title: Assessing the detection of human papillomavirus late mRNA in liquid base cytology samples for risk stratification of cervical disease.
abstract: Molecular human papillomavirus (HPV) testing is an important and developing tool for cervical disease management. However there is a requirement to develop new HPV tests that can differentiate between clinically significant and benign, clinically insignificant infection. Evidence would indicate that clinically significant infection is linked to an abortive HPV replication cycle. In particular the later stages of the replication cycle (i.e., production of late messenger (m) RNAs and proteins) appear compromised. Compared to current DNA-based tests which indicate only presence or absence of virus, detecting virus mRNAs by reverse transcriptase PCR (RT-PCR) may give a more refined insight into viral activity and by implication, clinical relevance. A novel quantitative (q)RT-PCR assay was developed for the detection of mRNAs produced late in the viral replication cycle. Initially this was validated on HPV-containing cell lines before being applied to a panel of 223 clinical cervical samples representing the cervical disease spectrum (normal to high grade). Samples were also tested by a commercial assay which detects expression of early HPV E6/E7 oncoprotein mRNAs. Late mRNAs were found in samples associated with no, low and high grade disease and did not risk-stratify HPV infection. The data reveal hidden complexities within the virus replication cycle and associated lesion development. This suggests that future mRNA tests for cervical disease may require quantitative detection of specific novel viral mRNAs.
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Institute of Infection Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, UK.
authors.forename: George
authors.initials: G
authors.lastname: Chambers
authors.forename: David
authors.initials: D
authors.lastname: Millan
authors.forename: Kate
authors.initials: K
authors.lastname: Cuschieri
authors.forename: Heather A
authors.initials: HA
authors.lastname: Cubie
authors.forename: Sheila V
authors.initials: SV
authors.lastname: Graham
mesh_terms: D036022:Capsid Proteins / Q000235:genetics; D002461:Cell Line, Transformed; D005260:Female; D005838:Genotype; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins / Q000235:genetics; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D002577:Uterine Cervical Diseases / Q000145:classification / Q000175:diagnosis*
chemical_list: D036022:Capsid Proteins; C068651:E6 protein, Human papillomavirus type 16; C063316:L2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; C063317:L1 protein, Human papillomavirus type 16
keywords: cervical intraepithelial neoplasia; gene expression; human papillomavirus; liquid based cytology; mRNA tests