Physical state of HPV16 and chromosomal mapping of the integrated form in cervical carcinomas

Using a procedure based on restriction enzyme cleavage, self-ligation, and inverse polymerase chain reaction (rliPCR), the authors investigated 18 cervical intraepithelial neoplasia III (CIN III) cases and 37 invasive squamous carcinomas for integration of human papillomavirus type 16 (HPV16). All eighteen CIN III cases (severe dysplasia or high-grade squamous intraepithelial lesion) were found to harbor episomal HPV, but one of the samples contained mixed episomal and integrated forms. Seventeen of 37 invasive cervical carcinoma samples were identified previously as containing the completely integrated HPV 16 genome by using PCR covering the entire E1/E2 gene, and this was confirmed by rliPCR in 16 cases. One case, however, showed a low level of episomal deoxyribonucleic acid in addition to the predominant integrated form. Of the remaining 20 carcinoma samples showing episomal forms in the previous analysis, 14 were found to contain integrated forms using rliPCR, and four contained multimeric episomal forms. Thus, in total, 31 of 37 of the carcinomas (84%) showed the integrated HPV16 genome. The rliPCR product from five carcinoma cases was cloned into a plasmid vector and used as a template for "primer walking" deoxyribonucleic acid sequencing to deduce human sequences flanking the integrated HPV genome. Based on this information, bacterial artificial chromosome (BAC) and pi-derived artificial chromosome (PAC) clones were obtained and used as probes in fluorescent in situ hybridization experiments on human metaphase chromosomes. The results of the fluorescent in situ hybridization experiments showed evidence for HPV16 integration in chromosome regions 1q25, 3q28, 6p25, 11p13, and 18q22. Sixteen carcinoma samples, containing episomal HPV16, were sequenced in the long control region. Evidence for changes in E2 binding or silencer YY1 sequences was found in only two samples.