The majority of viral-cellular fusion transcripts in cervical carcinomas cotranscribe cellular sequences of known or predicted genes.

Integration of human papillomavirus (HPV) DNA into the host genome is a frequent event in cervical carcinogenesis and is reported to occur at randomly selected chromosomal sites. However, as the databases are being up-dated continuously, the knowledge based on sequenced viral integration sites also expands. In this study, viral-cellular fusion transcripts of a preselected group of 74 cervical carcinoma or cervical intraepithelial neoplasia grade 3 (CIN3) biopsies harboring integrated HPV16, HPV18, HPV31, HPV33, or HPV45 DNA were amplified by 3'-rapid amplification of cDNA ends PCR and sequenced. Consistent with previous reports, integration sites were found to be distributed throughout the genome. However, 23% (17 of 74) of the integration sites were located within the cytogenetic bands 4q13.3, 8q24.21, 13q22.1, and 17q21, in clusters ranging from 86 to 900 kb. Of note is that clusters 8q24.21 and 13q22.1 are within 1.5 Mbp of an adjacent fragile site whereas clusters 4q13.3 and 17q21 are >15 Mbp distant to any known fragile sites. It is tempting to speculate that as yet unknown fragile sites may be identified on the basis of HPV integration hotspots. No correlation between HPV type and specific integration loci was found. Of 74 fusion transcripts, 28 contained cellular sequences, which were homologous to known genes, and 40 samples contained sequences of predicted genes. In 33 fusion transcripts, both viral and cellular sequences were in sense orientation, indicating that the gene itself or upstream sequences were affected by integration. These data suggest that the influence of HPV integration on host gene expression may not be a rare effect and should encourage more detailed analyses.

Type-dependent integration frequency of human papillomavirus genomes in cervical lesions.

Chromosomal integration of high-risk human papillomavirus (HR-HPV) genomes is believed to represent a significant event in the pathogenesis of cervical cancer associated with progression from preneoplastic lesions to invasive carcinomas. This hypothesis is based on experimental data suggesting that integration-dependent disruption of HR-HPV E2 gene functions is important to achieve neoplastic transformation and on clinical data gathered by analyzing lesions induced by human papillomavirus (HPV) 16 and 18 that revealed integrated viral genome copies in the vast majority of cervical cancer cells. However, a substantial fraction of cervical cancers is associated with other HR-HPV types for which virtually no data concerning their integration status have been reported so far. Here, we compared integration frequencies of the five most common oncogenic HPV types (HPV16, 18, 31, 33, and 45) in a series of 835 cervical samples using a specific mRNA-based PCR assay (Amplification of Papillomavirus Oncogene Transcripts). Most precancerous lesions displayed exclusively episomal viral genomes, whereas 62% of the carcinomas had integrated viral genomes. However, the frequency of integrated HR-HPV genomes showed marked differences for individual HR-HPV types. HPV16, 18, and 45 were found substantially more often in the integrated state compared with HPV types 31 and 33. The analysis of the median age of patients with high-grade precancerous lesions and invasive cancers suggests that precancers induced by HPV types 18, 16, and 45 progress to invasive cervical cancer in substantially less time compared with precancers induced by HPV types 31 and 33. These findings suggest that integration of oncogenic HPV genomes in cervical lesions is a consequence rather than the cause of chromosomal instability induced by deregulated HR-HPV E6-E7 oncogene expression. Distinct HR-HPV types apparently provoke chromosomal instability in their host cells to a different extent than is reflected by their integration frequencies in advanced lesions and the time required for CIN 3 lesions to progress to invasive cancer.

Human papillomavirus DNA and e6/e7 mRNA status in relation to survival of patients treated for cervical squamous cell carcinoma.

The prognostic significance of human papillomavirus (HPV) DNA and E6/E7 mRNA, the presence of specific types, and the physical state of HPV DNA, were studied in 202 cervical squamous cell carcinomas. Absence or non-detectable levels of high-risk (types 16, 18, 31, 33, 35, 45, 52 and 58) E6/E7 mRNA, using the real-time nucleic acid sequence based amplification (NASBA) assay, and absence of HPV high-risk/HPV 6, 26, 66, 69, 73 (all methods collectively) were associated with poor overall survival in univariate analysis (P = 0.04 and P = 0.03, respectively) and had independent prognostic value in multivariate analysis (P = 0.01 and P = 0.03, respectively) including FIGO stage and age. Based on the individual results of type-specific PCR and in situ hybridization (ISH), the presence of HPV DNA was not found to be a prognostic factor. Likewise, concerning the presence of specific HPV types and the HPV integration status (determined by ISH), no prognostic significance was found. Mutation analyses of the TP53 gene revealed mutations in 3 of the 6 HPV negative samples (50%).

PreTect HPV-Proofer: real-time detection and typing of E6/E7 mRNA from carcinogenic human papillomaviruses.

Monitoring human papillomavirus (HPV) E6/E7 mRNA expression may provide an accurate and informative diagnostic approach for detection of oncogene activity related to the development of severe dysplasia or cervical carcinoma. A multiplex nucleic acid sequence based amplification (NASBA) assay, utilizing molecular beacon probes for real-time detection was developed for the identification of E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45. The assay is called PreTect HPV-Proofer and this report describes the development and the analytical performance of the assay. The reproducibility of PreTect HPV-Proofer with regard to a positive result was found to be between 96 and 100%, depending on HPV type. The melting temperature for the different molecular beacons was in the range of 48-55 degrees C, indicating conformational stability, i.e. the molecular beacons will not get activated by the 41 degrees C annealing temperature, but will be activated by the annealing to the target itself. The limit of detection for HPV 16 was ten SiHa or CaSki cells and for HPV 18 one HeLa cell. No cross reactivity was observed with E6/E7 mRNA from the other tested HPV types. mRNA from cervical cells was also successfully amplified after more than one year of storage. In conclusion, the PreTect HPV-Proofer assay, individually identifying E6/E7 mRNA expression from five carcinogenic HPV types, is a reproducible assay that may serve as a valuable tool in monitoring HPV infections producing proteins with a transforming potential.

