eprintid: 15850
rev_number: 12
eprint_status: archive
userid: 931
dir: disk0/00/01/58/50
datestamp: 2013-12-06 11:13:48
lastmod: 2013-12-09 08:12:05
status_changed: 2013-12-06 11:13:48
type: doctoralThesis
metadata_visibility: show
creators_name: Höfler, Daniela
title: HPV16 RNA patterns as diagnostic marker for
cervical cancer precursor lesions: Validation by
newly developed high-throughput RT-qPCR
subjects: ddc-570
subjects: ddc-600
divisions: i-140001
adv_faculty: af-14
abstract: Cervical cancer (CxCa) is the second most common cancer among women world-wide. DNA of 14
high-risk human papillomavirus (hrHPV) types is found in almost all cervical cancers (>96.3%),
with HPV16 being the most prevalent type. Cervical cells can be screened for abnormalities using
the cytological Papanicolaou (Pap) test and for the presence of HPV DNA and HPV E6/E7 fulllength
mRNA. However, all of the available tests show considerable drawbacks exhibiting either
poor clinical sensitivity or specificity. Reduced clinical specificity leads to over-treatment,
additional costs and enormous anxiety for women concerned. A highly specific identification of
high-grade lesions is desirable since those require surgical therapy contrary to early lesions that are
likely to regress spontaneously. Recently described HPV16 RNA patterns comprising the
upregulated transcripts E6*II and/or E1C, and the downregulated transcripts E1^E4 and/or L1 in
HPV16-transformed cervical cells showed potential to substantially improve HPV-based cervical
precancer screening: in a small cytology-based pilot study these patterns identified severe HPV16-
induced cervical lesions with a clinical sensitivity of 74% and a specificity of 84%. However,
singleplex nucleic acid sequence-based amplification (NASBA) assays used in the previous work
was labour, time and cost intensive.
In this PhD thesis, novel quantitative multiplex high-throughput reverse transcription PCR were
developed for the detection of the HPV16 RNA patterns with detection limits below 100 transcript
copies per PCR. Cross-reactivity with unspliced HPV16 sequences and cellular background
DNA/RNA was excluded. The comparison of the newly developed assay and the established
singleplex NASBA assays using RNA from 32 HPV16-positive fresh-frozen oropharyngeal
squamous cell carcinomas indicated a good quantitative correlation. The concordance of HPV16
RNA patterns between both methods was 100%. In order to validate HPV16 RNA patterns as
diagnostic marker for cervical cancer and its severe precursor lesions 165 single HPV16 DNApositive
cervical cell samples were analysed. The sensitivity for CIN3 and CxCa was 88% and the
specificity 84%, even in archived specimens with low RNA quality. Several HPV16 RNA patterns
“false-positive” CIN1 lesions in follow-up had progressed to CIN2 or CIN3 lesions, raising the
possibility that HPV RNA patterns could be an earlier marker for developing severe lesions than
histology. Poor quality of RNA extracted from formalin-fixed paraffin embedded tissues strongly
limits HPV16 RNA patterns analysis. To extend the HPV RNA patterns to the further seven most
frequent hrHPV types 18, 31, 33, 35, 45, 52 and 58, unknown splice junctions were identified. For
HPV18 singleplex assays were developed. The detection limit ranged between 101 E6*I and E1C
and 100 E1^E4 copies per PCR and will be improved in future experiments.
date: 2013
id_scheme: DOI
id_number: 10.11588/heidok.00015850
ppn_swb: 774585412
own_urn: urn:nbn:de:bsz:16-heidok-158509
date_accepted: 2013-10-16
advisor: HASH(0x55fc36ad5820)
language: eng
bibsort: HOFLERDANIHPV16RNAPA2013
full_text_status: public
citation:   Höfler, Daniela  (2013) HPV16 RNA patterns as diagnostic marker for cervical cancer precursor lesions: Validation by newly developed high-throughput RT-qPCR.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/15850/1/Phd%20thesis.DELI.240713.pdf