Reliable Gene Expression Profiling from Small and Hematoxylin and EosineStained Clinical Formalin-Fixed, Paraffin-Embedded Specimens Using the HTG EdgeSeq Platform

Clinical biomarker studies are often hindered by the scarcity or suboptimal quality of biological specimens. EdgeSeq, a transcriptomics analysis platform, combines quantitative nuclease protection assay technology with next-generation sequencing, using small amounts of starting material and delivering reproducible gene expression profiles from challenging material, such as formalin-fixed, paraffin-embedded (FFPE) tissue. To evaluate EdgeSeq for analysis of archives of stained FFPE tissue, EdgeSeq was performed on unstained, hematoxylin and eosin (H&E)estained, and immunohistochemistry-stained slides from patients with small-cell and nonesmall-cell lung cancer. Pairwise comparisons of gene expression profiles from stained and unstained slides showed higher Pearson correlation coefficients with H&E staining (0.86 to 0.97) than with immunohistochemistry staining (0.21 to 0.56). A 25-gene interferon-g signature score from unstained slides showed a Pearson correlation coefficient of 0.92 with H&E-stained slides and a significant Spearman correlation (P=0.0025) with immune scores. To test gene expression profiling in small samples, FFPE sample equivalents were examined from 5.0 to 0.08 mm(2) of a section (5 mm thick); sample equivalents >= 0.31 mm(2) showed alignment rates >69% and pairwise Pearson correlation coefficients >= 0.87. EdgeSeq can, thus, be used to profile small and H&E-stained FFPE tumor specimens to obtain biomarker data from limited tissue in oncology clinical trials and enable research into tumor microenvironment and immune cell engagement with tumors at the locoregional level.