Molecular Papanicolaou tests in the twenty-first century: molecular analyses with fluid-based Papanicolaou technology.
OBJECTIVE
This study was undertaken to demonstrate the feasibility of performing molecular analyses at the deoxyribonucleic acid, ribonucleic acid and protein levels of cervical cytologic examination with a methanol fluid-based Papanicolaou specimen collection system.


METHODS
Genomic deoxyribonucleic acid and total ribonucleic acid were extracted from cell pellets obtained from the residual fluid-based Papanicolaou specimen collection buffer after clinical processing. Genomic and human papillomavirus deoxyribonucleic acid polymerase chain reaction and reverse transcriptase-polymerase chain reaction were performed. Messenger ribonucleic acid transcript analysis and human papillomavirus 16 E6 mutational analysis were also performed. Methylation-specific polymerase chain reaction was used to evaluate hypermethylation status of the p16 gene and the gene for E-cadherin. Immunohistochemical staining for protein expression was performed on the processed monolayer slides.


RESULTS
Cell pellets from the residual fluid-based cytologic specimen yielded good quality deoxyribonucleic acid and ribonucleic acid. Molecular analyses of genomic deoxyribonucleic acid were successful for the identification of human papillomavirus E6 and p53 polymorphism status by means of restriction enzyme digestion and direct sequencing. Methylation status of the promotor regions of the p16 tumor suppressor gene and the gene for E-cadherin were also successfully identified. Ribonucleic acid was used as the template for transcript analysis and mutational analysis of the corresponding complementary deoxyribonucleic acid of the p53 gene. Protein expression analysis was demonstrated by immunohistochemical staining for carcinoembryonic antigen.


CONCLUSIONS
It is feasible to conduct multiple molecular analyses at the deoxyribonucleic acid, ribonucleic acid, and protein levels of the cervicovaginal cell pellets from the residual fluid-based Papanicolaou cytologic specimen. This relatively simple and widely used collection system will allow significant advances in molecular epidemiology and eventual development of a molecular Papanicolaou test.
Lin
W M
WM
Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas 75235-9032, USA.
Ashfaq
R
R
Michalopulos
E A
EA
Maitra
A
A
Gazdar
A F
AF
Muller
C Y
CY
D002272:Carcinoembryonic Antigen / Q000032:analysis; D004247:DNA / Q000032:analysis; D019175:DNA Methylation; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D000432:Methanol; D065006:Papanicolaou Test; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014626:Vaginal Smears / Q000379:methods / Q000639:trends*
D002272:Carcinoembryonic Antigen; D004279:DNA, Viral; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D000432:Methanol