Measurement of complex gene expression molecular signatures from fixed tissue has been problematic, especially given the desire to correlate those measurements to existing histology techniques within the context of the tissue microenvironment. BioSpyder Technologies has developed the CellSensus assay, an in situ adaptation of the TempO-Seq® targeted sequencing assay, to carry out gene expression analysis on H&E and IHC stained slides from histologically defined regions of interest down to a spatial resolution of 20μm. FFPE sections are stained and infused with TempO-Seq probes. The antibody- or H&E-stained slides are then digitally imaged and TempO-Seq probes eluted from selected regions of interest using the CellSensus platform, followed by qPCR amplification, pooling of the samples, sequencing, and analysis using the TempO-SeqR™ software package. H&E staining not only enables identification of the areas of tissue histology to be profiled, but provides images that validate that the profiling data is tissue specific and not cross-contaminated. Using a pan-cancer panel of 5,207 genes that includes the S1500+ Whole Transcriptome Surrogate assay, immune, and Hallmark Cancer genes, BioSpyder has profiled discrete focal areas of stroma, normal epithelium, cancer epithelium, and pre-cancerous high-grade PIN lesions from H&E stained archived prostate cancer patient resections. The data demonstrate not only that the different tissue types within the FFPE section can be profiled without contamination by neighboring cell types, but demonstrate a level of biomarker specificity as a result of the spatial resolution the CellSensus assay provides.