Abstract 800: Simultaneous methylation and mutational analysis of breast cancer genomes using droplet-based targeted sequencing.
Authors: Samuels Michael L. , Roth Matt , Srinivasan Preethi , Riehle Kevin , Brufsky Adam , Hartmaier Ryan , Boone David , Harris R. Alan , Wu Chia-Chin , Oesterreich Steffi , Lee Adrian , Milosavljevic Aleksandar 
Published: August 2014

Targeted sequencing using droplet-based PCR provides an established method to selectively and uniformly amplify thousands of genomic regions of interest for deep, cost-effective next generation sequencing. Recently, this approach has been adapted for the analysis of bisulfite-treated DNA, enabling both quantitative epigenomic analysis (strand-specific determination of cytosine methylation status with single-base pair resolution) and genomic mutation detection. Simultaneous collection of both methylation and genomic aberration information with a single method makes efficient use of precious clinical material and allows both researchers and clinicians to use this information to sub-type cancers.

Here we report on our sequencing results using a single breast cancer targeted panel for sequence specific analysis of both epigenomic methylation and genomic mutation in breast tumor samples. The Breast Cancer MethylSeq Panel includes PCR primer pairs designed to amplify 1000 targets, leaving room for an additional 3000 target loci of interest to be added by individual researchers in the future. This single panel provides information about both loci-specific CpG methylation and genomic mutations, enabling analysis of multiple content types including: A) cell subtype (CELL); B) genomic mutation (MUT); C) surrogate gene expression subtypes using promoter CpG islands or other methylation marks (EXP); D) copy-number variation (CNV); E) Loss of Heterozygosity (LOH); and F) methylation-mediated gene silencing and loss of imprinting (IMP). In addition, miRNA associated with breast cancer will be targeted for analysis (MIR).

The Breast Cancer MethylSeq Panel was tested and validated with a set of 64 bisulfite-converted DNA samples that included breast cancer cell lines, a range of ER positive and negative tumors and TCGA samples, and a set of 8 metastatic tumors from a single individual. Overall Illumina HiSeq sequencing success metrics, concordance with metadata on the various target classes (A-F above) using validated controls, and results from the patient samples will be presented.

The Breast Cancer MethylSeq Panel allows simultaneous analysis of epigenomic and genomic sequence alterations associated with breast tumor samples, enabling tumor sub-typing and correlation with clinical outcomes. This ‘core’ panel provides a unique (expandable) screening tool for the breast cancer research community, and a paradigm for efficient analysis of multiple endpoints from a single assay for other diseases.

Citation Format: Michael L. Samuels, Matt Roth, Preethi Srinivasan, Kevin Riehle, Adam Brufsky, Ryan Hartmaier, David Boone, R. Alan Harris, Chia-Chin Wu, Steffi Oesterreich, Adrian Lee, Aleksandar Milosavljevic. Simultaneous methylation and mutational analysis of breast cancer genomes using droplet-based targeted sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 800. doi:10.1158/1538-7445.AM2013-800