Abstract 2424: The in-parallel whole-genome ChIP-Seq analysis of primary tissues, patient derived xenografts, and cancer cell lines from HPV-relative HNSCC samples
Authors: Kelley Dylan Z. , Flam Emily L. , Wulf Hildegard A. , Guo Theresa , Izumchenko Evgeny , Singman Dzov A. , Danilova Ludmila V. , Stavrovskaya Elena D. , Considine Michael , Bishop Justin A. , Westra William H. , Khan Zubair , Koch Wayne M. , Sidransky David , Wheelan Sarah , Califano Joseph A. , Favorov Alexander V. , Fertig Elana J. , Gaykalova Daria A. 
Published: July 2017

Abstract
This project develops a novel experimental technique to perform ChIP-Seq (chromatin immunoprecipitation with massively parallel DNA sequencing) analysis of chromatin structure in primary tumor tissues from high risk HPV-related head and neck squamous cell carcinomas (HPV+ HNSCC). Recent data suggest that chromatin structure is the central regulator and predictor of cancer-specific expression and mutagenesis landscape of diseased cells. Genome-wide gene expression dysregulation in many tumors, including HPV+ HNSCC, are incompletely described by current knowledge. Methods for study of chromatin structure in primary tumor tissue are needed to better understand the role global epigenetic changes may play in these tumors. However, ChIP-Seq, which is the state-of-the-art method of elucidating chromatin structure, until now, has not been reliably performed on any HNSCC samples. Because chromatin structure is disrupted at room temperature, ChIP-Seq is especially complicated for primary patient tissues, which are primarily obtained as surgical waste after pathology review. Snap freezing of leftover waste surgical tissues and further tissue thawing for the analysis decreases chromatin structure integrity necessary for highly sensitive ChIP-Seq methodology, especially for tumor samples with chromatin structure deformed during carcinogenesis. To improve the chromatin structure integrity in tumor sample we added a xenografting step and minimized the exposure of cancer tissue to room temperature conditions after mouse surgery. We also minimized patient non-cancer tissue preservation at ambient temperature after patient surgery. We successfully performed ChIP-Seq for H3K4me3, H3K9me3, and H3K9ac on frozen uvulopalatopharyngoplasty (UPPP) primary tissues, frozen patient derived xenograft tissues, and freshly-cultured head and neck squamous cell carcinoma cell lines, revealing comparable success rates between tissue type and sample preservation techniques. ChIP-Seq techniques were performed and cross validated using tried and true qRT-PCR methods to demonstrate data reproducibility. The biological relevance of the ChIP-Seq data was confirmed through massive RNA-Seq analysis of 47 HPV+ HNSCC samples and 25 non-cancer controls. Analysis revealed that most H3K9ac and H3K9me3 enrichment is similar in primary tissues, regardless of disease status. Only small portion of them showed differential histone enrichment, which correlated with differential expression of corresponding genes. On the other hand, H3K4me3 showed strong tissue specificity and were found differentially enriched especially in tumor samples. The proposed experimental pipeline demonstrates high reproducibility between biological replicates, diversity of tissue models, and low dependence of ChIP-Seq analysis on tissue preservation techniques.
Citation Format: Dylan Z. Kelley, Emily L. Flam, Hildegard A. Wulf, Theresa Guo, Evgeny Izumchenko, Dzov A. Singman, Ludmila V. Danilova, Elena D. Stavrovskaya, Michael Considine, Justin A. Bishop, William H. Westra, Zubair Khan, Wayne M. Koch, David Sidransky, Sarah Wheelan, Joseph A. Califano, Alexander V. Favorov, Elana J. Fertig, Daria A. Gaykalova. The in-parallel whole-genome ChIP-Seq analysis of primary tissues, patient derived xenografts, and cancer cell lines from HPV-relative HNSCC samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2424. doi:10.1158/1538-7445.AM2017-2424