Abstract 2831: Identification of cell specific expression signatures using NGS global gene expression profiles from LCM procured breast carcinoma and adjacent stromal cells
Authors: Schageman Jeoffrey J. , Lea Kristi , Andres Sarah A. , Bramlett Kelli S. , Wittliff James L. 
Published: July 2017

Abstract
Our goal is to determine relationships of gene expression profiles to patient-associated characteristics, breast carcinoma pathology and biomarker status with clinical outcome to improve assessment of prognosis and therapy selection.
Procedures: De-identified frozen tissue biopsies of human breast carcinomas which were collected and stored under stringently controlled conditions were employed. Patient-related features (e.g., age, race, menopausal status, nodal status, clinical treatment and response) and results from biochemical analyses of established tumor markers (e.g., estrogen and progestin receptors, EGF receptor, HER-2/neu oncoprotein) guided tissue selection. Structural integrity of a tissue was first evaluated by light microscopy of H&E stained sections. To decipher uniqueness and clinical utility of gene expression profiles from specific cell types, Laser Capture Microdissection (LCM) with a PixCell IIeTM (Arcturus®, Thermo Fisher Scientific) was used to non-destructively collect either carcinoma cells or adjacent stromal cells on CapSureTM LCM caps. A second intact serial tissue section was processed identically to that of the section used for LCM as a control. Each of the two independent cell captures from the same tissue section and the second intact tissue section were independently extracted for RNA. RNA was assessed for integrity, purified and subjected to novel analyses using the Ion AmpliSeqTM RNA Breast Cancer Research Panel containing 1174 genes. Sequencing workflow included templating on the Ion Chef TM System and multiplexed sequencing on the Ion S5TM XL sequencing system and Ion 540TM chip.
Results: Each sequencing library yielded approximately 4-5 million reads with a mapping rate of &gt;98% that was achieved after alignment to the human transcriptome. Each sample was sequenced in triplicate with technical reproducibility near 99% with a mean of 1035 genes classified as detected. Cluster analyses of gene expression profiles revealed clear distinctions between stromal and carcinoma cells procured by LCM compared to intact tissue driven by strongly differentially expressed genes such as vimentin (VIM), MPRIP, stanniocalcin-2 (STC2) and estrogen receptor beta (ESR2).
Conclusions: Collectively, the results indicate that molecular signatures of breast carcinoma cells procured by LCM reveal unique gene expression profiles compared to those of the intact tissue section or the adjacent stromal cells. These novel findings are likely to discern molecular features unique to a breast carcinoma that may be related to clinical outcome to improve assessment of patient prognosis and therapy selection. Supported in part by a grant from the Phi Beta Psi Charity Trust (JLW & SAA) and a CTSP Award from the Commonwealth of Kentucky (JLW). For research use only.
Citation Format: Jeoffrey J. Schageman, Kristi Lea, Sarah A. Andres, Kelli S. Bramlett, James L. Wittliff. Identification of cell specific expression signatures using NGS global gene expression profiles from LCM procured breast carcinoma and adjacent stromal cells [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 2831. doi:10.1158/1538-7445.AM2017-2831