Abstract 2216: Next-generation whole transcriptome sequencing of triple-negative breast tumors and normal tissues
Authors: Radovich Milan , Clare Susan E. , Pardo Ivanesa , Hancock Bradley A. , Kassem Nawal , Sledge George W. , Rufenbarger Connie , Storniolo Anna Maria V. , Mathieson Theresa , Sun Jie , Henry Jill E. , Lillemoe Heather A. , Hilligoss Eric E. , Elliott James S. , Richt Ryan , Hickenbotham Matthew , Glasscock Jarret , Liu Yunlong , Schneider Bryan P. 
Published: February 2011

Abstract
Background: Triple-negative breast cancer (TNBC) disproportionally affects pre-menopausal women and women of African-American descent, and has been plagued by the absence of targeted therapies leading to poor survival. The paucity of therapeutic targets in TNBC impels us to utilize new technologies that can determine novel targets on a global scale. Using next-generation sequencing, we embarked on a study to analyze the whole transcriptomes of TNBC tumors compared to normal breast tissues in order to comprehensively identify novel targets by analyzing all full length transcripts expressed in these tissues.
Methods: Normal breast tissues from healthy pre-menopausal volunteers with no history of disease were procured from the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. To eliminate bias from stromal tissue, epithelial cells were laser capture microdissected and RNA extracted from captured cells. cDNA libraries from 10 TNBC tumors and 10 normal breast tissues were subsequently sequenced on an ABI SOLiD3 sequencer using a 50bp fragment run. For gene expression, mapping of reads to the human genome was performed using the ABI Whole Transcriptome Pipeline and outputs were imported into Partek Genomics Suite for analysis. To analyze for gene fusions, reads were mapped to the genome using the SOLiD Analysis Pipeline Tool, followed by an alignment to Refseq to map reads crossing exon-exon junctions. A composite transcriptome was formed from areas of the genome with significant expression (17% of the genome sequence) and served as a concise search space for identifying fusions. Reads not mapping to the genome or to RefSeq (a rich source of fusion reads) were then mapped to the composite transcriptome using BLAT to facilitate a highly accurate split-read alignment. Using a custom developed pipeline, reads that spanned transcribed regions from two different chromosomes, or to loci farther than 200kb apart on the same chromosome, were considered as candidate fusions.
Results/Discussion: Sequencing of the 10 TNBC tumors and 10 normal samples produced 1.1 billion reads equaling 58.15GB of data. Mapping of the reads to the genome revealed 1.6 million transcribed regions (exons) of significant expression. A preliminary analysis of gene expression shows 55.2% of the transcribed loci to have significant differential expression between tumor and normal. Network-node, non-coding RNA, and statistical analyses are currently ongoing. In a further interim analysis, we bioinformatically identified several interchromosomal fusions that were present in a majority of the tumors but were absent in the normal samples. RT-PCR validation of these candidate fusions in a larger validation cohort of TNBC tumors and normal breast tissues is ongoing. A multitude of additional analyses including but not limited to: novel transcripts, alternative splicing, and presence of viral genes are also planned.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2216.