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:  numerous residual blood cells in processed samples,  loss of CTCs that do not express epithelial markers, and  very laborious process. Here, we developed a novel system capable of overcoming all of these problems, which is perfect for the analysis of CTCs.
Our CTC analysis system is composed of 5 steps:  filtering whole blood followed by immunostaining and magnetic labeling of cells trapped on the filter,  depletion of white blood cells (WBCs) in the cells recovered from the filter by magnetic separation,  trapping the resultant cells at an observation chamber in a microfluidic enrichment device using dielectrophoresis,  recovering the cells as a concentrated sample after fluorescence microscope observation, and  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).
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|
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
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All authors have declared no conflicts of interest.