Abstract 389: The identity of all nucleated cells enriched by CellSearch
Authors: van Dalum Guus , van Lin Simone , Barradas Ana M.C. , Hiltermann Jeroen T.N. , Groen Harry J.M. , Terstappen Leon W.M.M. 
Published: August 2015

Abstract
Purpose: The FDA cleared CellSearch assay for enumeration of Circulating Tumor Cells (CTC) detects CTC by fluorescence microscopy after immunomagnetic enrichment targeting Epithelial Cell Adhesion Molecule (EpCAM). The system generated thumbnail images of DNA+, Cytokeratin+ (CK+) events and presents these to a reviewer for scoring events as CTC. An event is a CTC when it is negative for CD45, positive for DNA and CK and is larger than a 4 × 4 μm2 with a cell-like morphology. The presence of CTC defined in such manner is strongly associated with poor outcome. The question arises whether and how many other tumor cells are present in the blood that do not have the phenotype defined by the CellSearch system. In this study we investigated the number of cells that are obtained after EpCAM enrichment and immunofluorescent labeling by CellSearch that could not be assigned to a cell lineage and explored the addition of other markers to increase the number of cells that could be assigned to a cell lineage.
Methods: Automated image analysis was used to reanalyze archived CellSearch images of 12572 patient samples with benign breast and colorectal disease, non-metastatic breast and colorectal cancer, metastatic colorectal and prostate cancer. For the evaluation of additional markers blood from healthy volunteers aged 20-55 were used. For the evaluation of CD16-PerCP as an additional leukocyte marker, blood from 30 patients with metastatic lung cancer was used.
Results: The median number of DNA+CK− cells found was 450 of which only 155 (34%) were identified as leukocytes (CD45-APC+). The number of DNA+CK− was higher in patients with metastatic disease as compared to those with non-metastatic or benign disease (median 2028 vs 293). The proportion of identified leukocytes (DNA+CK−CD45+) was however only slightly lower (median 35% vs 43%). The number of DNA+CK−CD45− cells was larger in patients with more CTC (0 CTC: 495, 1-10 CTC: 2024, 11-100 CTC: 8140, &gt;100 CTC: 8983). The number of DNA+CK− cells also increased with aging of the blood samples, but did not account for the observed differences. The use of both CD16 and CD45 decreased the number of DNA+CK− cells and increased the fraction of cells identified as leukocytes to 90% in healthy donors and 78% in lung cancer patients.
Conclusion: A large proportion of nucleated cells obtained after EpCAM enrichment is not accounted for. The majority of these cells are leukocytes not detected with the current reagents and microscope of the CellSearch system. The correlation between DNA+CK−CD45− cells and increasing number of CTC suggests an association with cancer and warrants further investigation.
Citation Format: Guus van Dalum, Simone van Lin, Ana M.C. Barradas, Jeroen T.N. Hiltermann, Harry J.M. Groen, Leon W.M.M. Terstappen. The identity of all nucleated cells enriched by CellSearch. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 389. doi:10.1158/1538-7445.AM2015-389