Recombination of human papillomavirus‐16 and host DNA in exfoliated cervical cells: A pilot study of L1 gene methylation and chromosomal integration as biomarkers of carcinogenic progression

Human papillomavirus‐16 DNA replicates in productive infections in circular form, but is found in most carcinomas integrated into the host cell DNA. Because this transition is essential for carcinogenesis, detailed research is desirable and may help to triage patients with abnormal Pap smears. Previous studies addressed the chromosomal integration of HPV‐16 DNA in biopsies of tumors by an indirect biomarker, methylation of the viral L1 gene and by reverse ligation polymerase chain reaction (rliPCR). The pilot study reported here asked whether these techniques can be targeted successfully at DNA prepared from exfoliated cervical cells. Abnormal Pap smears of 21 patients that were positive for HPV‐16 were analyzed for (i and ii) methylation of the L1 gene after bisulfite modification and PCR amplification by direct sequencing and indirectly in cloned DNA and (iii) recombination with chromosomal DNA by rliPCR. Four of these 21 patients contained highly methylated L1 DNA, which was integrated in three of the samples with sufficient DNA for rliPCR analysis. Seven patients contained sporadically methylated L1 DNA, which was integrated in two and episomal in three samples with sufficient DNA. Ten patients contained only unmethylated DNA, which was episomal in six but possibly integrated in two samples. It is concluded that HPV‐16 is found integrated chromosomally in a fraction of precancerous infections, and with higher frequency in methylated than in low or unmethylated samples. Since L1 gene methylation indicates integration, it has the potential to be used as a clinical marker of cancer progression. J. Med. Virol. 82:311–320, 2010. © 2009 Wiley‐Liss, Inc.