Abstract 3173: HPV integration and methylation patterns in cervical intraepithelial neoplasia and invasive cervical cancer via HPV capture and high-throughput sequencing
Authors: Iden Marissa , Fye Samantha , Huang Yi-Wen , Liu Pengyuan , Rader Janet S. 
Published: May 2015

Persistent infection with high-risk human papillomaviruses (HPV) types is essential for the development of cervical cancer and its immediate precursor, cervical intraepithelial neoplasia 3 (CIN3). However, HPV infection alone is not sufficient to promote cervical carcinogenesis, thus it is hypothesized that the progression to invasive disease is dependent on viral and host factors. For example, it is well established that HPV integration into the host genome is a signature of invasive cervical cancer (ICC). Also, HPV genomes have CpG sites scattered throughout their genes that, with advancing disease, are increasingly methylated by the host cell's DNA methyltransferases. This methylation may alter the expression patterns of viral genes that are relevant to transformation. The goal of the current study was to investigate the landscape of viral-host integration and associated methylation in CIN and ICC. DNA from the lesions of patients diagnosed with either CIN or ICC was captured utilizing HPV and target gene enrichment and next-generation sequencing. DNA libraries were prepared using SureSelectXT Methyl-Seq Target Enrichment Kit (Agilent Technologies) and hybridized with a custom SureSelectXT Library (Agilent Technologies) designed to target 63 HPV types and xGen Lockdown Probes (Integrated DNA Technologies) designed to target 6 cancer-relevant genes. Library samples were pooled and sequenced using the Illumina HiSeq 2000. Bisulfate treated sequence reads obtained from Illumina Hi-Seq 2000 were analyzed by MethylCoder that generates per-base resolution of methylation data. In MethylCoder, the Genomic short-read nucleotide alignment program (GSNAP) was used for aligning short reads into reference genomes (HPV and hg19). For each CpG site, the methylation rate was calculated as the percentage of unconverted cytosines in each sample. In support of previous work, we found that multiple HPV types exhibited integration into various host genes in ICC. Also, HPV integration occurs in different patterns within the same gene. For example, in one tumor, HPV16 exhibited three distinct integrations into the PVT1 oncogene. Global methylation patterns were found to be unique among HPV types and samples, yet preliminary analysis suggests that the percent methylation in HPV may track with that of the host genome. Further, the overall methylation pattern of the virus was significantly different in CIN versus ICC. Here we provide a discussion of specific nonrandom integration sites across multiple ICC samples and representative HPV methylation profiles from CIN and ICC samples. Our data provide novel insights into the HPV CpG methylation sites and gene targets at viral-cellular junctions of different HPV types from CIN through ICC, thus potentially identifying new biomarkers for use in the screening and diagnosis of cervical cancer.

Citation Format: Marissa Iden, Samantha Fye, Yi-Wen Huang, Pengyuan Liu, Janet S. Rader. HPV integration and methylation patterns in cervical intraepithelial neoplasia and invasive cervical cancer via HPV capture and high-throughput sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3173. doi:10.1158/1538-7445.AM2014-3173