title: HPV16 RNA patterns defined by novel high-throughput RT-qPCR as triage marker in HPV-based cervical cancer precursor screening.
abstract: OBJECTIVE
  Cervical cancer precursor screening by HPV testing has a low positive predictive value for advanced lesion. HPV16 RNA patterns characteristic for HPV16-transformed cells but based on laborious, cost-intensive singleplex NASBA reactions promised high value in triaging HPV16 DNA-positive women.
  
  
  METHODS
  We developed two high-throughput reverse transcriptase quantitative (RT-q) PCR assays for the HPV16 transcripts E6*I, E1^E4 and E1C and the cellular transcript ubiquitin C and analysed RNA of 158 singly HPV16 DNA-positive cervical cell samples archived in PreservCyt buffer for the presence of transformation-associated HPV16 RNA patterns, i.e., upregulation of E6*I relative to E1^E4 and/or presence of E1C.
  
  
  RESULTS
  HPV16 RNA pattern analyses classified 85% of 58 samples diagnosed ≤CIN1 (no cytologically and histologically detectable cervical lesion or CIN grade 1) as negative and 90% of 59 samples diagnosed as ≥CIN3 (CIN grade 3 or invasive cancer) as positive. Among 41 CIN grade 2 samples representing an intermediate lesion group, 49% were HPV16 RNA patterns-positive. Interestingly, 3 of 4 HPV16 RNA patterns-positive lesions initially diagnosed as ≤CIN1 at follow-up 5-24 months later had progressed to ≥CIN2.
  
  
  CONCLUSIONS
  We successfully developed and validated a second generation of HPV16 RNA patterns assay by rapid RT-qPCR as triage marker for HPV16 DNA-positive women offering clinical utility to distinguish between the need for immediate colposcopy and continued observation. Limited follow-up data suggests that HPV16 RNA patterns-positivity in ≤CIN1 lesions can predict disease progression.
authors.affiliation: Division of Molecular Diagnostics of Oncogenic Infections, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Electronic address: d.hoefler@dkfz.de.
authors.forename: Daniela
authors.initials: D
authors.lastname: Höfler
authors.affiliation: Deutsche Klinik Bad Münder, Zentrum für IVF und Reproduktionsmedizin, Hannoversche Straße 24, 31848 Bad Münder, Germany. Electronic address: boehmer@izd-hannover.de.
authors.forename: Gerd
authors.initials: G
authors.lastname: Böhmer
authors.affiliation: Deutsche Klinik Bad Münder, Zentrum für IVF und Reproduktionsmedizin, Hannoversche Straße 24, 31848 Bad Münder, Germany. Electronic address: reinhard.vonwasielewski@krh.eu.
authors.forename: Reinhard
authors.initials: R
authors.lastname: von Wasielewski
authors.affiliation: Deutsche Klinik Bad Münder, Zentrum für IVF und Reproduktionsmedizin, Hannoversche Straße 24, 31848 Bad Münder, Germany. Electronic address: heinrich.h.neumann@gmail.com.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Neumann
authors.affiliation: Division of Molecular Diagnostics of Oncogenic Infections, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Electronic address: ghalec@gmail.com.
authors.forename: Gordana
authors.initials: G
authors.lastname: Halec
authors.affiliation: Division of Molecular Diagnostics of Oncogenic Infections, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Electronic address: d.holzinger@dkfz.de.
authors.forename: Dana
authors.initials: D
authors.lastname: Holzinger
authors.affiliation: Division of Molecular Diagnostics of Oncogenic Infections, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Electronic address: bolor73@yahoo.com.
authors.forename: Bolormaa
authors.initials: B
authors.lastname: Dondog
authors.affiliation: Division of Molecular Diagnostics of Oncogenic Infections, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Electronic address: l.gissmann@dkfz.de.
authors.forename: Lutz
authors.initials: L
authors.lastname: Gissmann
authors.affiliation: Division of Molecular Diagnostics of Oncogenic Infections, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Electronic address: m.pawlita@dkfz.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Pawlita
authors.affiliation: Division of Molecular Diagnostics of Oncogenic Infections, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Electronic address: schmi.markus@gmail.com.
authors.forename: Markus
authors.initials: M
authors.lastname: Schmitt
mesh_terms: D000328:Adult; D000368:Aged; D004279:DNA, Viral / Q000235:genetics; D055088:Early Detection of Cancer / Q000379:methods; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D012367:RNA, Viral / Q000235:genetics*; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*; D055815:Young Adult; D002578:Uterine Cervical Dysplasia / Q000473:pathology / Q000821:virology*
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral
keywords: Cervical intraepithelial neoplasia; E1C; E1^E4; E6*I; HPV16 RNA patterns; Triage test