Abstract 1375: Multiplex gene expression using the HyCEAD assay in CTCs isolated with the Parsortix™ system
Authors: Englert David F. , Kolesnikova Mariya , Zaman Nishat , Hustler Arianna , Wishart Gabrielle , O'Shannessy Daniel J. 
Published: August 2019

Abstract
Background: Discrimination between benign and malignant pelvic masses by gene expression profiling of circulating tumor cells (CTCs) may provide information to assist in treatment decisions and improve outcomes. CTCs can be isolated and harvested from blood based on cell size and deformability using the Parsortix™ system. Because the number of CTCs in the blood of pelvic mass patients suspected of having ovarian malignancies is likely very low, gene expression profiling of these CTCs requires a highly sensitive detection system that is tolerant of the presence of normal nucleated blood cells.
Objective: To assess the suitability of the HyCEAD assay for gene expression profiling of cells in Parsortix harvests. HyCEAD (Hybrid Capture Enrichment Amplification and Detection) is a fast and simple method for the simultaneous analysis of 100 or more mRNA species captured from cell lysates. The products of the multiplex amplification with HyCEAD can be quantified by hybridization on a flow-through chip.
Methods: Multiple HyCEAD primer/probe sets were designed for 125 relevant genes, and purified total RNA or lysates from Parsortix harvests were processed with the HyCEAD assay. Primer/probe sets were screened using total RNA from cell lines and ovarian cancer tissues. Genes with significant expression in white blood cells were culled from the gene set. A set of non-human poly-adenylated mRNA molecules (separately quantified using digital droplet PCR) were used as spike-in standards to assess absolute sensitivity. CaOV3 cells spiked into blood from normal healthy volunteers (NHVs) were used as a model system for sensitivity analyses.
Results: Many genes were identified that yielded substantially greater signal intensities in harvests from HNV blood spiked with CaOV3 cells relative to the unspiked blood samples. The expression levels could be quantified over a signal intensity range of 2.5 log10 units. Small numbers of non-human standard molecules could be detected; stochastic detection failures were observed when five or fewer molecules of the standards were assayed (dropout level). CVs in repeatability experiments were typically less than 20% with numbers of standard molecules greater than the dropout level. CVs in repeatability measurements of human genes related to ovarian cancer in spiked Parsortix harvests averaged about 25%. CVs were greater between assays of Parsortix harvests of different HNVs, reflecting in part biological variability and variation of the number of cells actually spiked into the blood and the number of cells recovered in the individual harvests.
Conclusion: The HyCEAD assay provides adequate sensitivity to simultaneously profile expression levels of more than 100 genes in small numbers of CTCs directly from the lysates of Parsortix harvests. Expression profiling is limited to genes expressed at much higher levels in CTCs than in normal nucleated blood cells.
Citation Format: David F. Englert, Mariya Kolesnikova, Nishat Zaman, Arianna Hustler, Gabrielle Wishart, Daniel J. O'Shannessy. Multiplex gene expression using the HyCEAD assay in CTCs isolated with the Parsortix™ system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1375.