Epstein-barr virus (EBV) in cervical carcinoma detected by in situ hybridization targeting ebers and the viral genome.

High-risk human papillomavirus (HR-HPV) is etiologically associated with the development and progression of cervical cancer, although other factors are involved. Epstein-Barr virus (EBV) detection in premalignant and malignant tissues from uterine cervix has been widely reported; however, its contribution to cervical cancer development is still unclear. Here, a comprehensive analysis regarding EBV presence and its potential role in cervical cancer, the frequency of EBV/HR-HPV coinfection in uterine cervix and EBV infection in tissue-infiltrating lymphocytes were revised. Overall, reports suggest a potential link of EBV to the development of cervical carcinomas in two possible pathways: (1) Infecting epithelial cells, thus synergizing with HR-HPV (direct pathway), and/or (2) infecting tissue-infiltrating lymphocytes that could generate local immunosuppression (indirect pathway). In situ hybridization (ISH) and/or immunohistochemical methods are mandatory for discriminating the cell type infected by EBV. However, further studies are needed for a better understanding of the EBV/HR-HPV coinfection role in cervical carcinogenesis.

Introduction High risk human papillomaviruses (HR-HPVs) has a possible role in development and pathogenesis of breast cancer (BC) disease. Other oncogenic viruses showed to be linked to BC such as Epstein Barr virus (EBV), and Human mammary tumor virus (HMTV). Presence of high risk HPV genotypes, EBV or HMTV could affect the severity course of BC. Therefore, the aim of the current study is to explore distribution of HPV with regards to its genotypes and the presence of EBV and HMTV among Egyptian BC women with its correlation to age and hormonal status. Patients & Methods This study was conducted on 40 fresh tissues of BC patients and blood samples from 30 apparently healthy controls. All samples were investigated for detection of HPV, EBV and HMTV DNAs using qualitative PCR. Detection of HPV viral load was performed using quantitative real time PCR. HPV genotypes were determined on 10 purified positive PCR product using Sanger sequencing of HPV L1 region. The obtained sequences were compared to the known HPV DNA sequences at National center for biotechnology information (NCBI) and/or Papillomavirus Episteme (Pave) data bases using their online BLAST and taxonomy tools. Data were correlated with age and hormonal status. Results All the tested 3 oncogenic viruses were not detected into 30 controls. HPV, EBV and HMTV were detected in 20 (50%), 23 (57.5%), 28 (70%) respectively of the 40 BC patients by using PCR assay. HPV median load of the 20 positive samples was 4800 copies/μL. Seven different HPV genotypes were detected included high risk genotypes ((HPV 18 in 3 samples), 70 and 52), low risk genotype (11) detected into only one sample along with 3 other genotypes (102, 114 and 151). One sample showed no significant similarity with any sequence registered in the used databases. Presence of EBV and HMTV DNAs and high risk HPV genotypes was more associated with young age (lower than 50 years), P value = 0.015, 0.001 and 0.05, respectively but were not associated with hormonal status of BC patients. Conclusion HMTV was the most detected viral DNAs among BC patients, High risk HPVs were identified into 10% of studied Egyptian BC patients and were associated with young age. Further larger sample size study is needed to evaluate role of HPV genotypes on pathogenesis and outcome of BC disease. STDF Acknowledgment This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant No.22944. Citation Format: Nasra F Abdel Fattah, Shimaa A Metwally, Samah A Loutfy, Maha A Abo-Shadi, Ahmed B Barakat, Omar A Rabee, Ahmed M Osman, Amany M Helal, Tarek M Hashem, Manar M Moneer. Molecular detection and genotyping of human papillomavirus in presence of some oncogenic viruses among Egyptian breast cancer women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2264.

