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

• Published in: Gynecologic oncology 2007-09, Vol.106 (3), p.549-557
• Main Authors: Cricca, Monica, Morselli-Labate, Antonio Maria, Venturoli, Simona, Ambretti, Simone, Gentilomi, Giovanna Angela, Gallinella, Giorgio, Costa, Silvano, Musiani, Monica, Zerbini, Marialuisa
• Format: Article
• Language: English
• Subjects: Adult; Aged; Carcinoma, Squamous Cell - metabolism; Carcinoma, Squamous Cell - pathology; Carcinoma, Squamous Cell - virology; Cervical Intraepithelial Neoplasia - metabolism; Cervical Intraepithelial Neoplasia - pathology; Cervical Intraepithelial Neoplasia - virology; DNA, Viral - analysis; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; E2/E6 ratio; Female; Hematology, Oncology and Palliative Medicine; High-grade cervical lesions; HPV16 viral load; Human papillomavirus 16 - genetics; Human papillomavirus 16 - metabolism; Humans; Middle Aged; Obstetrics and Gynecology; Oncogene Proteins, Viral - genetics; Oncogene Proteins, Viral - metabolism; Papillomavirus Infections - metabolism; Papillomavirus Infections - pathology; Papillomavirus Infections - virology; Physical status; Repressor Proteins - genetics; Repressor Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Uterine Cervical Neoplasms - metabolism; Uterine Cervical Neoplasms - pathology; Uterine Cervical Neoplasms - virology; Viral Load
• ISSN: 0090-8258; 1095-6859
• DOI: 10.1016/j.ygyno.2007.05.004

Abstract 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 × 106 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 × 106 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.