Human papillomavirus and Epstein-Barr virus co-infection in cervical carcinoma in Algerian women

Abstract

Background: Despite 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.

Methods: Fifty 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.

Results: HR-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.

Conclusion: The 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.

Figure 1

Distribution of EBV and HR-HPV in patients with either cervical precancerous or cancerous lesions compared to normal subjects. High risk human papillomavirus were detected by PCR (using consensus primers) and by Hybrid Capture 2 (Digene). Epstein-Barr virus was detected by PCR using primers that amplify the entire BALF-1 gene. Percentage in ordinate; Normal cervix, cervical lesions and carcinoma cases in Abscissa. NC: normal cervixes; CIN-2/3: High-grade cervical lesions; CIN-1: Low-grade cervical lesions; SCC: squamous cell carcinoma.

Figure 2

Detection of EBV BALF1 gene by PCR. Amplified PCR products were electrophoresed and the presence of BALF1 sequence [39] was confirmed by hybridization with ³²P labeled BamH1-A fragment used as a probe. A: lanes 1-8 for tumor samples and B: lanes 9-16 for precancerous lesions. B95-8 DNA and H₂O were used as positive and negative controls respectively.

Figure 3

Transcriptional expression of LMP-1. A 406-bp PCR amplified fragment corresponding to the LMP-1 DNA sequence, and a 200-bp RT-PCR amplified fragment corresponding to the LMP-1 mRNA sequence. Lanes 1, 2 and 7 are positive signals. M: DNA fragments molecular markers. C-: negative control.

Figure 4

Detection of BARF-1 protein by Western blotting. Immunoblotting of BARF-1 on seven cervical tumor biopsies. Fifty micrograms of protein extract as measured by a Biorad protein assay were deposited onto 12% polyacrylamide gel and electrophoresed. Proteins were transferred onto nitrocellulose paper. BARF1 protein encoding 29 kDa (p29) was revealed by PepIII rabbit polyclonal anti-BARF-1. BARF-1 protein (p29) produced by the BARF-1 recombinant adenovirus system was used as a positive control indicated in first lane as BARF1.

Figure 5

Detection of HPV and EBV antigen by Immunohistochemistry. Immunohistochemistry analysis was performed on sections of cervix tissue samples embedded in paraffin. HPV and EBV antigens were detected by peroxidase mediated DAB-staining (Brown). All samples were counterstained with hematoxylin-eosin and then observed under a light microscope (original magnification 200x). Positive response is marked with a white arrow. A: Squamous-cell carcinoma observed by heamatoxylin-eosine staining. B: Squamous carcinoma cells positive to EBNA-1 revealed by monoclonal anti-EBNA-1 antibody (OT-1). C: Squamous carcinoma cells positive to LMP-1 revealed by monoclonal anti- LMP-1 antibody (S12). D: Cytoplasmic and nuclear HPV staining with monoclonal antibodies (K1H8), which recognize HPV type 6, 11, 16, 18, 31, 33, 42, 51, 52, 56 and 58. E: Squamous-cell carcinoma negative to EBNA-1. F: Squamous-cell carcinoma negative to LMP-1. Weak staining of rare lymphocytes, the tumor cells are negative for LMP1.

Figure 6

Flow chart showing the organization of HPV and EBV detection and expression in squamous cell carcinoma (SCC) cases by PCR using MY consensus primers and by Digene Hybrid Capture II (HC II). The Epstein-Barr virus (EBV) was detected by PCR using primers that amplify the entire BALF-1 gene. EBV expression was investigated by LMP-1 Reverse Transcription-PCR, BARF-1 Immunoblotting and LMP-1 and EBNA-1 Immunohistochemistry.