Viral DNA load, physical status and E2/E6 ratio as markers to grade HPV16 positive women for high-grade cervical lesions

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Objectives. Cervical intraepithelial neoplasias (CIN) associated with high-risk (HR) human papillomavirus infection, in addition to HR-HPV typing need other viral marker testing to distinguish a subset of lesions with clinical relevant infections. This study has evaluated the significance of viral markers, such as viral load, physical status and E2/E6 ratio, to stratify HPV16 infected women at a single point in time for grade of cervical lesions. Methods. One hundred sixty-six cytological specimens were selected from women with low ( n = 72) and high ( n = 94) grade squamous intraepithelial lesions (SIL), and positive to HPV16. All the 72 LSIL were CINI, 83 of the 94 HSIL were CINII/III and 11 SCC (Squamous Cervical Carcinoma). Cytological specimens were analysed by two different SYBR Green Real-time PCR assays (RT-PCR). Specific primers for both E2 and E6 viral genes and GAPDH cellular gene were designed to determine viral load, physical status and E2/E6 ratio. Results. The viral load was significantly higher in HSIL than in LSIL. In CINI episomal DNA was prevalent (72.2%), mixed forms (episomal and integrated) were 27.8%, suggestive of an early integration of viral DNA into cellular genome, no pure integrated forms were detected. However in CINII/III mixed DNA forms were prevalent (73.5%). In SCC pure integrated DNA was prevalent (81.8%) in absence of episomal forms. E2/E6 ratio decreased significantly from CINI to CINII/III and SCC with a linear trend. The logistic regression analysis showed that viral load higher than 1.38 × 10 6 genome copies per 300 ng of total DNA associated with E2/E6 ratio lower than 0.90 was highly significant in differentiating CINII/III versus CINI, while the only E2/E6 value lower than 0.17 was significant in differentiating SCC from CINI. Conclusions. Viral load higher than 1.38 × 10 6 genome copies per 300 ng of total DNA and E2/E6 ratio values allow HPV16 infected women with high grade cervical intraepithelial lesions to be recognized.