Gene Expression Profiling of Formalin-Fixed, Pariffin-Embedded (FFPE) Tissues from Triple-Negative (TN) Breast Cancer (BC) Patients.
Authors: Baumbach L. , Yan J. , Ahearn M. , Gomez C. , Jorda M. , Halsey T. , Mejias A. , Ellilson K. , Mulligan K. , Gluck S. , Pegram M. 
Published: December 2010

Abstract
Background: FFPE tumor samples present a technical challenge for gene expression profiling studies. Newer technologies are resulting in quality data and new insights from these archived tissues. TNBC is a sub-type of BC characterized by a lack of erbB2 gene amplification and estrogen and progesterone receptor expression. This clinically-defined BC sub-type carries a poor prognosis, is insensitive to hormonal or HER-2 targeted therapeutic agents, and has different incidence among ethnic groups. A better understanding of the genetic and molecular mechanisms underlying TNBC is critical to improving clinic outcomes and developing individualized therapies.Study Objective: We demonstrate the utility of FFPE samples in obtaining consistent, reproducible data from gene expression arrays, and apply this technology to the identification of differentially expressed genes between TNBC and normal breast tissue that are common or unique among selected ethnic groups.Methods: RNA isolation and labeled cDNA preparation from freshly cut FFPE blocks (marked by a pathologist as to normal vs. tumor tissue) was performed using the NuGEN™ WT-Ovation™ FFPE RNA Amplification System. Hybridization of tumor and normal cDNAs occurred to a breast cancer focused gene expression array (Breast Cancer DSA Research Tool, Almac Diagnostics Inc). Each patient' sample served as it's own control (tumor vs. normal). In total, 75 FFPE samples were profiled. The quality of each DSA chip was assessed on parameters selected automatically from GCOS report files per chip using MATLAB script based web application developed by Almac Dx. Data pre-processing used the Resolver Error Model. All parameters including Raw Q, Background, Scaling Factor and all controls met quality criteria set by Affymetrix and Almac Dx SOPs. Hybridization results were assessed using Principal Component Analysis and Clustering Analysis in Rosetta Resolver Gene Expression Data Analysis System 7.1 to identify potential outliers, contamination, or intra-tumor heterogeneity.Results: A Sign Agreement Matrix of FFPE and fresh frozen tissue samples during validation of the Breast Cancer DSA demonstrated that 98% of the probesets showed the same direction of fold change [p Spearman(FC)=0.84]. The FFPE samples had an average present call ∼ 43%, and more than 90% of the FFPE samples had present call rates greater than 25%. QC analysis demonstrated that the Almac Breast Cancer DSA was able to clearly and consistently separate tumor samples from normal in FFPE tissues and to identify samples of quality or integrity issues. Application of this methodology to analyses of differentially expressed transcripts between cancer and normal tissue samples across ethnicities in the TNBC samples, detected 1350 differentially expressed genes in the African-American group, 1220 genes in the Caucasian group and 1226 genes in the White Hispanic group. We also observed certain subtle ethnic-specific expression patterns across these three ethnic groups.Summary: The above-described methodology can be used to reliably measure gene expression in FFPE breast samples. Our study results are being validated in a larger data set.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6125.