High-risk human papillomaviruses (HR-HPVs) are known to contribute to cervical cancer (CC), but the role of Epstein-Barr virus (EBV) in this process remains unclear, despite EBV's widespread detection in premalignant and malignant cervical tissues. In this cross-sectional study of 258 cervical samples, including both formalin-fixed paraffin-embedded (FFPE) and fresh cervical tissues, the presence and viral load of HR-HPVs (HPV-16 and HPV-18) and EBV were evaluated in Iranian women with cervical intraepithelial neoplasia (CIN), squamous cell carcinoma (SCC), and a cervicitis control group using real-time PCR. The study revealed a significant correlation between disease severity and both increased HPV-16 positivity and HPV-16 and HPV-18 co-infection (p<0.001). Interestingly, the control group had a higher frequency of EBV-positive cases than SCC/CIN groups (p<0.001). HPV-16 DNA load increased with disease severity (P<0.001), while HPV-18 showed no significant difference (P=0.058). The control group had a higher EBV DNA load compared to SCC/CIN groups (P=0.033). HPV-16 increased the risk of CIN II, CIN III, and SCC, while HPV-18 increased the risk of CIN II and CIN III. Notably, EBV was associated with a lower risk of CIN groups and SCC. No significant difference in EBV co-infection with HPV-16/18 was found, failing to support the hypothesis that EBV is a cofactor in CC. However, high EBV viral load in the control group suggests a potential "hit and run hypothesis" role in CC progression. This hypothesis suggests that EBV may contribute briefly to the initiation of CC with an initial impact but then becomes less actively involved in its ongoing progression.

BackgroundDespite the fact that the implication of human papillomavirus (HPV) in the carcinogenesis and prognosis of cervical cancer is well established, the impact of a co-infection with high risk HPV (HR-HPV) and Epstein-Barr virus (EBV) is still not fully understood.MethodsFifty eight randomly selected cases of squamous cell carcinomas (SCC) of the uterine cervix, 14 normal cervices specimens, 21 CIN-2/3 and 16 CIN-1 cases were examined for EBV and HPV infections. Detection of HR-HPV specific sequences was carried out by PCR amplification using consensus primers of Manos and by Digene Hybrid Capture. The presence of EBV was revealed by amplifying a 660 bp specific EBV sequence of BALF1. mRNA expression of LMP-1 in one hand and protein levels of BARF-1, LMP-1 and EBNA-1 in the other hand were assessed by RT-PCR and immunoblotting and/or immunohischemistry respectively.ResultsHR-HPV infection was found in patients with SCC (88%), low-grade (75%) and high grade (95%) lesions compared to only 14% of normal cervix cases. However, 69%, 12.5%, 38.1%, and 14% of SCC, CIN-1, CIN-2/3 and normal cervix tissues, respectively, were EBV infected. The highest co-infection (HR-HPV and EBV) was found in squamous cell carcinoma cases (67%). The latter cases showed 27% and 29% expression of EBV BARF-1 and LMP-1 oncogenes respectively.ConclusionThe high rate of HR-HPV and EBV co-infection in SCC suggests that EBV infection is incriminated in cervical cancer progression. This could be taken into account as bad prognosis in this type of cancer. However, the mode of action in dual infection in cervical oncogenesis needs further investigation.

Epstein Barr virus (EBV) has been incriminated in the pathogenesis of Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study aimed to investigate the prevalence and the pattern of the expression of Epstein-Barr in HL and NHL tissue samples obtained from Omani patients attending Sultan Qaboos University Hospital (SQUH). Besides, to compare the sensitivity and specificity of immunohistochemistry(IHC) and in situ hybridization (ISH) for the detection of EBV in HL and NHL and finally to have more understanding of the pathogenesis of EBV in HL and NHL among patients in Oman. Formalin-fixed paraffin-embedded tissue samples consisting of 26 Hodgkin and 34 non-Hodgkin lymphomas were assessed for the presence of EBV by IHC to detect Latent membrane protein (LMP), expression and by using ISH to detect Epstein -Barr encoded RNAs (EBERs). The expression of LMP and EBERs were detected respectively in 46.2% and 57.7% of Hodgkin’s lymphoma cases and were detected in 11.8% and 14.7% respectively of non-Hodgkin’s lymphoma cases. The intensity of LMP-1 and EBER expression was significantly high in mixed cellularity compared to other subtypes. The expression of EBV was detected in transformed cells in both HL &amp; NHL. The expression of EBV in transformed cells in both HL and NHL indicates that EBV may play a pro vital role in the pathogenesis of HL and NHL among patients in Oman. Moreover, this study indicates that IHC is to some degree compatible in terms of sensitivity and specificity to ISH in the detection of EBV in HL and NHL.

