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Correlation between ebv co-infection and HPV16 genome integrity in Tunisian cervical cancer patients

Infection with high risk Human papillomavirus (HR-HPV) is necessary but not sufficient to cause cervical carcinoma. This study explored whether multiple HR-HPV or coinfection with Epstein-Barr virus (EBV) influence the integration status of HPV16 genome. The presence and typing of HPV in a series of 125 cervical specimens were assessed by polymerase chain reaction (PCR) using the specific primers for the HPV L1 region. As for EBV infection, the viral EBNA1 gene was used for its detection through PCR amplification. Disruption of the HPV E2 gene was assessed by amplification of the entire E2 gene with single set of primers, while E2 transcripts were evaluated by a reverse transcription PCR method (RT-PCR). The overall prevalence of HPVDNA was of 81.8% in cervical cancers versus 26.9% in benign lesions. In HPV positive cases, HPV16 and HPV18 were the most prevalent types, followed by HPV types 33, 31. EBV EBNA1 prevalence was statistically more frequent in cervical carcinomas than in benign lesions (29.5%, vs 9.6%; P=0.01). No viral infection was detected in healthy control women. The uninterrupted E2 gene was correlated with the presence of E2 transcripts originating from the HPV episomal forms. It was observed that integration was more common in HPV18 and EBV coinfection. The presence of EBV caused a five-fold [OR= 5; CI= 1.15-21.8; P = 0.04] increase in the risk of HPV16 genome integration in the host genome. This study indicates that EBV infection is acting as a cofactor for induction of cervical cancer by favoring HPVDNA integration.

have been suspected to participate in cervical carcinogenesis (15,33,31,13). For example, latent infection with EBV has been shown to act as a carcinogenic cofactor in several epithelial cell malignancies (38). The viral EBNA1 regulator of EBV transcription and replication is typically expressed in most forms of EBV-positive malignancies (24). On the other hand, integration of HPV DNA into the human genome is considered an essential step in the progression of HPVassociated cervical infection to invasive cervical carcinoma (18,41). Upon integration the viral E2 gene, encoding a transcriptional repressor, is often disrupted with loss of its repressor function thus leading to up regulation of the transcription of the E6 and E7 oncogenes (30,12). High expression of the viral oncogenes leads to disturbance of cell cycle controls and genomic instability that increase the risk of malignant transformation (25,29). The main goal of our study was to explore frequency distribution of HR HPV genotypes and EBV infection in cervical cancer and premalignant lesions in order to correlate them with HPV 16 physical state.

Sample Collection and Study Population
This study enrolled 44 patients with cervical carcinoma recruited from the Radio-oncology department of Salah Azaiez Institute (Tunis, Tunisia). The mean patient age was 57.  (Tunis, Tunisia) for reasons other than cervical cancer. Also twenty nine Healthy controls women, counseling for routine reproductive healthcare, were used as reference. The range of ages varied from 27 to 56 years old, with an average of 41 years. Controls had no past or current history of cervical abnormalities. Biopsy specimens were transferred into cryotubes, suspended in 1 ml PBS (phosphate-buffered saline, pH 7.4) and stored frozen at -20°C until analysis.

DNA and RNA isolation
DNA extraction was performed using the QIAamp system, QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to standard procedures as described in the kit, with detergent lysis buffer and proteinase K digestion. Total DNA was eluted in 100 µl of elution buffer and stored frozen at -20°C. RNA from frozen tissues was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) as recommended by the supplier and dissolved in 30µl RNase free water.
The quality and quantity of the target nucleic acids were assessed by agarose gel electrophoresis and optical density measurement (absorbance at 260 nm).

HR-HPV typing and EBV detection
Purified genomic DNA was amplified using PCR technique for the HR HPVL1 and EBV EBNA1 genes (synthesized by GeneCust Europe, Dudelange, Luxembourg) using specific primers, as well as for the internal reference βglobin gene, to determine whether the isolated DNA was suitable for amplification. Thermal cycles were performed in a programmable PCR thermal cycler (Applied Biosystem, Foster City, USA), as previously described (46,21). Each PCR experiment was performed with positive (HPV plasmids, cervical carcinoma previously proved to contain EBV) and negative (Blank water) controls. Samples tested positive for the Kahla, S. e t a l .
Correlation between EBV co-infection and HPV16 genome β-globin DNA control, confirming that none of the samples was inhibitory for PCR or lacked DNA. The Primer sequences, amplicon sizes and cycling programs are illustrated in Table 1.

Frequencies of HR-HPV DNA and EBV in cervical samples
By using the primers directed against the highly conserved   Figure 1A and Figure 1B Presence of the intact HPV16 E2 DNA, flanking the 1026-bp segments of the ORF fragment, was finally confirmed by HPV16 E2 RNA using primers giving an amplified product of 149 bp. In general amplification of E2 with RT-PCR appeared more sensitive than amplification of the DNA-PCR method for detection of intact HPV E2 gene, because E2 DNA signals from RT-PCR are usually stronger than those from DNA-PCR (Figure 1 A, Figure 1B).

Disruption of the E2 gene together with multiple HR HPV or EBV coinfections predict carcinogenic progression
To analyze whether viral factors might have an influence on the integration status, we correlated disruption of the HPV16 genome with multiple HR HPV and EBV coinfections.
Such analysis revealed that HPV18 coinfection was found more often associated with HPV16 integration, while in contrast, HPV33 coinfection was only associated with HPV16 episomal forms (Figure 2). We also correlated the presence of EBV to the status of the HPV16 genome in coinfected lesions.
Interestingly, EBV was more frequently present in samples with integrated forms of HPV16 than in the episomal forms.    (15). However, other authors did not reach to the same conclusion, confirming that EBV was acting in cervical tumor pathogenesis (33). This, together with the fact that EBV infection may contribute in the integration of HPV16 genome occurrence, suggests that, although they might not be involved in the oncogenic processes directly, they might play a role as cofactor. Accordingly, the viral EBV EBNA1 protein has been shown to be able to decrease apoptosis and DNA repair in EBV-associated lesions thus potentially contributing to malignant transformation of cotransfected EBV/HPV lesions (16).
In summary, further knowledge of markers of HPV genomic integration has the potential to improve objective management of cervical neoplasia. The data presented here might also be beneficial to our understanding of the biology of