DNA Aneuploidy and Integration of Human Papillomavirus Type 16 E6/E7 Oncogenes in Intraepithelial Neoplasia and Invasive Squamous Cell Carcinoma of the Cervix Uteri

Abstract

Purpose: Increasingly deregulated expression of the E6-E7 oncogenes of high-risk human papillomaviruses (HRHPVs) has been identified as the major transforming factor in the pathogenesis of cervical dysplasia and derived cancers. The expression of these genes in epithelial stem cells first results in chromosomal instability and induces chromosomal aneuploidy. It is speculated that this subsequently favors integration of HR-HPV genomes into cellular chromosomes. This in turn leads to expression of viral cellular fusion transcripts and further enhanced expression of the E6-E7 oncoproteins. Chromosomal instability and aneuploidization thus seems to precede and favor integration of HR-HPV genomes.Experimental Design: To prove this sequential concept, we analyzed here the sequence of events of DNA aneuploidization and integration in a series of HPV-16-positive cervical dysplastic lesions and carcinomas. Eighty-five punch biopsies of HPV-16-positive cervical lesions (20 CIN1/2, 50 CIN3, and 15 CxCa) were analyzed for DNA ploidy by DNA flow cytometry and for integration of HPV E6/E7 oncogenes using the amplification of papillomavirus oncogene transcripts assay, a reverse transcription-PCR method to detect integrate-derived human papillomavirus oncogene transcripts.Results: DNA aneuploidy and viral genome integration were both associated with increasing dysplasia (P < 0.001, 2 test for trend). In addition, DNA aneuploidy was associated with increased viral integration (P < 0.01, Fisher's exact test). Nineteen of 20 (95%) lesions with integrated viral genomes had aneuploid cell lines; however, only 19 of 32 (59%) lesions with aneuploid cell lines had integrated viral genomes.Conclusions: These data support the hypothesis that aneuploidization precedes integration of HR-HPV genomes in the progression of cervical dysplasia. Accordingly, deregulated viral oncogene expression appears to result first in chromosomal instability and aneuploidization and is subsequently followed by integration of HR-HPV genomes in the affected cell clones.

Type‐specific human papillomavirus oncogene messenger RNA levels correlate with the severity of cervical neoplasia

“…24 There was no differences in testing performance with duration of storage (data not shown). A QRT-PCR target to human GAPDH gene (GenBank J04038) was applied for all sample RNAs to validate the quality of RNA extract.…”

Chromosomal Biomarkers for Detection of Human Papillomavirus Associated Genomic Instability in Epithelial Cells of Cervical Cytology Specimens

“…Accumulation of copy number imbalances using the comparative genomic hybridization approach was demonstrated on 70 snap-frozen cervical squamous intraepithelial lesions. 23 DNA flow cytometry analysis of 85 punch biopsies of HPV16-positive cervical lesions in the study of Melsheimer et al 24 revealed a statistically significant trend of aneuploidy associated with increasing severity of dysplasia. The 8q24 region has also been shown to be a frequent site of HPV integration and is often amplified in cervical cell lines and cervical lesions.…”

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

“…In an in vitro study acquisition of high levels of genomic instability in cervical keratinocytes in monolayer culture occurred after integration of HPV16 (Pett et al, 2004). On the other hand, Melsheimer et al (2004) have shown that in CIN and cervical carcinomas aneuploidization precedes integration of HPV DNA in the progression of cervical dysplasia. However, irrespective of a temporal association between integration and genetic instability, the current concept of cervical carcinogenesis suggests that integration provides the cell with a selective growth advantage (Duensing and Munger, 2004;Wentzensen et al, 2004).…”