Detection of high‐risk human papillomavirus E6 and E7 oncogene transcripts in cervical scrapes by nested RT‐polymerase chain reaction

Abstract

The oncogenic potential of the high-risk human papillomavirus (HPV) genotypes (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) depends on the expression of the two viral oncogenes E6 and E7. Thus, the detection of HPV E6/E7 oncogene transcripts could serve as a factor in the evaluation of a risk of development of cervical intraepithelial neoplasia (CIN) and its progression to cervical cancer. A nested RT-PCR assay for the detection of E6/E7 oncogene transcripts of all known high-risk HPV genotypes was established. In the study described, 779 high-risk HPV-DNA-positive cervical scrapes exhibiting all grades of CIN, including non-dysplastic cervical mucosa (CIN 0), were examined. Spliced E6/E7 oncogene transcripts of all the high-risk HPVs were detected in numerous samples, with an overall detection rate of 47%. In 227 cases with agreement between the cytologic and histologic findings, the prevalence increased with lesion severity: CIN 0, 18%; CIN I, 58%; CIN II, 77%; CIN III, 84%. Multiple transcriptionally active high-risk HPVs were detected in 12% (33/279) of patients with multiple high-risk HPV infections. This work sets the stage for a prospective follow-up study currently being undertaken to evaluate the prognostic relevance of the detection of high-risk HPV E6/E7 oncogene transcripts for the persistence of a high risk HPV infection, and the possible evolution and further development of a CIN. Future applications of the assay described may include the monitoring of women in studies investigating antiviral treatment or vaccination.

Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts

“…The high levels of E6/E7 expression in CIN which we observed appear to be contradictory to the data obtained in several other studies (Falcinelli et al, 1993;Riethdorf et al, 2001;Sotlar et al, 2004;Molden et al, 2005;Andersson et al, 2006). The most probable explanation for this is the sampling strategy used (biopsies versus cervical scrapes).…”

Biological activity of probable/possible high‐risk human papillomavirus types in cervical cancer

“…1,2 Epidemiological studies have demonstrated that 20 mucosal HPV types from five phylogenetically related species of the genus alpha (a5, a6, a7, a9 and a11-termed as ''high-risk clade'') are consistently found as single HPV infections in CxCa worldwide. [3][4][5][6] Based on their frequency in CxCa and available biological data, 12 of these types, i.e., types 16,18,31,33,35,39,45, 51, 52, 56, 58 and 59 have been defined as carcinogens (IARC Group 1A)-hereafter referred to as high-risk (HR)-HPV. For eight other types, in the high-risk clade, i.e., types 26, 53, 66, 67, 68, 70, 73 and 82, the combination of their low frequency, lack of data on their active transcription and their transforming potential in model systems, has led them to be classified as only probable/possible carcinogens (IARC Groups 2A and 2B)-hereafter referred to as possible highrisk (pHR)-HPV types.…”

“…9 The presence of splice sites within the E6 ORF, giving rise to the E6*I/E7 transcript, is considered a biological feature of HR-HPV, but not low-risk types (LR-HPV). 8,10 E6*I splice sites have been described for all 12 HR- [11][12][13][14][15][16] and for pHR-HPV types 66 and 68. 16 For the maintenance of the transformed phenotype of HPV-infected CxCa cells continuous expression of both E6 and E7 genes is required.…”

“…8,10 E6*I splice sites have been described for all 12 HR- [11][12][13][14][15][16] and for pHR-HPV types 66 and 68. 16 For the maintenance of the transformed phenotype of HPV-infected CxCa cells continuous expression of both E6 and E7 genes is required. 17 The oncoproteins encoded by the E6 and E7 genes ensure cell proliferation by binding to the cellular p53 and pRb proteins, disrupting their functions and leading to cellular transformation.…”

“…These are predominantly available as formalin-fixed paraffin-embedded (FFPE) tissues, which are suitable for IHC analysis, but due to the degradation and cross-linking present a technical challenge for nucleic acid analysis. 24 Several commercially available RNA assays based on detection of full-length E6/E7 transcripts, 25,26 as well as ''in house'' RNA assays based on detection of spliced E6*I/E7 transcripts, 15,16,27 have been described. These assays aim to amplify rather long cDNA sequence from cervical smear samples (up to 620 bp for full-length and up to 280 bp for spliced transcripts, respectively) and therefore are not suited for FFPE tissue analysis.…”