Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer

Abstract

Background Enumeration of circulating tumor cells (CTCs) has proven clinical significance for monitoring patients with metastatic cancers. Multiplexed gene expression profiling of CTCs is a potential tool for assessing disease status and monitoring treatment response. The Parsortix® technology enables the capture and harvest of CTCs from blood based on cell size and deformability. The HyCEAD™ (Hybrid Capture Enrichment Amplification and Detection) assay enables simultaneous amplification of short amplicons for up to 100 mRNA targets, and the Ziplex™ instrument quantifies the amplicons for highly sensitive gene expression profiling down to single cell levels. The aim of the study was to functionally assess this system. Methods The HyCEAD/Ziplex platform was used to quantify the expression levels for 72 genes using as little as 20 pg of total RNA or a single cultured tumor cell. Assay performance was evaluated using cells or total RNA spiked into Parsortix harvests of healthy donor blood. The assay was also evaluated using total RNA obtained from Parsortix harvests of blood from metastatic breast cancer (MBC) patients or healthy volunteers (HVs). Results Using genes with low expression in WBC RNA and/or in unspiked Parsortix harvests from HVs, the assay distinguished between the different breast cancer and ovarian cancer cell lines with as little as 20 pg of total RNA (equivalent to a single cell) in the presence of 1 ng of WBC RNA. Single cultured cells spiked into Parsortix harvests from 10 mL of HV blood were also detected and distinguished from each other. CVs from repeatability experiments were less than 20%. Hierarchical clustering of clinical samples differentiated most MBC patients from HVs. Conclusion HyCEAD/Ziplex provided sensitive quantification of expression of 72 genes from 20 pg of total RNA from cultured tumor cell lines or from single cultured tumor cells spiked into lysates from Parsortix harvests of HV blood. The HyCEAD/Ziplex platform enables the quantification of selected genes in the presence of residual nucleated blood cells in Parsortix harvests. The HyCEAD/Ziplex platform is an effective tool for multiplexed molecular characterization of mRNA in small numbers of tumor cells harvested from blood.

Advancements in Circulating Tumor Cell Research: Bridging Biology and Clinical Applications

“…The challenges in sequencing the CTC genome and transcriptome include low abundance in the bloodstream, making their isolation difficult [ 173 ], significant genetic heterogeneity compared to the primary tumor, which can affect the capture of the full spectrum of mutations present [ 15 ], and the need for sophisticated bioinformatics tools and computational resources for data analysis and interpretation [ 174 ]. However, improvements in the isolation and capture of CTCs, the ease of use of different analytical tools, and the removal of biases introduced during sample and sequencing library preparation are increasing the possibility of detecting rare mutations in CTCs [ 175 , 176 ]. By targeting mutational hotspots, a combination of whole-genome amplification, PCR, and Sanger sequencing was used to examine point mutations in the KRAS, BRAF, and PIK3CA genes in CTCs of patients with CRC.…”