Abstract 125: Digital Spatial Molecular Profiling evidence from H&E stained FFPE that the tumor microenvironment confers gene expression phenotype specificity

Abstract

Abstract Identifying the molecular phenotype of cells within the context of their tumor tissue microenvironment may be critical to understanding cancer and improving patient treatments and outcomes. TempO-Seq® (DOI:10.1371/journal.pone.0178302, doi.org/10.1093/toxsci/kfx153) can be used to profile gene expression from lysates of scraped FFPE tissue down to 2 mm2 areas of 5 μm thick sections. We report use of an in situ TempO-Seq transcriptomic assay carried out on slide-mounted FFPE by an automated stainer (Leica BOND), followed by H&E staining and imaging to perform standard histology. Regions of interest (ROIs) down to 20 μm in diameter are selected at the same time. Probes from these ROIs are automatically eluted, obtaining a detailed gene expression profile. This process is called Digital Spatial Molecular Profiling (DSMP). DSMP was used to profile gene expression from histologically discrete areas within tumor sections. A pan cancer assay of 5,207 genes was used, containing biomarkers for cancer, immune responses and interactions, as well as the S1500v2 whole transcriptome surrogate assay (doi.org/10.1371/journal.pone.0191105), permitting all known molecular pathways to be mapped. Data analysis was carried out using the automated TempO-SeqR™ software package. Replicate areas of cancer, high grade PIN (prostatic intraepithelial neoplasia), normal epithelium, and stroma within prostate cancer FFPE sections were profiled and compared to the profiling of a 2 mm2 scraped area of tissue defined as 100% cancer. What was notable from the DSMP data was that key cancer and immune cell biomarkers were not detected at all in normal, key normal biomarkers were not detected at all in cancer, PIN expressed a collection of normal, cancer and immune biomarkers, and stromal biomarkers were not detected in normal, PIN or cancer glands, while housekeepers were detected in all tissue types. In contrast, housekeeper genes and key biomarkers of cancer, normal, stroma, and immune cells, were all expressed in the 100% pure area of cancer, lacking the biomarker specificity afforded by histologically focused DSMP profiling. The DSMP specificity obtained from the FFPE tissue was striking, leading us to hypothesize that factors within the tumor microenvironment (whether it is adjacent cells, the structural effects of tissue, or sequestration/gradients of extracellular modulators within the tissue) exert molecular specificity on cells within the tissue not present in cultured or dissociated cells devoid of context. Citation Format: Elliot Imler, Raymond Nagle, Bruce Seligmann. Digital Spatial Molecular Profiling evidence from H&E stained FFPE that the tumor microenvironment confers gene expression phenotype specificity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 125.