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 perf orming molecular analyses at the deoxyribonucleic acid, ribonucleic acid an d protein levels of cervical cytologic examination with a methanol fluid-ba sed Papanicolaou specimen collection system. STUDY DESIGN: Genomic deoxyribonucleic acid and total ribonucleic acid were extracted from cell pellets obtained from the residual fluid-based Papanic olaou specimen collection buffer after clinical processing. Genomic and hum an papillomavirus deoxyribonucleic acid polymerase chain reaction and rever se transcriptase-polymerase chain reaction were performed. Messenger ribonu cleic acid transcript analysis and human papillomavirus 16 E6 mutational an alysis were also performed. Methylation-specific polymerase chain reaction was used to evaluate hypermethylation status of the p16 gene and the gene f or E-cadherin. Immunohistochemical staining for protein expression was perf ormed on the processed monolayer slides. RESULTS: Cell pellets from the residual fluid-based cytologic specimen yiel ded good quality deoxyribonucleic acid and ribonucleic acid. Molecular anal yses of genomic deoxyribonucleic acid were successful for the identificatio n of human papillomavirus E6 and p53 polymorphism status by means of restri ction enzyme digestion and direct sequencing. Methylation status of the pro motor regions of the p16 tumor suppressor gene and the gene for E-cadherin were also successfully identified. Ribonucleic acid was used as the templat e for transcript analysis and mutational analysis of the corresponding comp lementary deoxyribonucleic acid of the p53 gene. Protein expression analysi s was demonstrated by immunohistochemical staining for carcinoembryonic ant igen. CONCLUSION: It is feasible to conduct multiple molecular analyses at the de oxyribonucleic acid, ribonucleic acid, and protein levels of the cervicovag inal cell pellets from the residual fluid-based Papanicolaou cytologic spec imen. This relatively simple and widely used collection system will allow s ignificant advances in molecular epidemiology and eventual development of a molecular Papanicolaou test.