In Situ RNA-FISH with Localized Profiling by Sequencing from FFPE

DESCRIPTION provided by applicant We will develop and demonstrate the feasibility of a technology designed to profile hundreds of RNA targets in select regions as small as single cells of fixed tissues while maintaining morphological context FFPE tissues will be subjected to a combined RASL Seq RiboMap protocol followed by in situ imaging of RNAs using the commercial RiboMap assay to identify regions and or single cells of interest RASL Seq probes will be recovered from those regions of interest or single cells providing profiles of gene expression level of a large set of genes e g in this Phase I RASL Seq is a novel gene expression profiling assay which exploits the hybridization of two probes to target RNA and their subsequent successful ligation for specificity and then use of sequencing to quantify the probes as a measure of RNA abundance and to provide a second level of specificity counting only perfect probe matches The novelty is use of probe hybridization ligation for in situ imaging and localization and the ability to provide a morphological context to expression profiling of large sets of genes comprising biomarker signatures In Phase I we will demonstrate the feasibility of the assay on sections of FFPE cancer cells prepared from cell lines and mixtures of cell lines and clinical tissue benchmarked against laser capture microscopy We will demonstrate feasibility of the combined in situ RASL Seq RiboMap assay and recovery of probes from sub areas of tissue and single cells within the tissue and show that it is feasible for a pathologist o measure the Oncotype DX r and MammaPrint r signatures in situ and to identify cells with an epithelial mesenchymal transition EMT or stem cell phenotype and expression of therapeutic targets in non invasive versus invasive breast cancer tissue In Phase II we will expand the assay to a combined in situ RASL Seq RiboMap IHC assay optimize the protocol and its performance sensitivity reproducibility expand the protocol to include measurement of DNA and measurement of mutations and develop validate a commercial method and associated hardware to efficiently and simply release and recover the RASL Seq probes for sequencing from a localized area of the FFPE tissue as small as a single cell We will then expand on the clinical tissue feasibility data to produce a demonstration of its utility and begin marketing the assay directly or through a partnership s PUBLIC HEALTH RELEVANCE In this Phase I project we will develop and demonstrate the feasibility of an assay that will enable clinical investigators to first identify cells and areas o interest down to single cells within FFPE biopsy sections of solid tumors based on imaging RNAs and what is novel and significant measure the expression of large numbers of RNA genes that make up a diagnostic or functional signature from this localized area maintaining the association of an expression profile with the tissue structure This will enable researchers to identify and validate signatures within a morphologic context without the interference of tissue that is not diseased enable the measurement of differential expression of large sets of genes between single cells of different types within the context of the tissue in the pursuit of understanding cancer mechanisms and progression and enable pathologists to focus powerful gene signatures for the first time on just the cancer tissue or worst acting cancer cells within th whole tissue to give better cleaner diagnosis and prognosis