Abstract A34: A preclinical study of a novel FACS-based rare cell capture platform for the detection, capture and molecular characterization of circulating tumor cells.
Authors: Watanabe Masaru , Serizawa Masakuni , Sawada Takeshi , Fujimura Yuu , Koizumi Fumiaki , Koh Yasuhiro 
Published: August 2014

Abstract
Background: Recent advances in molecular targeted cancer therapy offer a wide variety of therapeutic strategies. The presence or absence of particular actionable genomic aberrations has been shown to predict response to molecularly targeted treatments as a companion diagnostics. Conventionally this strategy relies on analysis of primary tumor samples, whereas circulating tumor cells (CTCs) offer an alternative source for the detection of genetic alterations as a “liquid biopsy”. To acquire genetic profile of CTCs, development of reliable platform and workflow to detect and capture a small number of mutation-bearing CTCs from a blood sample is necessary. However, attempts to isolate rare CTCs for further molecular analysis remain technically challenging by the currently available enrichment methods.
Methods: We have developed a rare cell capture platform with On-Chip Sort (On-Chip Biotechnologies), a novel benchtop cell sorter equipped with a disposable microfluidic chip. Spike-in experiments using lung cancer cell lines with varying EpCAM expression levels were conducted to assess capture and purification efficiency of our platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by fixation and labeling with cytokeratin (CK)-FITC, vimentin-PE, CD45-Alexa Fluor 700 antibodies, and/or Hoechst 33342 for nuclei staining. The enriched and labeled samples were then sorted on On-Chip Sort based on CK, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification (WGA) followed by mutation analysis using deep targeted sequencing. Amplicon libraries were generated using Illumina TruSeq Amplicon Cancer Panel and sequenced on a MiSeq sequencer. Copy number analysis of the WGA samples were performed by quantitative PCR (qPCR).
Results: Spike-in experiments revealed excellent overall mean detection, recovery and purity rates of 85.0 ± 9.9%, 70.9% ± 10.6 and 72.9% ± 15.1, respectively (n=27). These results were obtained from samples containing as few as 5 tumor cells per 4 mL of blood, independent of EpCAM expression. A 100% success rate of mutation detection in EGFR, KRAS and BRAF genes from captured cells (n=27) was achieved with pyrosequencing as well as deep targeted sequencing. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number change of EGFR gene from captured tumor cells.
Conclusions: We described an efficient and convenient platform for the capture of CTCs utilizing a novel cell sorter. We also conducted a proof-of-principle preclinical study for a workflow capable of molecular characterization of captured CTC population. Further evaluation using clinical samples is warranted.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A34.
Citation Format: Masaru Watanabe, Masakuni Serizawa, Takeshi Sawada, Yuu Fujimura, Fumiaki Koizumi, Yasuhiro Koh. A preclinical study of a novel FACS-based rare cell capture platform for the detection, capture and molecular characterization of circulating tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A34.