Expression of small sets of genes in carcinoma and stromal cells predict clinical behavior of human breast cancer.
Authors: Andres SA , Kerr II DA , Englert DF , Wilson DJ , Wittliff JL 
Published: December 2010

Abstract
Abstract #6146
Background: The Human Genome Project has provided opportunities to develop precise tests for diagnostics, therapy selection and monitoring. From reiterative analyses of various microarray studies, 14 genes expressed in human breast carcinoma cells procured from de-identified tissue sections by LCM and 18 genes predicted in adjacent stromal cells were identified, whose expression appears related to clinical outcome. An additional 11 genes were identified in estrogen receptor positive (ER+) breast cancer exhibiting different survival probabilities. These genes have been evaluated with qPCR and with a disposable TipChip array (Xceed Molecular) as candidates for development of a prognostic test measuring clinically relevant gene expression profiles.&#x2028; Materials and Methods: RNA was isolated from 118 frozen tissue biopsies using the RNeasy® Mini kit (Qiagen) and analyzed for quality and quantity using the Bioanalyzer (Agilent). cDNA for qPCR measurements was prepared in Tris-HCl buffer containing KCl, MgCl2, DTT (Invitrogen), dNTPs (Invitrogen), RNasin® (Promega) and Superscript® RT III (Invitrogen). qPCR reactions were performed using Power Sybr® Green PCR Master Mix (Applied Biosystems), forward/reverse primers and cDNA obtained from the reverse transcription reaction. Relative gene expression was calculated with the ddCt method, using β-actin as the housekeeping gene and Universal Human Reference RNA (Stratagene) as a calibrator. Prior to gene expression analyses, extensive QC experiments were conducted, e.g., qPCR reactions were performed in triplicate with duplicate wells in each 384-well plate, to ensure reproducibility.&#x2028; Results: Gene candidates were analyzed on the Ziplex® Automated Workstation (Xceed Molecular) with a TipChip array containing probes for 61 genes including normalization controls. Preliminary gene expression results by qPCR and the TipChip were comparable for many of the 43 candidate genes (r2 ≥ 0.5 with ESR1 = 0.6). These data were correlated with disease-free and over-all survival outcomes stored in our extensive IRB-approved database. Expression of SLC39A6, FUT8 and CX3CL1 in the carcinoma/stromal subset correlated independently with disease-free survival using Cox Regression analyses (p &lt; 0.02). Using the 13-gene subset derived from ER+ breast cancers, CAXII, ERBB4 and RERG correlated independently with recurrence (p &lt; 0.03). These genes, as well as BCL2, correlated independently with overall survival (p &lt; 0.02).&#x2028; Conclusion: Expression profiles of 21 of the 32 candidate genes from this gene subset predicted early breast cancer recurrence. In an ER+/PR+ subpopulation, ERBB4 and HER-2 gene expression correlated best with recurrence in a multivariate Cox model (p = 0.001). Collectively, results suggest that an expression test based on these genes will predict clinical outcome. Supported in part by grants from Phi Beta Psi Charity Trust, University of Louisville's Office of Technology Transfer and from Xceed Molecular Corp. SAA & DAK II are recipients of IPBIS Graduate Fellowships.
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6146.