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Poster display

1197 - Genetic testing by a novel high-purity concentration system for circulating tumor cells independent of epithelial markers

Date

10 Oct 2016

Session

Poster display

Presenters

Hiroshi Ito

Citation

Annals of Oncology (2016) 27 (6): 401-406. 10.1093/annonc/mdw380

Authors

H. Ito1, H. Takagi1, M. Kozuka1, S.H. Kim2, M. Hirai1, T. Fujii2

Author affiliations

  • 1 Reserch & Development Division, Arkray, Inc., 602-0008 - Kyoto/JP
  • 2 Institute Of Industrial Science, The University of Tokyo, 153-8505 - Tokyo/JP
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Resources

Abstract 1197

Background

Genetic analysis of circulating tumor cells (CTCs) is useful as liquid biopsy. However, there are 3 challenging issues in processing of CTC samples for clinical use of the analysis: [1] numerous residual blood cells in processed samples, [2] loss of CTCs that do not express epithelial markers, and [3] very laborious process. Here, we developed a novel system capable of overcoming all of these problems, which is perfect for the analysis of CTCs.

Methods

Our CTC analysis system is composed of 5 steps: [1] filtering whole blood followed by immunostaining and magnetic labeling of cells trapped on the filter, [2] depletion of white blood cells (WBCs) in the cells recovered from the filter by magnetic separation, [3] trapping the resultant cells at an observation chamber in a microfluidic enrichment device using dielectrophoresis, [4] recovering the cells as a concentrated sample after fluorescence microscope observation, and [5] detecting genetic mutation of the cells without nucleic acid purification using the whole sample by quenching probe method. Using whole blood spiked with cultured cancer cell lines (NCI-H2228, NCI-H1650, NCI-H1975, SW620 or MCF-7), we demonstrated our system and detected their genetic mutations (EML4-ALK, EGFR, KRAS or PIK3CA).

Results

Our system successfully detected EML4-ALK, EGFR, KRAS or PIK3CA mutations of the cell lines spiked in 8 mL of whole blood. The detection sensitivity of our method was 1 cell/mL, and the average number of residual nucleated cells was 87 (Table 1). Our system could report the results of genetic mutation detection and each enumeration of cancer cells and residual nucleated cells within 9 hours of starting the processing of whole blood.

Sensitivity and reproducibility results for spiking test

test cell line expected count [cells in 8 mL] detected count [cells] residual nucleated cell [cells] detected EGFR mutation
1 NCI-H1650 8 (SD ± 1) 2 98 ex19del
2 8 (SD ± 1) 3 121 ex19del
3 8 (SD ± 3) 2 25 ex19del
4 8 (SD ± 3) 2 48 ex19del
5 12 (SD ± 3) 2 30 ex19del
6 NCI-H1975 11 (SD ± 4) 5 80 L858R
7 7 (SD ± 3) 4 129 L858R
8 12 (SD ± 3) 9 79 L858R
9 7 (SD ± 3) 1 165 L858R
10 10 (SD ± 3) 3 91 L858R

Conclusions

Our system would be useful for the analysis of gene mutations in wide-ranging CTC subsets independent of expression of a certain antigen, and may be particularly effective for CTCs that have changed their phenotype.

Clinical trial identification

Legal entity responsible for the study

Arkray, Inc.

Funding

Arkray, Inc.

Disclosure

All authors have declared no conflicts of interest.

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