Abstract 2216: Next-generation whole transcriptome sequencing of triple-negative breast tumors and normal tissues

• Published in: Cancer research (Chicago, Ill.) Ill.), 2010-04, Vol.70 (8_Supplement), p.2216-2216
• Main 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.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM10-2216

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 inter