included 208 FFPE breast tissue samples obtained from female patients aged ≥18 years, diagnosed with invasive breast cancer, fibrocystic changes, or normal breast tissue at the Pathology Laboratory of Ankara Etlik City Hospital between November 1, 2022, and June 30, 2023. Based on histopathological diagnosis, patients were allocated into two groups: the study group (n=108, breast cancer cases) and the control group (n=100, comprising fibrocystic changes and normal tissue). Fibrocystic changes were classified as non-neoplastic and included in the control group due to their benign nature and lack of infectious etiology.

Clinical parameters, including age, tumor laterality, histopathological diagnosis, tumor grade, and BI-RADS score, were retrieved from the hospital information system. Tumor laterality was noted as right or left breast, and tumor grading was conducted using the Modified Bloom-Richardson system.

Exclusion criteria comprised pending pathology reports, insufficient FFPE material, duplicate samples, non-breast-origin metastatic tumors, prior history of breast cancer treatment, or post-treatment biopsies. Cases diagnosed with conditions other than breast cancer, fibrocystic changes, or normal breast tissue were also excluded.

All histological slides were re-evaluated by an experienced pathologist, and representative paraffin blocks were selected from the archive. For PCR analysis, 5 µm-thick sections were obtained using the HistoCore Multicut microtome (Leica Biosystems, Germany), transferred into microcentrifuge tubes, and stored at room temperature.

In Situ Hybridization (ISH) was performed exclusively on PCR-confirmed EBV DNA positive samples. For ISH, freshly cut 4 µm sections were prepared and processed on the same day. To avoid cross-contamination, the microtome blade was decontaminated with 70% ethanol and wiped with sterile gauze between each sample.

Molecular analysis

Deparaffinization process: FFPE breast biopsy specimens were treated with 1 mL of xylene, vortexed vigorously, and centrifuged at 10,000 rpm (11,180 × g) for 5 minutes. The supernatant was carefully discarded, and the xylene wash was repeated twice. The resulting pellet was resuspended in 1,000 µL of ethanol and centrifuged at 10,000 rpm for 3 minutes. After centrifugation, tube caps were opened, and residual ethanol was allowed to evaporate completely at room temperature over 10-15 minutes [6].

DNA extraction: Following deparaffinization, tissue pellets were incubated at 95°C for 3 minutes in a heating block (Four E’s Scientific Co. Ltd., China) according to the protocol of the Bio-Speedy Rapid Nucleic Acid Extraction Kit (Bioeksen R&D Technologies Inc., Istanbul, Türkiye). Subsequent DNA extraction was carried out using the Zybio EXM3000 Nucleic Acid Isolation System (Zybio Inc., China) in accordance with the manufacturer’s instructions.

DNA amplification: Genomic DNA samples were amplified using Bio-Speedy Quantitative qPCR Kits specific for EBV, HHV-8, BKPyV and JCPyV (Bioeksen R&D Technologies Inc., Türkiye), on the Magnetic Induction Cycler (Mic) PCR platform (Bio Molecular Systems - BMS, Australia), following the manufacturer’s protocols. The targeted gene regions and their associated viral species are summarized in Table 1.

Table 1: PCR targets.

Specifically, the assays amplified the BALF-5 gene (DNA polymerase) for EBV, ORF73 (LANA) for HHV-8, VP1-VP2 (capsid proteins) for BKPyV, and the agnoprotein gene for JCPyV. These loci were selected based on their high sequence conservation and diagnostic significance.

Each qPCR run included both positive and negative control samples. The adequacy of DNA content, presence of PCR inhibitors, and efficiency of DNA extraction in clinical samples were assessed using amplification of the RNAseP internal control.

Epstein-Barr enconded RNA in situ hybridization (EBER-ISH): In tissue sections where EBV DNA positivity was identified, EBER-ISH analysis was performed using the FORM EBER Probe and the VENTANA ISH/VIEW Blue Detection Kit (Roche Diagnostics GmbH, Germany). The analysis was conducted on the BenchMark IHC/ISH automated staining system. The procedure was carried out in accordance with the manufacturer’s protocol and included the following steps: deparaffinization of tissue sections, denaturation of nucleic acids, hybridization using a fluorescently labeled probe specific to EBER RNA, background staining, and the removal of non-specific probe binding.

