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 cer vical intraepithelial neoplasia III (CIN III) cases and 37 invasive squamou s carcinomas for integration of human papillomavirus type 16 (HPV16). All e ighteen CIN III cases (severe dysplasia or high-grade squamous intraepithel ial lesion) were found to harbor episomal HPV, but one of the samples conta ined 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 t his was confirmed by rliPCR in 16 cases. One case, however, showed a low le vel of episomal deoxyribonucleic acid in addition to the predominant integr ated form. Of the remaining 20 carcinoma samples showing episomal forms in the previous analysis, 14 were found to contain integrated forms using rliP CR, and four contained multimeric episomal forms. Thus, in total, 31 of 37 of the carcinomas (84%) showed the integrated HPV16 genome. The rliPCR prod uct 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 informati on, bacterial artificial chromosome (BAC) and pi-derived artificial chromos ome (PAC) clones were obtained and used as probes in fluorescent in situ hy bridization experiments on human metaphase chromosomes. The results of the fluorescent in situ hybridization experiments showed evidence for HPV16 int egration in chromosome regions 1q25, 3q28, 6p25, 11p13, and 18q22. Sixteen carcinoma samples, containing episomal HPV16, were sequenced in the long co ntrol region. Evidence for changes in E2 binding or silencer YY1 sequences was found in only two samples.