Presence of E6 and E7 mRNA from human papillomavirus types 16, 18, 31, 33, and 45 in the majority of cervical carcinomas.

The oncogenic potential of the human papillomavirus (HPV) early genes E6 and E7 is well established and a source of interest with regard to HPV testing for cervical carcinoma. Here we present a study performed with 204 histologically confirmed invasive cervical squamous cell carcinomas (SCCs) in which we evaluated the HPV E6 and E7 mRNA detection assay PreTect HPV-Proofer for detection of high-risk HPV types 16, 18, 31, 33, and 45. For further evaluation, detection of E6 and E7 mRNA from HPV types 35, 52, and 58 by real-time multiplex nucleic acid sequence-based amplification was also included. For comparison and to assess the overall prevalence of various HPV types, samples were also tested for HPV DNA by both consensus and type-specific PCR, reverse line blotting, sequencing, and in situ hybridization. The overall prevalence of HPV was 97%. HPV E6 and E7 transcripts were detected in 188 of 204 (92%) biopsy specimens, of which 181 contained one of the following HPV types: 16, 18, 31, 33, or 45. Consensus PCR and type-specific PCR detected HPV in 187 of 204 and 188 of 204 (92%) specimens, respectively. In conclusion, this study verifies the presence of HPV E6 and E7 mRNA in SCCs and demonstrates that HPV infections among Norwegian women with SCCs are limited mainly to the five high-risk types, 16, 18, 31, 33, and 45. This, together with the fact that PreTect HPV-Proofer detects the HPV oncogenic transcripts, suggests that the assay is a valuable approach in the field of HPV detection in cervical carcinoma.

Comparison of human papillomavirus messenger RNA and DNA detection: a cross-sectional study of 4,136 women >30 years of age with a 2-year follow-up of high-grade squamous intraepithelial lesion.

The purpose of this study was to compare the detection of human papillomavirus (HPV) DNA with detection of mRNA. The study included 4,136 women >30 years of age. E6/E7 mRNA expression from the carcinogenic HPV types 16, 18, 31, 33, and 45 was detected by the PreTect HPV-Proofer assay, whereas the presence of HPV DNA was detected by Gp5+/6+ consensus PCR followed by type-specific PCR. A total of 4.0% had an abnormal cytologic diagnosis, 3.0% were positive by PreTect HPV-Proofer, 4.4% by type-specific PCR, and 10.4% by consensus PCR. For detection of HPV in high-grade squamous intraepithelial lesion (HSIL), no significant difference was observed between PreTect HPV-Proofer and consensus PCR. For women with a cytologic normal, atypical squamous cell of uncertain significance, and low-grade SIL diagnosis, the detection rate of HPV was significantly higher by Gp5+/6+ consensus PCR (P < 0.005) than by PreTect HPV-Proofer. Histology confirmed 14 of 23 cytologic HSIL as cervical intraepithelial neoplasia grade >2. Of these women, PreTect HPV-Proofer and type-specific PCR detected 12, whereas consensus PCR detected 13. In conclusion, for HSIL, detection of E6/E7 transcripts from HPV types 16, 18, 31, 33, and 45 are present to the same degree as DNA detected by consensus PCR. Equally important, only a small proportion of the HPV DNA-positive women with a normal, atypical squamous cell of uncertain significance or low-grade SIL diagnosis had a detectable mRNA expression. HPV E6/E7 mRNA detection by PreTect HPV-Proofer represents a new promising test as an adjunct to cytology.

Predicting CIN2+ when detecting HPV mRNA and DNA by PreTect HPV-proofer and consensus PCR: A 2-year follow-up of women with ASCUS or LSIL Pap smear.

It has been suggested that human papillomavirus (HPV) testing improves follow-up of atypical cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) in cervical cancer screening programs. To evaluate the prognostic value of including HPV testing as an adjunct to cytology, we carried out a 2-year follow-up study of 77 women with ASCUS or LSIL Papanicolaou (Pap) smear in the Norwegian Cervical Cancer Screening Program (NCCSP) for detection of histological cervical intraepithelial neoplasia (CIN) 2+. The study includes a comparison between viral mRNA and DNA detection. PreTect HPV-Proofer was used for HPV E6/E7 mRNA detection from the 5 high-risk types 16, 18, 31, 33 and 45, and Gp5+/6+ consensus PCR was used for HPV DNA detection. Twice as many women were positive for HPV DNA (54.6%) than for HPV mRNA (23.4%). PreTect HPV-Proofer and consensus PCR had a sensitivity of 85.7% (95% confidence interval [CI] = 42.1-99.6) for detecting CIN2+ during follow-up. The specificity was significantly higher for PreTect HPV-Proofer, 84.9% (95% CI = 73.9-92.5), than for consensus PCR, 50.0% (95% CI = 37.4-62.6). PreTect HPV-Proofer positive women were 69.8 times (95% CI = 4.3-1137.3) more likely to be diagnosed with CIN2+ within 2 years than PreTect HPV-Proofer negative women. Consensus PCR-positive women were 5.7 times (95% CI = 0.6-52.0) more likely to be diagnosed with CIN2+ within 2 years than PCR-negative women. With equal sensitivity and higher specificity than consensus PCR, the PreTect HPV-Proofer might offer an improvement for the triage of women with ASCUS or LSIL Pap smear.