Abstract C98: Evaluation of the PAXgene tissue system: Preservation of morphology and gene expression in human melanoma.

Abstract

Abstract Introduction: PreAnalytiX has recently developed the PAXgene® Tissue System, a formalin-free tissue fixation method for simultaneous preservation of tissue morphology and biomolecules. In this study, PAXgene Tissue was used to stabilize human melanoma specimens. Mirrored samples of melanoma were fixed with either neutral buffered formalin or PAXgene Tissue, or snap frozen in liquid nitrogen (LN2) and were compared for preservation of morphology, antigenicity, RNA and noncoding small RNAs. Materials and Methods: Three cases of melanoma were equally divided into two parts: one part was fixed with PAXgene Tissue reagents, the second part of cases 1 and 2 were fixed with formalin and the second part of case 3 was snap-frozen in LN2. Formalin and PAXgene Tissue fixed samples were processed and embedded in paraffin (resulting in FFPE and PFPE tissue respectively). Tissue sections were stained with H&E or immunohistochemically for Ki-67 and S100. RNA or total RNA including miRNA was purified from all samples and analyzed for purity, integrity and performance in RT-qPCR assays on a TaqMan® PCR Array for expression analysis of 32 different human endogenous control genes and in 26 different single assays specific for non-coding, small RNAs. Summary of results: H&E morphology was well preserved in sections of FFPE and PFPE. After optimization of the heat induced epitope retrieval step for PFPE samples, antigen localization, staining intensities, and number of stained cells were equivalent in FFPE and PFPE tissue for Ki67 and S100. RNA purity was generally high with A260/A280 values >1.9 for all samples. RNA integrity as measured on Agilent Bioanalyzer ranged from 7.7 to 9.5 for LN2 samples, between 3.7 to 6.7 for PFPE and 2.1 to 2.8 for FFPE samples. Correlation of gene expression results in qRT-PCR was high between RNA from PFPE and LN2 (R2 = 0.99). Correlation between mRNA from PFPE and FFPE tissue was much lower (R2 =0.86). In contrast, when all the results from 26 different single non-coding, small RNA specific qRT-PCR assays were compared, there was a high correlation between miRNAs from PFPE with LN2 (R2 = 0.99) as with PFPE and FFPE (R2 = 0.99). Conclusion: The PAXgene Tissue System preserves morphology, antigenicity, RNA and miRNA profiles in human melanoma specimens enabling morphological and multimodal biomarker testing with one sample. Disclaimer: For research use only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C98.