Viral DNA load, physical status and E2/E6 ratio as markers to grade HPV16 positive women for high-grade cervical lesions.
OBJECTIVE
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 1.38x10(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.


CONCLUSIONS
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.
Cricca
Monica
M
Department of Clinical and Experimental Medicine, Microbiology Section, University of Bologna, and Department of Obstetrics and Gynecology, S. Orsola-Malpighi Hospital, Italy.
Morselli-Labate
Antonio Maria
AM
Venturoli
Simona
S
Ambretti
Simone
S
Gentilomi
Giovanna Angela
GA
Gallinella
Giorgio
G
Costa
Silvano
S
Musiani
Monica
M
Zerbini
Marialuisa
M
D000328:Adult; D000368:Aged; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D030361:Papillomavirus Infections / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D019562:Viral Load; D002578:Uterine Cervical Dysplasia / Q000378:metabolism / Q000473:pathology / Q000821:virology*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C054855:E2 protein, Human papillomavirus type 16; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins