Focal gene expression profiling of counterstained FFPE with correlation to morphology using TempO-Seq

We previously reported on use of the whole transcriptome TempO-Seq® assay to profile gene expression from 1 mm2 focal areas of 5 µm thick FFPE sections of normal and cancerous tissue to identify disease biomarkers and mechanistic pathways. We are now presenting data demonstrating that the TempO-Seq assay can be performed as an “in situ” assay on slides by an automated slide stainer, followed by counterstaining. Then, using an automated digital imaging platform, areas as small as 30 μm in diameter within the FFPE section can be profiled, permitting the gene expression data to be correlated directly to the specific morphology of that focal area. The pathologist uses a computer interface to select areas for profiling during the course of the histologic examination of the section, and then the TempO-Seq probes are automatically recovered from those region(s) of interest. After depositing them into PCR tubes, the assay is completed with PCR amplification and sample barcoding before pooling the samples into a library for sequencing. Analysis of the sequencing data is carried out automatically to report results. There is no use of laser capture or destruction of the tissue. The TempO-Seq processed slides can be archived and additional areas may be sampled at a later date. In this study, we sample replicate areas of matched normal vs. cancerous tissue, measuring gene biomarkers of clinical utility, gene fusions, and SNPs. We present gene expression profiles for replicate areas of pure stroma, single normal glands, single high-grade PIN glands, and single cancerous glands from prostate FFPE, demonstrating the statistical power of the TempO-Seq in situ assay as well as the correlation to focal histology and the use of focal differential expression to elucidate molecular pathways involved in the transition from normal tissue to cancer. We report measurements of clinically relevant biomarkers and signatures from clinical FFPE samples, plus therapeutic targets and drug resistance genes. This new approach demonstrates that complex molecular tests can be carried out by any pathologist in their own lab, and makes moot the issues of “% cancer” and amount of tissue required for testing. The approach we present brings extraction-free complex molecular testing of FFPE into the pathology lab and takes molecular pathology to a new level of simplicity, focal precision and correlation to morphology. Citation Format: Elliot Imler, Milos Babic, Deanna Adams, Peter Shepard, Joanne Yeakley, Raymond B. Nagle, Bruce Seligmann. Focal gene expression profiling of counterstained FFPE with correlation to morphology using TempO-Seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2106.