Enrichment methods to detect bone marrow micrometastases in breast carcinoma patients: clinical relevance

Abstract

Introduction: Improving technologies for the detection and purification of bone marrow (BM) micrometastatic cells in breast cancer patients should lead to earlier prognosis of the risk of relapse and should make it possible to design more appropriate therapies. The technique used has to overcome the challenges resulting from the small number of target cells (one per million hematopoietic cells) and the heterogeneous expression of micrometastatic cell markers. In the present study, we have assessed the clinical relevance of current methods aimed at detecting rare disseminated carcinoma cells.

Methods: BM aspirates from 32 carcinoma patients were screened for the presence of micrometastatic cells positive for epithelial cell adhesion molecule and positive for cytokeratins, using optimized immunodetection methods. A comparison with data obtained for 46 control BM aspirates and a correlation with the clinical status of patients were performed.

Results: We developed a sensitive and efficient immunomagnetic protocol for the enrichment of BM micrometastases. This method was used to divide 32 breast carcinoma patients into three categories according to their epithelial cell adhesion molecule status. These categories were highly correlated with the recently revised American Joint Committee on Cancer staging system for breast cancer, demonstrating the clinical relevance of this simple and reliable immunomagnetic technique. We also evaluated immunocytochemical detection of cytokeratin-positive cells and cytomorphological parameters. Immunocytochemistry-based methods for the detection of BM micrometastases did not provide any information about the clinical status of patients, but helped to refine the immunomagnetic data by confirming the presence of micrometastases in some cases. We also tested a new density gradient centrifugation system, able to enrich the tumor fraction of BM specimens by twofold to threefold as compared with standard Ficoll methods.

Conclusion: These improved methods for the detection of micrometastatic cells in patient BM should help clinicians to predict the clinical status of breast cancer patients at the time of surgery or treatment.

Figure 1

Prognostic significance of the immunomagnetic (IM) detection of bone marrow (BM) micrometastases in patients. The number of MOC31-positive cells detected was compared in 46 control patients (group1), and in 10 patients with 'localized' breast cancer (group 2) and 22 patients with 'advanced' breast cancer (group 3). Data are expressed as the number of MOC31-positive cells found in 50 million crude nucleated BM cells for each patient. The median and mean number of cells ± standard deviation of the mean are presented for each patient group.

Figure 2

Classification of patients according to immunomagnetic detection of MOC31-positive cells in bone marrow (BM) specimens. BM samples from control patients (white), and from patients with 'localized' breast cancer (gray) and patients with 'advanced' breast cancer (black) were subjected to the immunomagnetic technique for the detection of MOC31-positive cells. These patients were then divided into three groups on the basis of the number of cells trapped: group A includes patients with more than 150 cells, group B includes those with 50–150 trapped cells, and group C includes those with less than 50 cells trapped among 50 million nucleated BM cells.

Figure 3

Immunocytochemistry targeting cytokeratins (CKs) in bone marrow (BM) aspirates from breast cancer patients. (a) Distribution of CK-positive cells for 32 patients. (b) CK-positive cells labeled (purple) by immunocytochemistry performed on BM mononuclear cell cytospots of one breast cancer patient with 'advanced disease' (PM634).

Figure 4

Immunomagnetic (IM) sorting and immunofluorescence characterization of bone marrow (BM) micrometastatic cells in 'advanced disease' breast cancer patients. BM aspirates were immunopurified using MOC31-conjugated Dynabeads. Following IM sorting, immunofluorescence experiments were performed using anti-cytokeratin (a) and anti-CD45 (b) antibodies (green labeling), and were analyzed by light/fluorescence microscopy. Magnification, × 400 (a) and × 200 (b). These data are representative of experiments performed with BM aspirates from several patients.