Detection of Disseminated Tumor Cells in Bone Marrow of Patients with Breast Cancer: Comparison of Two Enrichment Techniques
S Krishnamurthy, J Reuben, M Cristofanilli, A Lucci. U.T. M. D. Anderson Cancer Center, Houston, TX
Background: Presence of disseminated tumor cells (DTCs) in bone marrow (BM) of patients with breast cancer is associated with a poor prognosis. Currently, there is no standardization of the methods for the detection of DTCs in BM. We sought to compare the performance of two enrichment techniques for detecting DTCs in BM, including density gradient separation(DGS) and immunomagnetic separation(IMS) using EpCam beads for detection of DTCs.
Design: BM (10 ml) was aspirated from bilateral iliac crests in EDTA primed tubes at the time of primary breast surgery. One half of the specimen was layered with lymphoprep, centrifuged at 1,500 rpm for 10 minutes, the buffy coat was separated, washed in PBS, centrifuged and from the cell pellet, 10 cytospin smears were made; one of which was stained by Papanicalaou method and the others(9) immunostained with pancytokeratin antibody. The other half of the BM aspirate were enriched with affinity columns containing EpCAM (epithelial marker) magnetic beads (Miltenyi Biotech). The purified cell populations were pelleted onto two slides, one of which was stained with Pap method and the other immunostained with pancylokeratin antibody. The overall sensitivity of both methods for detecting CTCs was calculated.
Results: We included 40 BM specimens for a direct comparison of DGS and IMS. Pap stained cytospin smears of DGS separated cells showed abundant hematopoietic cells (1000) without any recognizable tumor cells in any of the cases. Rare isolated CK positive cells, morphologically consistent with tumor cells were detected in 9 of the 40 cases (22.5%). IMS yielded cells ranging from 5-50 in each slide. Pap stained cytospin smears showed few cells (3-10) in 3 of the 40 cases (7.5%) with morphological features, consistent with tumor cells. CK immunostaining of the destained Pap stained smears confirmed the presence of CK positive cells in these three cases. The overall sensitivity of the DGS was higher than IMS 22.5% vs7.5% in this study. The difference in sensitivity between the two techniques reached statistical significance (p=.009, Fishers exact test).
Conclusions: 1. The sensitivity of DGS was significantly higher than IMS for isolation of DTCs in BM.2. IMS yields more DTCs, which can allow further phenotypic characterization beyond identification in some if not all cases.3. The decreased sensitivity of IMS is most likely due to loss of cells in the process of enrichment and expression of EpCAM in some but not all DTCs.
Wednesday, March 11, 2009 9:30 AM
Poster Session V # 9, Wednesday Morning