Plasmablastic lymphoma (PBL) of the oral cavity is an aggressive neoplasm derived from B cell, considered to be the second more common among human immunodeficiency virus (HIV)-associated malignancies. As Epstein-Barr virus (EBV) infection has been associated with this neoplasm, the aim of the present study was to assess the presence of EBV in 11 cases of oral HIV-related PBL and investigate the controversial issue of the presence of Human herpesvirus-8 (HHV-8) in these tumors. DNA was extracted from nine cases of HIV-associated oral lymphomas, diagnosed as PBL, and genomic material was amplified by polymerase chain reaction to verify the presence of EBV. In situ hybridization (ISH) for EBV was performed in five cases. Immunohistochemical analysis was conducted to confirm previous diagnosis and verify HHV-8 infection. The 11 cases had diagnosis confirmed by immunohistochemical analysis. Only nine cases presented an adequate amount of DNA for analysis, and EBV was detected in seven of them. The five cases tested for EBV viral infection by ISH showed positive signals. All 11 cases were negative for HHV-8. The presence of EBV in all cases studied favors a direct role of this virus in the development of HIV-related PBL, and this finding could be considered when dealing with HIV patients.

Epstein-Barr virus detection in ductal carcinoma of the breast.

Epstein-Barr virus (EBV) is detected in Hodgkin and Reed-Sternberg (HRS) cells in up to 50% of patients with Hodgkin's disease (HD). HD patients have been reported to express high serum titers against EBV antigens, even prior to the diagnosis of HD. Patients with high serum titers have a poorer prognosis. The aim of this study was to examine the relationship between the presence of EBV in HRS cells and the antibody titers reactive with different EBV antigens. Frozen serum and histopathological tissues were available from 107 untreated HD patients diagnosed between 1979 and 1991. The presence of EBV in the HRS cells was evaluated with immunohistochemistry directed against the LMP-1 antigen and/or with in situ hybridization of EBER-1. Analyses were performed of serum titers against early antigen (EA), diffuse (IgA and IgG) and restricted (IgG), virus-capsid antigen (VCA) (IgA and IgG), and EBV-encoded nuclear antigens (EBNA, EBNA 1, EBNA 2A, EBNA 2B, EBNA 6). EBV was detected in 27/107 (25%) tumor specimens, with a higher proportion in the MC group 8/13 (62%) (p < 0.01). IgG VCA and EBNA were detected in 99/107 (93%), evidence of a previous EBV infection. There were no significant relationships between antibody titers reactive with different EBV antigens and detectable EBV in HRS cells. Furthermore, there did not appear to be any relationship between EBV serology or the presence of EBV in HRS cells and clinical outcome. The role of EBV in the development of HD, especially its relationship to the immunological response, remains unclear.

Introduction: It is suggested that Epstein-Barr virus (EBV) may play an important role in cervical cancer development. Most studies found a higher rate of EBV in cervical cancer samples in comparison to premalignant and normal groups. In this regard, this study aimed to investigate the prevalence of EBV in cervical samples. Methods: In total, 364 samples from 179 healthy subjects, 124 women with premalignant lesions, and 61 patients with cervical cancer were investigated using nested-PCR. Results: The mean age ± SE was 54.1 ± 13.4 in women with cervical cancer, 36.1 ± 9.4 among women with premalignant lesions, and 36.6 ± 11.5 in healthy individuals. In total, 290 out of 364 samples were human papillomavirus (HPV) positive and the following HPV genotypes were detected among them: HPV 16/18 was found in 43.1%, 23.9%, and 65.5% of normal, premalignant, and malignant samples, respectively, and other high-risk types were detected in 56.9% of normal, 76.1% of premalignant, and 34.5% of malignant samples. The prevalence of EBV was found to be 9.8%, 2.4%, and 2.8% in cervical cancer, premalignant lesions, and normal specimens, respectively, and the difference was statistically significant (p = 0.028). The overall frequency of coinfection between EBV and HPV was shown to be 3.6%. The coinfection was more prevalent among HPV 16/18-infected samples than other high-risk HPVs (6.6 vs. 2.9%) although the difference was not reached a statistically significant difference (p = 0.23). Conclusion: Our findings indicated that EBV could play an important role as a cofactor in the progression of cervical cancer. However, future studies with larger sample sizes and the expression analysis of EBV transcripts or proteins are mandatory.

