Enrichment and enumeration of circulating tumor cells by efficient depletion of leukocyte fractions

Background: Enumeration and characterization of circulating tumor cells (CTCs) can provide information on patient prognosis and treatment efficacy. However, CTCs are rare, making their isolation a major technological challenge. We developed a technique for enrichment, and subsequent characterization of CTCs based on efficient depletion of human leukocytes. Methods: The technique (CanPatrol (TM) CTC enrichment) we developed is based on red blood cell lysis to remove erythrocytes, followed by depletion of CD45+ leukocytes using a magnetic bead separation method, and subsequent isolation of CTCs by virtue of their larger size, compared with leukocytes. We also demonstrated that fluorescence in situ hybridization (FISH) and genetic abnormalities analysis could be performed on the isolated CTCs. Results: The spiking experiments showed that the average efficacy of leukocytes depletion was 99.98% and the average tumor cells recovery was not lower than 80%. FISH could be used to perform ALK gene rearrangement analysis on the collected NCI-H2228 cells, and EGFR Exon 19 deletion was detected by PCR-based analysis in isolated HCC827 cells. The in vivo feasibility of this technique had been demonstrated in patients with non-small cell lung cancer, breast, colon, and esophageal cancers. CTCs were detected in 13 of 59 blood samples. Tumor microemboli was also detected in three breast cancer samples. Conclusions: The technique we developed allowed isolation and characterization of circulating epithelial tumor cells that do not express classical epithelial antigens. This potentially leads to a more accurate enumeration of the number of CTCs and is suitable for application to a broad range of cancers.