Abstract LB-142: Preservation of gene expression profile and histomorphology in human breast tumor tissue with the new PAXgene® tissue system

Abstract

Abstract Introduction: PreAnalytiX has developed a system for preservation of histomorphology and nucleic acids in paraffin embedded tissue samples. The system is comprised of a collection container for formalin-free fixation and stabilization of tissue specimens and purification kits for isolation of DNA, RNA or microRNA (miRNA) from PAXgene Tissue fixed, paraffin embedded (PFPE) tissue samples. In this case study, a tissue specimen of human infiltrating ductal carcinoma (HIDC) of the breast was divided into three parts after resection and fixed either in neutral buffered formalin (NBF) or the PAXgene Tissue Container, or snap frozen in liquid nitrogen (LN2). The three, different preparations of the tumor sample were compared for preservation of histomorphology, RNA integrity, and gene expression profile. Materials and Methods: The part of the tumor specimen treated with PAXgene Tissue reagents (PFPE) was fixed for two hours and stored for 20 days at 4°C before processing and paraffin embedding according to manufacturer's instructions. The NBF fixed sample (FFPE) was fixed for eight hours, processed, and embedded in paraffin. PFPE and FFPE samples were stained with hematoxylin and eosin (H&E) or immunohistochemical stains for HER2 and estrogen receptor α (ERα). RNA was purified from sections of PFPE, FFPE and from frozen tissue using the PAXgene Tissue RNA Kit (PFPE), the RNeasy® FFPE Kit (FFPE) and the RNeasy® Mini Kit (LN2). RNA was analyzed on the Agilent Bioanalyzer for purity and integrity. Expression levels for 92 genes associated with cancer and 4 housekeeping genes were quantified in real-time RT-PCR using the Applied Biosystems TaqMan® Array Human Molecular Mechanisms of Cancer 96-Well Plate. Results: H&E stained sections of PFPE were similar to, or indistinguishable from, FFPE tissue. However, the differentiation of both chromatin structure and nuclear substructure were preserved better in PFPE than in FFPE tissue. Immunohistochemical staining of HER2 and ER gave comparable staining intensities in PFPE and FFPE samples. RNA from frozen and PFPE samples were of high integrity with average RIN values of 9.5 and 5.6 respectively, compared to RIN value of 2.2 from FFPE. Gene expression analysis of 96 genes in real time RT-PCR showed a high correlation of CT (threshold cycle) values for the samples from frozen and PFPE tissue (r2=0.98), and a poor correlation between frozen and FFPE (r2=0.64). Conclusion: The PAXgene Tissue System preserves morphology and gene expression profile in paraffin embedded breast cancer tissue samples. Histomorphology is preserved similarly to that seen in FFPE tissue, while the gene expression profile shows a high correlation to snap frozen tissue. For research use only, not for use in diagnostic procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-142.