Abstract 3997: Circulating tumor cell expression analysis using highly multiplexed qPCR to quantify a panel of 624 common cancer genes
Authors: Hu Brian , Xu Yucheng , Xu Tong , Goldkorn Amir 
Published: May 2015

Abstract
Introduction: Circulating tumor cells (CTCs) have the potential to advance personalized care by yielding a ready source of cancer tissue for characterization. While genomic analysis of CTCs has gained momentum, efforts to measure gene expression have been hampered by the need capture “ultra-pure” CTCs (i.e. no white blood cell contamination) and reliably amplify and measure transcript levels from these rare cells. To address this need, we developed a cell micromanipulation technique coupled with multiplexed qPCR analysis to isolate ultra-pure CTCs for amplification and measurement of a large panel of common cancer genes.
Methods: Experiments were conducted in parallel, under IRB approval, using peripheral blood from cancer patients (prostate, kidney) as well as LNCaP prostate cancer (PCa) cells spiked into blood samples from healthy donors. Blood samples were depleted of white and red blood cells using the EasySep and RosetteSep kits (Stem Cell Tech, Inc.). After depletion, epithelial cells were identified by staining for cell surface epithelial cell adhesion molecule (EpCAM) and recovered under fluorescent microscopy using an xyz motorized micropipette device configured for this purpose (Zeiss, Eppendorf). Recovered cells were lysed for RNA extraction and reverse transcription. Pooled primer multiplexed preamplification was performed, followed by qPCR expression profiling by Taqman Open Array human cancer panel of 624 cancer-related genes. Gene expression relative quantities (RQ) were normalized to unspiked LNCaP PCa cells.
Results: Multiplexed preamplification from as little as 100pg LNCaP RNA yielded a gene expression profile with correlation coefficient (R2) of 0.71 when compared with gene expression from abundant, unamplified LNCaP RNA. Ultrapure CTCs were recovered from a patient with PCa (1 CTC) and a patient with kidney cancer (6 CTCs), and their respective RNA quality was confirmed by cancer-specific single-gene preamplification and qPCR measurement: androgen receptor for PCa (Ct 25.7) and HIF1-α for kidney cancer (Ct 19.0). Then, high throughput parallel cancer gene expression was measured using the Open Array 624 Cancer Gene Panel. The RQ profile from the PCa CTC closely resembled that of LNCaP PCa cells spiked and recovered from normal blood, whereas the RQ profiles of the kidney cancer CTCs and of white blood cells were progressively less similar, as expected.
Conclusions: These results demonstrate the feasibility of querying gene expression in a high throughput manner from rare, ultrapure CTCs. This capability, once validated in expanded studies, can be leveraged to identify gene pathways which drive cancer resistance and progression in individual patients. Such real time transcriptional profiling, in conjunction with genomic analysis, can harness the full “liquid biopsy” potential of CTCs to guide personalized cancer treatment.
Citation Format: Brian Hu, Yucheng Xu, Tong Xu, Amir Goldkorn. Circulating tumor cell expression analysis using highly multiplexed qPCR to quantify a panel of 624 common cancer genes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3997. doi:10.1158/1538-7445.AM2014-3997