Characterization of viral-cellular fusion transcripts in a large series of HPV16 and 18 positive anogenital lesions

Abstract

Persistent high risk type human papillomavirus (HR ± HPVs) infections induce dysplasia or cancer of the anogenital tract, most notably of the uterine cervix. The viral genome usually persists and replicates as an episomal molecule in early dysplasia, whereas in advanced dysplasia or cervical cancer HPV genomes are frequently integrated into the chromosomal DNA of the host cell. Previous studies suggested that modi®cation of critical cellular sequences by integration of HPV genomes might signi®cantly contribute to the neoplastic transformation of anogenital epithelia (insertional mutagenesis). This prompted us to characterize the integration loci of high risk HPV genomes in a large set of genital lesions. We ampli®ed E6/E7 oncogene transcripts derived from integrated HPV16 and HPV18 genomes and characterized in detail the co-transcribed cellular sequences of 64 primary genital lesions and ®ve cervical cancer cell lines. Database analyses of the cellular parts of these fusion transcripts revealed 51 dierent integration loci, including 26 transcribed genes (14 known genes, 12 EST sequences with unknown gene function). Seventeen sequences showed similarity to repetitive elements, and 26 sequences did not show any database match other than genomic sequence. Chromosomal integration loci were distributed over almost all human chromosomes. Although we found HPV sequences integrated into cancer related genes and close to fragile sites, no preferential site or integration motif could be identi®ed. These data demonstrate that target directed insertional mutagenesis might occur in few HPV-induced anogenital lesions, however, it is rather the exception than the rule.

MYC activation associated with the integration of HPV DNA at the MYC locus in genital tumors

“…HPV18 is frequently associated with adenocarcinoma (Lombard et al, 1998) and preferentially integrates at the MYC locus (Ferber et al, 2003b). However, we and others (Brink et al, 2002;Wentzensen et al, 2002) have also found HPV16 DNA at this locus. It is well established that cervical neoplasia start at the squamous columnar junction of the cervix epithelium, a part of the mucosa in which the basal cells, exposed to infections, have the potential to differentiate into either squamous or glandular cells.…”

“…Up to 181 different sites corresponding to 243 cases have been observed (Brink et al, 2002;Klimov et al, 2002;Wentzensen et al, 2004;Kraus et al, 2005;and our unpublished results). In spite of this scattering, it is remarkable that the chromosome band 8q24, to which MYC maps, is the most recurrent site for insertion of HPV DNA, as it is found in 10.7% (26/243) of the all cases (Du¨rst et al, 1987;Popescu et al, 1987;Couturier et al, 1991;Hori et al, 1991;Brink et al, 2002;Wentzensen et al, 2002;Ferber et al, 2003b;Ziegert et al, 2003;Kraus et al, 2005). A low level of MYC overexpression has been found in HeLa cells harboring HPV18 sequences in chromosome band 8q24 (Du¨rst et al, 1987;Lazo et al, 1989), but no evidence of MYC activation directly related to HPV insertion at the MYC locus has been published.…”

“…Besides MYC, a number of genes, potentially involved in carcinogenesis, such as CEACAM5, FANCC (Wentzensen et al, 2002;Ferber et al, 2003b), TP63 (Wentzensen et al, 2002), TERT (Ferber et al, 2003a), MYOF, Ribosomal protein gene RPS27 (Klimov et al, 2002), MYCN (Sastre-Garau et al, 2000), NFIB (Ziegert et al, 2003), have also been found to correspond to targets for the insertion of HPV DNA. In spite of this diversity, only a few cases with an integration pattern compatible with a mechanism of insertional mutagenesis have been observed (Wentzensen et al, 2002).…”

“…In spite of this diversity, only a few cases with an integration pattern compatible with a mechanism of insertional mutagenesis have been observed (Wentzensen et al, 2002). Cellular genome deletion, observed at the site of integrated HPV DNA (Wilke et al, 1996), may also lead to the inactivation of cellular genes with tumor suppressive properties.…”

“…These data suggest that MYC activation could be secondary to the insertion of HPV DNA sequences at the MYC locus. Although a large diversity of integration sites has been reported (Wentzensen et al, 2004), MYC chromosome region is the most recurrent localization for HPV insertion, found in about 10% of genital neoplasias, observed in chromosome band 8q24 by fluorescence in situ hybridization (FISH) (Du¨rst et al, 1987;Couturier et al, 1991;Hori et al, 1991;Brink et al, 2002) or at the MYC locus by DNA/RNA analysis (Wentzensen et al, 2002;Ferber et al, 2003b;Ziegert et al, 2003;Kraus et al, 2005). However, despite this recurrence, no evidence of MYC overexpression directly associated with the insertion of HPV DNA at the MYC locus has been shown so far.…”