Histopathological findings in cases of hairy leukoplakia (HL) are not exclusive to this lesion. A total of 36 tissue samples from patients previously diagnosed with HL based solely on morphological aspects were used in this study. Our purpose was to confirm the presence of Epstein-Barr virus (EBV) in these tissue samples by in situ hybridization (ISH), and to compare the detection of EBV with specific histopathological findings observed in each case. Among the 36 specimens, 80.55% were EBV positive, confirming the previous clinical and histhophatological diagnosis. None of the histopathological findings analyzed correlated with the presence or absence of EBV. This shows that a definitive diagnosis of HL cannot be established based on histopathological findings alone. Because there are many important implications on the establishment of definitive diagnosis of HL, the detection of EBV by ISH is obligatory.

Cervical cancer, one of the most common cancers in women, is primarily driven by high-risk human papillomaviruses (HPV) infections, particularly HPV-16. Co-infection with Epstein-Barr virus (EBV) has been reported to exacerbate disease progression by influencing HPV genome integration. This study examines HPV-16 integration status, p16INK4a expression, and their relationship with EBV co-infection and viral load in cervical cancer cases. In this study, 134 HPV-16-positive formalin-fixed, paraffin-embedded cervical samples were collected and analyzed for HPV-16 viral load, genome integration and EBV co-infection, followed by p16INK4a immunohistochemistry. Statistical analysis was performed to examine the association between viral markers and cervical cancer progression. HPV-16 viral loads varied significantly by histological grade, with the highest loads observed in cervical intraepithelial neoplasia 2 (CIN 2) lesions. HPV integration status revealed episomal forms in 32.8% of samples, mixed forms in 56%, and fully integrated forms in 11.2%. p16INK4a expression correlated with disease progression, increasing with CIN grade and in squamous cell carcinoma (SCC). EBV was detected in 13.4% of samples, but no significant associations were found between EBV infection and HPV integration, viral load, or p16INK4a expression levels. HPV-16 viral load and integration status are strongly associated with cervical lesion severity, while p16INK4a expression increases with lesion grade, indicating its utility as a diagnostic marker. EBV co-infection did not significantly impact lesion progression, suggesting that its role in cervical cancer remains unclear.

PurposePrevious studies have found that Epstein-Barr virus (EBV) is associated with periodontitis, though some controversy remains. This meta-analysis aimed to clarify and update the relationship between EBV and periodontitis as well as clinical parameters.MethodsA comprehensive search was conducted in the PubMed and Scopus databases in December 2020. Original data were extracted according to defined inclusion and exclusion criteria. Outcomes were analyzed, including overall odds ratios (ORs) and 95% confidence intervals (CIs). A random-effects model was used, and publication bias was assessed by Egger’s and Begg’s tests. Sensitivity analysis was used to evaluate the stability of the outcome.ResultsTwenty-six studies were included in the present meta-analysis, involving 1354 periodontitis patients and 819 healthy controls. The included studies mostly showed high quality. The overall quantitative synthesis for the association between EBV and periodontitis was an increased odds ratio when subgingival EBV was detected OR = 7.069, 95% CI = 4.197–11.905, P<0.001). The results of subgroup analysis suggested that the association of EBV with periodontitis was significant in Asian, European, and American populations (P<0.001; P = 0.04; P = 0.003, respectively) but not in African populations (P = 0.29). Subgroup analysis by sample type showed that subgingival plaque (SgP), tissue and gingival crevicular fluid GCF were useful for EBV detection (P<0.001). EBV detection amplification methods included nested PCR, multiplex PCR and PCR (P<0.001; P = 0.05, P<0.001, respectively), but EBV detection by real-time PCR and loop-mediated isothermal amplification presented no significant result (P = 0.06; P = 0.3, respectively). For the clinical parameters of periodontitis, pocket depth (PD) and bleeding of probing (BOP) percentages were higher in the EBV-positive sites than in the EBV-negative sites (MD 0.47 [0.08, 0.85], P = 0.02; MD 19.45 [4.47, 34.43], P = 0.01).ConclusionsA high frequency of EBV detection is associated with an increased risk of periodontitis. The EBV association was particularly significant in all populations except in African populations. Subgigival plaque (SgP), tissue and GCF were not significantly different useful material for detecting EBV in periodontitis. Nested PCR and multiplex PCR are reliable methods for this purpose. In the presence of EBV, PD and BOP are reliable clinical parameters for gingival inflammation. However, some caution in such interpretation is justified due to heterogeneity among studies. A suggested extension could assess the parallel influence of other human herpesviruses.