During microscopic evaluation, the presence of a blue chromogenic reaction product within the target cells was interpreted as a positive result for EBV. To confirm the specificity of the hybridization signal and to exclude non-specific probe binding, a known EBV-negative breast tissue sample was used as a negative control. All positive and negative controls were processed concurrently with the study specimens under identical conditions.

Data analysis

Statistical analyses were conducted using SPSS (version 25). Data normality was assessed with the Kolmogorov Smirnov test, and descriptive statistics were reported as frequency (n), percentage (%), mean, minimum-maximum range, and Standard Deviation (SD).

Qualitative variables were analyzed using Pearson’s Chi-Square and Fisher’s exact test, while quantitative variables were assessed with the independent samples t-test. To evaluate the association between EBV and breast cancer, binary logistic regression analysis was performed, calculating the Odds Ratio (OR) with a 95% Confidence Interval (CI). Statistical significance was set at p<0.05.

In this study, FFPE breast tissue samples from a total of 208 female patients aged between 23 and 91 years were examined. Of the total biopsy specimens, 48.9% (100/208) were classified as benign, comprising 97 cases of fibrocystic changes and 3 samples of normal breast tissue. The remaining 51.1% (108/208) were diagnosed as invasive breast carcinoma.

All samples tested positive for internal controls, and the presence of EBV, HHV-8, BKPyV and JCPyV DNA was evaluated. Among the investigated viruses, only EBV DNA was detected, with a positivity rate of 2.9% (6/208; 95% CI: 1.1-6.2). No positivity was observed for HHV-8, BKPyV, or JCPyV.

As shown in Figure 1, EBV DNA was detected in 4.6% of malignant breast tissue samples and in only 1% of benign tissues, though the difference was not statistically significant (p = 0.214). Although the relative odds of EBV presence were elevated in patients with malignant tumors compared to the control group (OR: 4.80; 95% CI: 0.55 41.87), the association was not statistically significant (p = 0.155).

Table 2. Comparison of patient characteristics based on EBV DNA status.

The clinical and demographic characteristics of EBV-positive and EBV-negative patients are detailed in Table 2. Among EBV DNA-positive cases with malignant histopathology (n=5), all were over the age of 40 and diagnosed with invasive ductal carcinoma (IDC).

To determine the specific cell type harboring EBV (epithelial cells vs. lymphocytes), ISH was performed on the six EBV DNA-positive samples. However, no EBER expression was detected in any of the examined specimens (Figure 2).

Figure 2 Representative images of EBER-ISH. (a) Breast tissue section negative for EBER expression (40x magnification). (b) Positive control: Hodgkin lymphoma tissue demonstrating strong nuclear EBER expression (100x magnification)

The oncogenic potential of viruses in breast cancer remains a subject of debate, with EBV, HHV-8, BKPyV and JCPyV frequently investigated in this context. However, reported prevalence rates vary widely due to differences in detection techniques, tissue preservation methods, targeted genomic regions, and population-based factors.

EBV, a widespread herpesvirus infecting over 90% of the global population, is estimated to contribute to approximately 0.5-2% of all malignancies globally [7]. Its involvement in breast cancer is controversial, with reported positivity rates ranging from 4.6% to 60.7% [8,9]. In Turkey, EBV was detected in 16.2% and 23% of invasive breast carcinoma samples in two separate studies [10,11] while others have found no positivity at all [12, 13]. These discrepancies may arise from sample type (fresh-frozen vs. FFPE), methodological sensitivity (PCR vs. ISH), and variability in the amplified genomic regions (e.g., BALF5 vs. EBER or LMP1) [14,15].

