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.

Key words: HPV; human papillomavirus; DNA; mRNA; PreTect HPV-Proofer; NASBA; PCR; ASCUS; LSIL Cytological cervical cancer screening programs have been successful in reducing the incidence of cervical cancer, even though a single conventional Papanicolaou (Pap) smear is only moderately accurate and does not achieve concurrent high sensitivity and specificity. 1,2 The management of women with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) is problematic because only a small proportion will progress to cervical intraepithelial neoplasia (CIN) 3 and invasive cervical carcinoma (ICC). Histologically verified CIN has been found in 10 -60% of women with an ASCUS diagnosis, with CIN2/3 present in more than 5%. 3-11 Pap smear follow-up of women with an ASCUS smear fails to identify all women at higher risk of CIN2ϩ, suggesting that cervical cancer screening programs might benefit from implementing new diagnostic tests in the triage of women with equivocal Pap smears. 12 Infection with high-risk (HR) types of HPV is necessary for the development of ICC 13-16 and the expression of the E6/E7 oncogenes is necessary for conversion to and maintenance of malignancy in cervical tissue. [17][18][19] Therefore, detection of the E6/E7 mRNA of HR-HPV types might serve as a better risk evaluation factor than mere DNA detection for the development of high-grade squamous intraepithelial lesion (HSIL) and ICC. 20 The combination of cytology and HPV testing seems to save additional life at a reasonable cost compared to Pap testing alone. 21,22 Detection of E6/E7 mRNA can be achieved by using the commercial PreTect HPV-Proofer assay (NorChip AS, Klokkarstua, Norway), that utilizes nucleic acid sequence based amplification (NASBA).The aim of our study was to assess whether a positive HPV mRNA or DNA test at the time of an ASCUS or LSIL Pap-smear identifies women diagnosed with a histological CIN2ϩ after 2 years of follow-up. Material and methods Study subjectsThe study subjects comprise a subgroup from 4,136 women older than 30 years of age that visited a selection of gynecologists in Oslo, Norway, and have been tested in 2001 for the presence of HPV DNA by Gp5ϩ/6ϩ consensus PCR and E6/E7 transcripts by real-time multiplex NASBA (PreTect HPV-Proofer, NorChip AS) in addition to cytology. 35 PreTect HPV-Proofer detects mRNA from HPV types 16, 18, 31, 33 and 45, whereas Gp5ϩ/6ϩ consensus PCR detects HPV DNA from the L1 region in Ͼ20 HPV types. We included all women with an index Pap smear diagnosis of ASCUS or LSIL (n ϭ 77). The index Pap smear refers to the smear taken together with the HPV testing. Information on Pap smears in the 10-year period before the inclusion in our study was obtained from CRN registers. Former abnormal smears mean any smear that is not normal or unsatisfactory and has been taken before the index smear in 2001. Follow-upSeventy-seven women were followed up for 24 months in the registers of the Cancer Registry of Norway (CRN) with subsequent Pap smears...

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.The presence of human papillomavirus (HPV) infection in the majority of cases of cervical neoplasia has been considered evidence of an etiological role of HPV in cervical cancer. The association is strong, consistent, and specific to a limited number of viral types (2,5,32,46). On the other hand, the lifelong risk of HPV infection is 80%, and only a small proportion of women infected with HPV will develop high-grade cervical neoplasia (42). Among HPV-positive women, it is therefore of the utmost importance to identify those with an increased risk of developing cervical carcinoma by either methods that reveal persistent HPV infection (38), methods that detect viral oncogene expression (40), or the use of additional human markers for cervical carcinogenesis (45).The most frequent HPV types found in cervical intraepithelial neoplasia and squamous cell carcinoma (SCC) are HPV type 16 (HPV-16) and HPV-18, which in 1995 were classified as human carcinogens by the International Agency for Research on Cancer. In total, based on their high frequencies in carcinomas, 13 HPV types are now considered carcinogenic types (6). In the largest multinational studies performed so far, HPV types 16, 18, 31, 33, and 45 were shown to be the most prevalent types associated with cervical carcinomas (2, 22, 32), with HPV-16 alone found in more than 50% of the cases. The oncogenic potential of these high-risk HPV types lies in the oncoproteins E6 and E7, which bind to and modulate a number of different gene products, in particular, the ...

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 HPVProofer assay, whereas the presence of HPV DNA was detected by Gp5+/6+ consensus PCR followed by typespecific 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.

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. [Cancer Res 2008;68(7):2514-22]