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Efficient isolation and genetic analysis of circulating tumor cells (CTCs) from cancer patients’ blood is a critical step for clinical applications using CTCs. Here, we report a novel CTC-isolation method and subsequent genetic analysis. CTCs from the blood were complexed with magnetic beads coated with antibodies against the epithelial cell adhesion molecule (EpCAM) and separated vertically on a density-gradient medium in a modified well-plate. The recovery rate of model CTCs was reasonable and the cell purity was enhanced dramatically when compared to those parameters obtained using a conventional magnetic isolation method. CTCs were recovered from an increased number of patient samples using our magnetic system vs. the FDA-approved CellSearch system (100% vs. 33%, respectively). In 8 of 13 cases, targeted deep sequencing analysis of CTCs revealed private point mutations present in CTCs but not in matched tumor samples and white blood cells (WBCs), which was also validated by droplet digital PCR. Copy-number alterations in CTCs were also observed in the corresponding tumor tissues for some patients. In this report, we showed that CTCs isolated by the EpCAM-based method had complex and diverse genetic features that were similar to those of tumor samples in some, but not all, cases.

Vertical Magnetic Separation of Circulating Tumor Cells for Somatic Genomic-Alteration Analysis in Lung Cancer Patients