A relevant immunomagnetic assay to detect and characterize epithelial cell adhesion molecule‐positive cells in bone marrow from patients with breast carcinoma

Abstract

BACKGROUND The efficient detection and characterization of micrometastatic cells in the bone marrow of patients with breast carcinoma are of prognostic and therapeutic importance. The technique used must overcome the challenges that result from the small number of target cells (1 per 1 million hematopoietic cells) and the heterogeneous expression of micrometastatic cell markers. In this study, the authors assessed and improved the current methods for purifying and characterizing rare disseminated carcinoma cells. METHODS The authors developed a single‐step assay that does not require density‐gradient separation. This assay can be performed directly on crude human bone marrow aspirates and is based on the use of immunomagnetic beads coated with an antibody that recognizes an epithelial cell‐surface epitope, the epithelial cell adhesion molecule (EpCAM). To determine the specificity of the assay, the authors evaluated bone marrow specimens from 46 control patients. RESULTS The novel method was highly reproducible and was capable of detecting as few as 10 carcinoma cells among 50 million hematopoietic cells. The yield was nearly 100%, with only 0.01% nonspecific cell draining. The authors found that 68 ± 51 cells were trapped per 50 million cells in control crude aspirates and that density‐gradient separation increased this number by 2‐fold to 29‐fold. These trapped cells expressed EpCAM, represented 1.4 × 10⁻⁴ % of the sample, and were characterized as of hematopoietic cell origin (CD45 positive) or progenitor cell origin (CD34 positive). CONCLUSIONS The authors developed a highly efficient and reproducible, single‐step immunomagnetic assay that may be performed directly on crude human bone marrow aspirates. The authors believe the current study is the first to demonstrate that some rare bone marrow cells (CD45‐positive or CD34‐positive cells) may express EpCAM and, to some extent, may contaminate the purified micrometastatic cell fraction. Thus, a universal marker for micrometastatic cells remains to be discovered. Cancer 2004. © 2004 American Cancer Society.