IN SITU RNA-FISH WITH LOCALIZED PROFILING BY SEQUENCING FROM FFPE


Year of Award:
2015
Award Type:
R43
Project Number:
CA187787
RFA Number:
PAR-13-327
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
SELIGMANN, BRUCE E.
Other PI or Project Leader:
N/A
Institution:
BIOSPYDER TECHNOLOGIES, INC.
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 2-3 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. 250 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 theassay directly or through a partnership(s).