Detection of circulating tumor cells in breast cancer may improve through enrichment with anti-CD146

Abstract

PurposeMost assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells.Recently, our group reported that in contrast to other molecular breast cancer subtypes, "normallike" cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology [1]. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. Methods CD146and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch™ technology, 7.5 mL whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. ResultsEight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146.Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. ConclusionsCells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients.3

Improved Circulating Tumor Cell Detection by a Combined EpCAM and MCAM CellSearch Enrichment Approach in Patients with Breast Cancer Undergoing Neoadjuvant Chemotherapy

“…All samples were processed within 96 hours at the central laboratory, Erasmus MC Cancer Institute, Rotterdam, the Netherlands. Two CTC enumerations, both from 7.5 mL of blood, were done using the CellSearch System as described before (9). In brief, EpCAMpositive and MCAM-positive CTCs were enumerated in two separate runs using the CellSearch Epithelial Cell Kit (Janssen Diagnostics).…”

“…We found melanoma cell adhesion molecule (MCAM; CD146) to be expressed on EpCAM-negative breast cancer cell lines and tested its use as enrichment marker next to EpCAM. In a small series of patients with mBC, MCAM-positive CTCs were detected in 9 of 20 patients (45%), suggesting that CTC detection can be improved using this dual enrichment approach (9).…”

“…The CellSearch System relies on the expression of the epithelial cell adhesion molecule (EpCAM; CD326) on CTCs and misses EpCAM-negative CTCs (8)(9)(10)(11). We showed that particularly breast cancer cell lines with epithelial-to-mesenchymal transition (EMT) features lack expression of EpCAM and are therefore not detected by the CellSearch System (9,12). Because cells that have undergone EMT probably represent an aggressive, clinically relevant subpopulation of CTCs (10), we aimed to detect EpCAMnegative CTCs by alternative approaches.…”

Immunomagnetic detection of cancer cells in pleural effusion of generalized cancer

“…The recent reports have shown how variable the EpCAM expression can be, and, in conclusion, how cocktail of antibodies is eligible [15][16][17][18]. A combination of antibodies bound to the beads would enable the capturing of cancer cells, which do not necessarily express EpCAM as used in the Adnatest™ protocols.…”

Minimal Residual Disease and Circulating Tumor Cells in Breast Cancer

“…As an example, some available technologies detect only EpCAM+ CTCs (Table 1). However, it has been shown that BC cell lines with low EpCAM expression and high expression of mesenchymal markers cannot be effi ciently captured using a purely EpCAM-based mechanism [33,43,44]. Some other technologies are using enrichment based on red blood cell lysis or leukocyte depletion (CD45-negative depletion) aiming at a less biased CTC enrichment (Table 1).…”