Cricca Monica, Morselli-Labate Antonio Maria, Venturoli Simona, Ambretti Simone, Gentilomi Giovanna Angela, Gallinella Giorgio, Costa Silvano, Musiani Monica, Zerbini Marialuisa
Department of Clinical and Experimental Medicine, Microbiology Section, University of Bologna, and Department of Obstetrics and Gynecology, S. Orsola-Malpighi Hospital, Italy.
Gynecol Oncol. 2007 Sep;106(3):549-57. doi: 10.1016/j.ygyno.2007.05.004. Epub 2007 Jun 13.
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.
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.
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 1.38x10(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.
Viral load higher than 1.38x10(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.
与高危（HR）人乳头瘤病毒感染相关的宫颈上皮内瘤变（CIN），除了进行HR-HPV分型外，还需要其他病毒标志物检测，以区分具有临床相关感染的病变亚组。本研究评估了病毒载量、物理状态和E2/E6比值等病毒标志物在某一时刻对HPV16感染女性的宫颈病变分级进行分层的意义。
从患有低级别（n=72）和高级别（n=94）鳞状上皮内病变（SIL）且HPV16呈阳性的女性中选取166份细胞学标本。所有72份低级别鳞状上皮内病变均为CINI，94份高级别鳞状上皮内病变中有83份为CINII/III，11份为鳞状宫颈癌（SCC）。通过两种不同的SYBR Green实时荧光定量PCR检测（RT-PCR）分析细胞学标本。设计E2和E6病毒基因以及GAPDH细胞基因的特异性引物，以确定病毒载量、物理状态和E2/E6比值。
高级别鳞状上皮内病变中的病毒载量显著高于低级别鳞状上皮内病变。在CINI中，游离型DNA占主导（72.2%），混合形式（游离型和整合型）占27.8%，提示病毒DNA早期整合到细胞基因组中，未检测到纯整合形式。然而，在CINII/III中，混合DNA形式占主导（73.5%）。在鳞状宫颈癌中，纯整合DNA占主导（81.8%），不存在游离型。从CINI到CINII/III和鳞状宫颈癌，E2/E6比值呈线性显著下降。逻辑回归分析表明，每300 ng总DNA中病毒载量高于1.38x10(6)基因组拷贝且E2/E6比值低于0.90，在区分CINII/III与CINI方面具有高度显著性，而仅E2/E6值低于0.17在区分鳞状宫颈癌与CINI方面具有显著性。
每300 ng总DNA中病毒载量高于1.38x10(6)基因组拷贝以及E2/E6比值，可识别出感染HPV16且患有高级别宫颈上皮内病变的女性。
