Abstract 3789: Simultaneous visual assessment of RNA and protein expression in circulating tumor cells using the AccuCyte-Cytefinder system

Background. Circulating tumor cells (CTCs) provide real-time information regarding patient tumor phenotype, including RNA expression. This information may be valuable in guiding care of cancer patients. Simultaneous RNA and protein assessment has been reported previously in large populations of cells. Here, we describe methods to simultaneously evaluate RNA and protein expression on rare single CTCs using the AccuCyte – CyteFinder system (RareCyte). Methods. To establish RNA detection in model CTCs (mCTCs), we used SkBr3 cells spiked into blood and the isolated buffy coat was processed by AccuCyte onto microscope slides. RNAscope® in-situ hybridization (ISH, Advanced Cell Diagnostics) was performed to detect Her2, UBC (positive control) and dapB (negative control) expression in mCTCs. A process to simultaneously stain for RNA and protein was then developed to allow identification of mCTCs by protein expression of cytokeratin (CK) and EpCAM and measurement of gene expression with RNAscope. Various blood collection tubes were tested to measure gene expression, protein expression, and cell recovery up to 48 hours after blood draw. Image analysis software was developed to automatically analyze and count RNA dot number from 40x z-stack images of mCTCs. Finally, clinical samples were stained with the combined RNA/protein assay. Results. Using RNAscope, Her2 and UBC expression could be detected in all SkBr3 cells and was negative in surrounding white blood cells (WBC). Using blood collected into EDTA tubes and processed immediately after spike-in, there were an average of 29 Her2 mRNA dots and 20 UBC dots/cell; negative control dapB gave 1 dot/cell. Using CK and EpCAM expression for mCTCs identification, recovery of mCTCs was over 95%. The RNA/protein assay was compared using blood collected and stored in EDTA, CellSave®, Cell-Free DNA®, Cell-Free RNA®, Cyto-Chex®, and RareCyte BCT tubes. At 48 hours, Cell-Free RNA tubes had the highest number of RNA dots/cell – about half as many dots/cell compared to fresh EDTA samples. Clinical samples were successfully stained with the RNA/protein assay. Conclusions. We have developed a protocol that identifies rare mCTCs using protein staining, and measures RNA expression by RNAscope ISH. This assay may be a useful clinical tool for the real-time investigation of CTC gene expression in cancer patients. Citation Format: Arturo B. Ramirez, Sarah B. Crist, Tyler Yeats, Jeffrey L. Werbin, Jackie L. Stilwell, Eric P. Kaldjian. Si