In our cohort, EBV DNA was detected in 4.6% of malignant breast tumors and in 1.0% of benign controls, representing one of the lowest prevalence rates in the literature. Notably, all EBV DNA positive malignant cases occurred in patients over 40 years of age and were histologically classified as IDC. Despite positive PCR findings, EBER-ISH failed to demonstrate viral transcript expression in any case. As EBER-ISH is considered the benchmark for identifying latent EBV infection in tumor cells, this discrepancy suggests that the detected viral DNA may be transcriptionally inactive or localized in non neoplastic cells, such as infiltrating lymphocytes [16-18].

Moreover, unlike other EBV-associated malignancies, EBER expression in breast cancer cells is typically very low or absent, suggesting that EBV, if involved in breast carcinogenesis, may act through a distinct biological mechanism [19,20]

The limitations of FFPE-derived nucleic acids, which are often fragmented and degraded, may have further impacted detection sensitivity [21,22]. Additionally, the genomic region targeted in PCR assays significantly affects detection efficiency; for example, EBER-2 tends to be more sensitive than LMP1 [22]. Although IDC has been the primary focus of most research, including recent studies, no significant association has been identified between tumor grade and EBV positivity [15]. Regional differences also play a role, with higher detection rates frequently observed in Asian and African cohorts compared to North American populations [22].

In this study, HHV-8, BKPyV, and JCPyV DNA were not detected in malignant breast tumor tissues or in the control group. Although HHV-8 has been associated with certain malignancies, its relationship with breast cancer has been investigated in only a limited number of studies. In two studies conducted using FFPE tissues, HHV-8 positivity rates were reported as 3.7% and 12.3%, respectively [23,24]. Similar to our study, some other investigations have also failed to detect the presence of HHV-8 [25]. These findings indicate a lack of strong molecular evidence supporting a direct association between HHV-8 and breast cancer. In our study, HHV-8 DNA was not detected; this result may be related to the low prevalence of HHV-8 in Turkey [26]. Similarly, BKPyV and JCPyV were not observed, consistent with the generally low and inconsistent detection rates reported in breast cancer research [4,27-29]. Polyomaviruses may evade detection due to genetic variability that affects primer binding, highlighting the need for precise molecular assay design. Additionally, their low presence in tumors may be explained by a “hit-and-run” mechanism, where the virus initiates transformation but is no longer detectable afterward [30].

This study is subject to several limitations. The small number of EBV DNA positive cases limited the statistical power, despite a moderately elevated odds ratio (OR: 4.80, p = 0.155). Additionally, reliance on FFPE tissues may have reduced sensitivity, especially in samples with low viral loads. ISH was applied only to PCR-positive samples, and our single-gene, single-center design limits the generalizability of the findings.

Nonetheless, our data contribute to the growing body of evidence suggesting that EBV is infrequently present in breast cancer tissue, and that HHV-8, BKPyV, and JCPyV are likely not involved in breast cancer pathogenesis. The exclusive detection of EBV in older patients with IDC suggests possible histotype- or age-related patterns that warrant further investigation. Large-scale, multi institutional studies incorporating fresh tissue samples, multiplex detection platforms, and transcriptomic or proteomic analyses are needed to clarify the role of viral infections in breast tumorigenesis.

In this study, only EBV DNA was detected in breast cancer tissues, with low prevalence and no evidence of other viral agents. In EBV DNA positive samples, EBER expression could not be demonstrated by ISH. These findings suggest that the potential role of EBV in the pathogenesis of breast cancer may be limited. Further studies with larger samples and comprehensive molecular and histopathological analyses are needed to more accurately assess the relationship between viral infections and breast cancer.

The clinical research protocol received approval from the No. 1 Clinical Research Ethics Committee of Ankara Etlik City Hospital, with the decision dated 26.07.2023 and numbered AESH-EK1-2023-397.

GK: Conceptualization, Resources, administration, Methodology, Investigation, Data curation, Formal analysis, Writing - original draft, Writing - review and editing, Visualization, Supervision. AB: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Resources, Writing - original draft, Writing - review and editing, Visualization, Supervision, Project administration. ATA: Conceptualization, Methodology, Investigation, Data curation, Resources, Writing - original draft, Writing - review and editing, Supervision. All authors have read and approved the final version of the manuscript.

We would like to thank Bioksen R&D Technologies Inc., Türkiye, for providing PCR kit support in the study.

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