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Sensitive cytometry based system for enumeration, capture and analysis of gene mutations of circulating tumor cells.

Sawada T, Watanabe M, Fujimura Y, Yagishita S, Shimoyama T, Maeda Y, Kanda S, Yunokawa M, Tamura K, Tamura T, Minami H, Koh Y, Koizumi F - Cancer Sci. (2016)

Bottom Line: We compared the number of CTC detected using our system with those detected using the CellSearch system in 46 patients with various cancers.The percentage of samples with detected CTC was significantly higher with our system (65.2%) than with CellSearch (28.3%).The number of detected CTC per patient using our system was statistically higher than that using CellSearch (median 5, 0; P = 0.000172, Wilcoxon test).

View Article: PubMed Central - PubMed

Affiliation: Shien-Lab, National Cancer Center Hospital, Tokyo, Japan.

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Related in: MedlinePlus

The results of cell sorting using On‐chip Sort in spike‐in experiments. (a) A microscopic image of captured cells using a BIOREVO BZ‐9000 fluorescence microscope (Keyence, Osaka, Japan). The spiked tumor cells were stained with EpCAM‐PE and CK‐FITC to distinguish them from leukocytes. (b) Linearity of captured tumor cell counts with spiked cell counts using 4 mL of spiked peripheral blood. PC‐9 cells (100, 20 and 5 cells) or BT20 cells (100, 50, 25 and 10 cells) were spiked into the blood. The captured cell counts were visually calculated using a BIOREVO BZ‐9000. (c) The mean capture rate (%) of PC‐9 (blue) and BT‐20 (red) cells. Error bars represent SE of the mean.
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cas12868-fig-0004: The results of cell sorting using On‐chip Sort in spike‐in experiments. (a) A microscopic image of captured cells using a BIOREVO BZ‐9000 fluorescence microscope (Keyence, Osaka, Japan). The spiked tumor cells were stained with EpCAM‐PE and CK‐FITC to distinguish them from leukocytes. (b) Linearity of captured tumor cell counts with spiked cell counts using 4 mL of spiked peripheral blood. PC‐9 cells (100, 20 and 5 cells) or BT20 cells (100, 50, 25 and 10 cells) were spiked into the blood. The captured cell counts were visually calculated using a BIOREVO BZ‐9000. (c) The mean capture rate (%) of PC‐9 (blue) and BT‐20 (red) cells. Error bars represent SE of the mean.

Mentions: PC‐9 cells (5, 20 or 100 cells) and BT‐20 cells (10, 25, 50 or 100 cells) were spiked into 4 mL of peripheral blood, and the spiked cells were then recovered from the blood using On‐chip Sort. Recovered cells were visually distinguished from leukocytes (Fig. 4a) and were then counted under a fluorescent microscope. Regression analysis of the number of captured cells versus the number of spiked cells produced a correlation coefficient (R2) of 0.9029 (Fig. 4b). The mean capture rates were 101.35 ± 15.3% for PC‐9 and 77.0 ± 8.1% for BT‐20 (Fig. 4c). To estimate the minimal number of CTC that are required for detection of mutations using our method, we performed further spike‐in experiments. PC‐9 cells (5, 10 or 100 cells) and H1650 cells (5, 10 or 30 cells), which have the EGFR exon 19 deletion mutation, and BT20 cells (10, 20 or 50 cells) that express the PIK3CA variant H1047R mutation were spiked into 4 mL of peripheral blood. Following processing of each sample in a manner similar to that described for CTC enrichment (Fig. 2), the spiked cells were sorted from the peripheral blood using On‐chip Sort, which successfully sorted spiked cells from all samples. EGFR deletion mutation was detected in samples spiked with 5, 10 or 100 PC‐9 cells and 5, 10 or 30 H1650 cells using the method described in the Materials and Methods. The PIK3CA variant H1047R mutation was detected in samples spiked with 20 or 50 BT20 cells. The same results were obtained twice in independent experiments.