Epstein-Barr virus (EBV), one of the most significant causes of lymphoid and epithelial cancers, has been linked to oral carcinogenesis; however, this etiological association remains controversial. To investigate this association, the present study aimed to determine the prevalence of EBV in cancerous and non-cancerous oral tissues from Ahvaz, Iran. In total, 164 blocks of formalin-fixed paraffin-embedded tissues from oral squamous cell carcinoma (OSCC), including 76 tongue squamous cell carcinomas and 88 non-cancerous tongue tissues, were collected from Ahvaz Imam Khomeini Hospital, Ahvaz, Iran, from December 2014 to March 2019, for this case-control study. The tissues were cut into 15-μm-thick sections, and DNA was extracted using a solution of Phenol, Chloroform, and Isoamyl Alcohol. The EBV detection and typing were performed using nested polymerase chain reaction. The EBV was detected in 9 (5.48%) out of the 164 samples studied, including 4 (5.26%) of the 76 SCC cases and 5 (5.68%) of the 88 samples in the control group (P&gt;0.05). The EBV was positive in 2.40% of the 83 male and 8.6% of the 81 female samples (P&gt;0.05). In terms of the histological grades of the case group, 3 (3/57) and 1 (1/13) of the EBV-positive samples were well and moderately differentiated, respectively (P&gt;0.05). For EBV typing, the 9 EBV-positive samples were tested, and it was found that 2 and 7 of the cases were EBV type I and II, respectively. Results of the current study demonstrated the low frequency of EBV in Iranian patients with OSCC, with EBV type II predominating. Further studies are required to clarify the association between EBV and OSCC.

Epstein-Barr virus (EBV), one of the most significant causes of lymphoid and epithelial cancers, has been linked to oral carcinogenesis; however, this etiological association remains controversial. To investigate this association, the present study aimed to determine the prevalence of EBV in cancerous and non-cancerous oral tissues from Ahvaz, Iran. In total, 164 blocks of formalin-fixed paraffin-embedded tissues from oral squamous cell carcinoma (OSCC), including 76 tongue squamous cell carcinomas and 88 non-cancerous tongue tissues, were collected from Ahvaz Imam Khomeini Hospital, Ahvaz, Iran, from December 2014 to March 2019, for this case-control study. The tissues were cut into 15-μm-thick sections, and DNA was extracted using a solution of Phenol, Chloroform, and Isoamyl Alcohol. The EBV detection and typing were performed using nested polymerase chain reaction. The EBV was detected in 9 (5.48%) out of the 164 samples studied, including 4 (5.26%) of the 76 SCC cases and 5 (5.68%) of the 88 samples in the control group (P>0.05). The EBV was positive in 2.40% of the 83 male and 8.6% of the 81 female samples (P>0.05). In terms of the histological grades of the case group, 3 (3/57) and 1 (1/13) of the EBV-positive samples were well and moderately differentiated, respectively (P>0.05). For EBV typing, the 9 EBV-positive samples were tested, and it was found that 2 and 7 of the cases were EBV type I and II, respectively. Results of the current study demonstrated the low frequency of EBV in Iranian patients with OSCC, with EBV type II predominating. Further studies are required to clarify the association between EBV and OSCC.

High prevalence of a 30-base pair deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 gene and of strain type B EBV in Mexican classical Hodgkin's disease and reactive lymphoid tissue