Quantitation of human papillomavirus 16 E6 and E7 DNA and RNA in residual material from ThinPrep Papanicolaou tests using real-time polymerase chain reaction analysis.
BACKGROUND
The detection of specific human papillomavirus 16 (HPV-16) E6 and E7 oncogene transcripts may be a sensitive indicator of the direct involvement of viral oncogenes in the development of cervical neoplasia and carcinoma. The goal of this study was to determine the potential clinical uses of real-time polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) methods for evaluating HPV-16 E6 and E7 oncogene expression.


METHODS
ThinPrep cervical samples were tested for expression of oncogenes of HPV-16 by real-time PCR or RT-PCR analysis and were compared with detection of expression by conventional PCR and RT-PCR analysis. Both sets of results were correlated with the cytologic diagnosis of the cervical samples.


RESULTS
The presence of HPV-16 E6 and E7 DNA and RNA was observed only in HPV-16 positive cervical carcinoma cell lines but not in HPV-18 positive or HPV negative cell lines. The percentage positive for HPV-16 E6 or E7 DNA in a series of ThinPrep cervical cytologic samples (n = 348 samples) was 0% for negative samples (n = 45 samples), 9.7% for atypical squamous cells of undetermined significance (ASCUS; n = 144 samples), 16.9% for low-grade squamous intraepithelial lesion (LSIL; n = 118 samples), and 51.2% for high-grade intraepithelial lesion (HSIL; n = 41 samples). The copy numbers per nanogram for both DNA and RNA E6 and E7 were increased significantly as severity of the lesions progressed from ASCUS to HSIL, and RNA copy numbers were a more sensitive indicator of HPV-16 E6 and E7 expression than DNA copy numbers. The increase in copy numbers took place in a stepwise fashion from ASCUS, to LSIL, to HSIL.


CONCLUSIONS
The detection of HPV-16 E6 and E7 expression by real-time RT-PCR or PCR analysis in ThinPrep cervical cytologic specimens may serve as a quick, reliable, and sensitive tool to identify a subset of patients who express HPV-16 oncoproteins.
Wang-Johanning
Feng
F
Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-3300, USA. fengi@uab.edu
Lu
Danielle W
DW
Wang
Yueying
Y
Johnson
Martin R
MR
Johanning
Gary L
GL
D000293:Adolescent; D000328:Adult; D000368:Aged; D002294:Carcinoma, Squamous Cell / Q000821:virology*; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006801:Humans; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000235:genetics; D065006:Papanicolaou Test; D027383:Papillomaviridae / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000821:virology*; D012367:RNA, Viral / Q000032:analysis*; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014412:Tumor Virus Infections / Q000821:virology*; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D014626:Vaginal Smears
D017931:DNA Primers; D004279:DNA, Viral; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012367:RNA, Viral; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16