Abstract 5554: Tumor tissue dissection for gene expression assessments in translational research studies
Authors: Kotoula Vassiliki , Kalogeras Konstantine T. , Televantou Despina , Kronenwett Ralf , Wirtz Ralph M. , Bobos Mattheos , Fountzilas George 
Published: August 2012

Abstract
Background - aim: Translational research studies increasingly employ routine histologic material (FFPE) for the investigation of gene expression with extract-based methods, like quantitative real time PCR (QPCR). Statistically significant differences in the expression levels of mRNA transcripts in tumor vs neighbouring non-tumor tissues have led to the consensus of including as high as possible tumor cell rates in the extract. Herein, we investigated whether the same mRNA markers yield different clinically relevant values when examined in macrodissected (MD) and non-macrodissected (NMD) FFPE breast carcinoma tissues. Methods: Matched NMD and MD samples from primary tumors (P, n=98 sample pairs) and from metastatic lymph nodes (LN, n=72, overlapping but not matching P samples) were recruited from a cohort of 357 early high-risk breast cancer patients that had been adjuvantly treated with epirubicin-CMF regimens with or without paclitaxel. FFPE sections were evaluated for tumor cell content (TCC) and were used as whole sections and upon manual macrodissection for the same tumor. All samples were assessed for ESR1, ERBB2, MAPT, MMP7 and RACGAP1 mRNA expression with QPCR, and the obtained relative quantification (RQ) values were compared as continuous variables and upon hierarchical clustering in matched sample series with respect to patient outcome. Results: Mean, median (±SD) values for TCC% in P-NMD / P-MD samples were 27.0, 25.0 (±14.8) / 68.1, 67.5 (±20.5) and in LN-NMD / LN-MD samples 30.1, 35.0 (±18.0) / 82.0, 90.0 (±18.2), respectively. Minimum TCC% was 2.5 in P-NMD and in LN-NMD samples, 35.0 in P-MD and 27.5 in LN-MD. In comparison to matched NMD samples, mRNA expression of ESR1 (p&lt;0.001) and MMP7 (p=0.016) was significantly lower in P-MD samples; MMP7 was also lower (p=0.006) but ESR1 (p=0.008), ERBB2 (p&lt;0.001), MAPT (p=0.005) and RACGAP1 (p&lt;0.001) were higher in LN-MD samples. A three-cluster approach yielded 68% concordance between P-NMD and P-MD, and 61% concordance between LN-NMD and LN-MD samples. Upon testing in each one of the four sample series, significant associations were observed for the above markers individually and in clusters with respect to patient disease-free and overall survival. However, the results obtained for P-NMD and LN-NMD samples did not differ from the ones in P-MD and in LN-MD samples, respectively. Conclusions: Although TCC% largely affects the obtained RQ values reflecting mRNA expression, the obtained results for ESR1, ERBB2, MAPT, MMP7 and RACGAP1 mRNA expression in MD samples did not practically add to, nor did they alter the evaluation of these markers with respect to patient outcome. For the five markers examined in this study, and probably for additional markers as well, the necessity of performing tissue macrodissection for enrichment in tumor cells in translational research and, potentially, diagnostics may need to be revisited.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5554. doi:1538-7445.AM2012-5554