Abstract 1056: Impact of host methylome on cervical cancer pathogenesis
Authors: Sen Shrinka , Bhattacharjee Samsiddhi , Mukhopadhyay Indranil , Mandal Paramita , Sharma Sweta , Roy Chowdhury Rahul , Sengupta Sharmila 
Published: August 2015

Identification of episomal HPV16 in cervical cancers (CaCx) besides the integrated forms, prompted us to investigate the global CpG methylation signatures among the different categories of CaCx samples, since alterations of CpG methylation in host genomes, common in many cancers, are likely to influence host pathogen interactions and the disease pathogenesis. We determined global DNA methylation patterns of the host genomes, in HPV16 (+) CaCx cases (11 episomal and 12 integrated) and controls (12 HPV16 (-) and 12 HPV16 (+), employing the Illumina Infinium 450K Bead Chip assay. Using Genome Studio software, we extracted the raw signal intensities of each probe. With statistical methods developed in-house, we performed color adjustment and normalization of the data using control probes. Differentially methylated CpG loci were identified by comparing methylation estimates (M-values) using a two-sample t-test followed by adjustment of p-values based on False Discovery Rate threshold of 0.01 among (i) CaCx cases, irrespective of HPV16 physical status, (ii) CaCx cases with episomal HPV16 and (iii) CaCx cases with integrated HPV16, compared to controls irrespective of HPV, since no difference was recorded among these two groups. Differentially methylated CpGs were identified and mapped using Illumina annotation to various genic regions (TSS1500, TSS200, 1st Exon, Gene Body, 5′ UTR and 3′ UTR). Irrespective of viral physical status, hypermethylation was more prominent across all genic regions than hypomethylation, albeit hypermethylation being lower in Gene Body and 3′-UTR regions only. Subsequently employing a Δβ threshold of 0.3 i.e. difference of β between two groups as 30%, differentially methylated CpGs mapped to 2536, 3222 and 3003 genes among total CaCx set, episomal and integrated CaCx cases, respectively. Ingenuity Pathway Analysis revealed a number of pathways that were significantly enriched among total CaCx cases, episomal or integrated, while some enriched pathways were recorded exclusively among episomal (Gap Junction Signaling, FGF Signaling, Huntington's Disease Signaling) and integrated (Chemokine Signaling etc.) CaCx cases. Expression of FGF2 gene (FGF Signaling pathway), PIK3R1 gene (Gap Junction Signaling pathway) and CXCL12 gene (Chemokine Signaling pathway) was found to be down regulated, based on Illumina -HT12_V4 array analysis on same set of samples. Significant negative correlation between hypermethylation in the promoter region, involving 5 CpGs of FGF and 1 CpG of CXCL12, were recorded (Pearson Correlation p-value&lt;0.001). In case of PIK3R1, downregulated expression was negatively correlated with hypermethylation of 2 CpGs in the Gene Body. The study highlights the relevance of host methylation in cervical cancer pathogenesis involving Gene Body methylation in addition to promoters, and provides novel insights into HPV16 related CaCx pathogenesis involving episomal and integrated viral genomes.

Citation Format: Shrinka Sen, Samsiddhi Bhattacharjee, Indranil Mukhopadhyay, Paramita Mandal, Sweta Sharma, Rahul Roy Chowdhury, Sharmila Sengupta. Impact of host methylome on cervical cancer pathogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1056. doi:10.1158/1538-7445.AM2015-1056