Introduction Different methodologies have been developed to analyse integration. Most of them are expensive and laborious. Since the most frequent disrupting happen in E1 or E2 genes we believe that amplify fragments covering theses genes could be associated to virus integration status.
Methods Hence, in order to evaluate the physical state (episomal or integrated) of HPV 16 and 18 genomes, a PCR combining 10 primers pairs for HPV16 and 11 primers pairs for HPV18 were used, covering the E1-E2 region, adapted as described by Vernon et al. (1997)1 and Collins Constandinou-Williams et al. (2009).2 CASKI and HeLa lineage were used as control.
Results Our preliminary results involved 93 samples cervical samples from patients infected by HPV16 and 37 by HPV18, harbouring cervical lesions in different stages of progression, except HPV18, exclusively associated with cancer lesions. Among HPV16, 26 (28%) were presented episomal, 30 (32%) mixed and 37 (40%) integrated forms. The upper region E1a were the most frequently absent (disrupted) (n = 30, 32%), followed by the downstream E2c region, (n = 21, 22,6%). Among HPV 18 cervical cancers, 5 (13,5%) were presented as episomal forms, 8 (21,5%) mixed and 24 (65%) were integrated. The downstream region E2P3 was the most frequently disrupted, (n = 30, 81%).
Conclusion It is interesting to observe that literature points out a predominance of disruption in E2 region but our results suggested a highly prevalent E1 inactivation. The approach here described is a very specific methodology that can successfully map the HPV 16 and 18 genome fragile areas. Data are being analysed in order to search for statistical correlation between integration and severity of the lesion but it has been observed that E1-E2 absences were suggestively frequent in HSIL and cancer. In a few cases, episomal forms were observed in cancer samples, suggesting additional biomarkers as responsible for carcinogenesis.
Disclosure of interest statement PCR, Genomic integration, episomal, HPV16.
Financial support: Bolsa Capes, Faperj APQ1.
Vernon SD, Unger ER, Miller DL, et al. Association of human papillomavirus type 16 integration in the E2 gene with poor disease-free survival from cervical cancer. Int J Cancer 1997;74:50–6
Collins SI, Constandinou-Williams C, Wek K, et al. Disruption of the E2 gene is a common and early event in the natural history of cervical human papillomavirus infection: a longitudinal cohort study. Cancer Res. 2009;69:3828–32
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