Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells

Human papillomavirus (HPV) genomes usually persist as episomal molecules in HPV associated preneoplastic lesions whereas they are frequently integrate d into the host cell genome in HPV-related cancers cells. This suggests tha t malignant conversion of HPV-infected epithelia is linked to recombination of cellular and viral sequences. Due to technical limitations, precise seq uence information on viral-cellular junctions were obtained only for few ce ll lines and primary lesions. In order to facilitate the molecular analysis of genomic HPV integration, we established a ligation mediated PCR assay f or the detection of integrated papillomavirus sequences (DIPS-PCR), DIPS-PC R was initially used to amplify genomic viral-cellular junctions from HPV-a ssociated cervical cancer cell lines (C4-I, C4-II, SW756, and HeLa) and HPV -immortalized keratinocyte lines (HPKIA, HPKII), In addition to junctions a lready reported in public data bases, various new fusion fragments were ide ntified. Subsequently, 22 different viral-cellular junctions were amplified from 17 cervical carcinomas and I vulval intraepithelial neoplasia (VIN II I). Sequence analysis of each junction revealed that the viral El open read ing frame (ORF) was fused to cellular sequences in 20 of 22 (91%) cases. Ch romosomal integration loci mapped to chromosomes 1 (2n), 2 (3n), 7 (2n), 8 (3n), 10 (1n), 14 (5n), 16 (1n), 17(2n), and mitochondrial DNA(1n), suggest ing random distribution of chromosomal integration sites. Precise sequence information obtained by DIPS-PCR was further used to monitor the monoclonal origin of 4 cervical cancers, 1 case of recurrent premalignant lesions and 1 lymph node metastasis. Therefore, DIPS-PCR might allow efficient therapy control and prediction of relapse in patients with HPV-associated anogenit al cancers. (C) 2001 Wiley-Liss, Inc.