Abstract 3061: A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of circulating tumor cells
Authors: Koh Yasuhiro , Watanabe Masaru , Sawada Takeshi , Kenmotsu Hirotsugu , Serizawa Masakuni , Ishige Masayuki , Takeda Kazuo , Yamamoto Nobuyuki , Takahashi Toshiaki , Koizumi Fumiaki 
Published: May 2015

Abstract
Background: Personalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis as “liquid biopsy”; however, attempts to isolate rare CTCs for further molecular analysis remain technically challenging by the currently available enrichment methods. In this study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology.
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 (H1975, A549 and H1755) 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 by pyrosequencing as well as by deep targeted sequencing. The mutant variant detection rates were markedly higher than those obtained with the CellSearch profile kit. qPCR analysis of amplified
DNA demonstrated reproducible detection of copy number changes of the EGFR gene n captured tumor cells.
Conclusions: Using a novel cell sorter, we established an efficient and convenient platform for the capture of CTCs. Results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients. Further evaluation using clinical samples is warranted.
Citation Format: Yasuhiro Koh, Masaru Watanabe, Takeshi Sawada, Hirotsugu Kenmotsu, Masakuni Serizawa, Masayuki Ishige, Kazuo Takeda, Nobuyuki Yamamoto, Toshiaki Takahashi, Fumiaki Koizumi. A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of circulating tumor cells. [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 3061. doi:10.1158/1538-7445.AM2014-3061