Sensitive cytometry based system for enumeration, capture and analysis of gene mutations of circulating tumor cells.

Sawada T, Watanabe M, Fujimura Y, Yagishita S, Shimoyama T, Maeda Y, Kanda S, Yunokawa M, Tamura K, Tamura T, Minami H, Koh Y, Koizumi F - Cancer Sci. (2016)

The results of cell sorting using On‐chip Sort in spike‐in experiments. (a) A microscopic image of captured cells using a BIOREVO BZ‐9000 fluorescence microscope (Keyence, Osaka, Japan). The spiked tumor cells were stained with EpCAM‐PE and CK‐FITC to distinguish them from leukocytes. (b) Linearity of captured tumor cell counts with spiked cell counts using 4 mL of spiked peripheral blood. PC‐9 cells (100, 20 and 5 cells) or BT20 cells (100, 50, 25 and 10 cells) were spiked into the blood. The captured cell counts were visually calculated using a BIOREVO BZ‐9000. (c) The mean capture rate (%) of PC‐9 (blue) and BT‐20 (red) cells. Error bars represent SE of the mean.
© Copyright Policy - creativeCommonsBy-nc-nd
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814266&req=5

cas12868-fig-0004: The results of cell sorting using On‐chip Sort in spike‐in experiments. (a) A microscopic image of captured cells using a BIOREVO BZ‐9000 fluorescence microscope (Keyence, Osaka, Japan). The spiked tumor cells were stained with EpCAM‐PE and CK‐FITC to distinguish them from leukocytes. (b) Linearity of captured tumor cell counts with spiked cell counts using 4 mL of spiked peripheral blood. PC‐9 cells (100, 20 and 5 cells) or BT20 cells (100, 50, 25 and 10 cells) were spiked into the blood. The captured cell counts were visually calculated using a BIOREVO BZ‐9000. (c) The mean capture rate (%) of PC‐9 (blue) and BT‐20 (red) cells. Error bars represent SE of the mean.
Mentions: PC‐9 cells (5, 20 or 100 cells) and BT‐20 cells (10, 25, 50 or 100 cells) were spiked into 4 mL of peripheral blood, and the spiked cells were then recovered from the blood using On‐chip Sort. Recovered cells were visually distinguished from leukocytes (Fig. 4a) and were then counted under a fluorescent microscope. Regression analysis of the number of captured cells versus the number of spiked cells produced a correlation coefficient (R2) of 0.9029 (Fig. 4b). The mean capture rates were 101.35 ± 15.3% for PC‐9 and 77.0 ± 8.1% for BT‐20 (Fig. 4c). To estimate the minimal number of CTC that are required for detection of mutations using our method, we performed further spike‐in experiments. PC‐9 cells (5, 10 or 100 cells) and H1650 cells (5, 10 or 30 cells), which have the EGFR exon 19 deletion mutation, and BT20 cells (10, 20 or 50 cells) that express the PIK3CA variant H1047R mutation were spiked into 4 mL of peripheral blood. Following processing of each sample in a manner similar to that described for CTC enrichment (Fig. 2), the spiked cells were sorted from the peripheral blood using On‐chip Sort, which successfully sorted spiked cells from all samples. EGFR deletion mutation was detected in samples spiked with 5, 10 or 100 PC‐9 cells and 5, 10 or 30 H1650 cells using the method described in the Materials and Methods. The PIK3CA variant H1047R mutation was detected in samples spiked with 20 or 50 BT20 cells. The same results were obtained twice in independent experiments.

Bottom Line: We compared the number of CTC detected using our system with those detected using the CellSearch system in 46 patients with various cancers.The percentage of samples with detected CTC was significantly higher with our system (65.2%) than with CellSearch (28.3%).The number of detected CTC per patient using our system was statistically higher than that using CellSearch (median 5, 0; P = 0.000172, Wilcoxon test).

View Article: PubMed Central - PubMed

Affiliation: Shien-Lab, National Cancer Center Hospital, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus