IMMUNOMAGNETIC TECHNIQUES FOR THE ENRICHMENT AND DETECTION OF ISOLATED BREAST-CARCINOMA CELLS IN BONE-MARROW AND PERIPHERAL-BLOOD

Detection of isolated tumor cells (TC) in bone marrow (BM) from patien ts with breast cancer is usually accomplished by immunocytochemical (I CC) analysis of up to 2 x 10(6) mononuclear cells (MNC). However, this method is cumbersome if large numbers of BM cells (i.e. >1 x 10(7) ce lls) are to be analyzed. This emphasizes the need for TC enrichment st rategies. This report describes immunomagnetic separation (IMS) techni ques for enrichment and detection of viable breast carcinoma cells in BM and peripheral blood (PB). The positive IMS technique was performed by incubation of MNC with 2.8 mu m magnetic particles (rat antimouse IgG1 M280-Dynabeads) coated with monoclonal antibody (mAb) against epi thelial surface antigens. The rosetted tumor cells were then visualize d by ICC staining using alkaline phosphatase-conjugated A45-B/B3 antic ytokeratin mtib (Fab). The negative TMS technique was performed by inc ubation of MNC with anti-CD45-coated M450-Dynabeads (4.5 mu m), follow ed by ICC staining of the nonrosetted cells. When 1000, 100, and 10 br east carcinoma cells were mixed with 1 x 10(7) MNC, an average of 748 (n = 9), 70 (n = 10), and 7.8 TC (n = 8), respectively, were detected with the positive IMS technique. With the negative IMS technique, 648 (n = 8), 57.8 (n = 6), and 7.3 TC (n = 6), respectively, were detected . The analysis of 1 x 10(7) MNC with the IMS techniques was compared w ith the ICC analysis of 2 x 10(6) unseparated MNC. A mean 3.7-fold (ra nge 1.5-6.4) to 4.2-fold (2.5-8.2) (positive IMS) and 3.1-fold (range 2.0-5.0) to 3.8-fold (2.0-6.0) (negative IMS) higher TC detection freq uency was achieved after enrichment by LMS in experiments with 100 and 1000 TC/10(7) MNC. The IMS techniques were used for examination of BM samples from locally advanced breast cancer patients. A 5.3-fold mean increase (range 2.1-13.3) in the number of TC detected was obtained w hen the use of positive and negative IMS together was compared with th e direct ICC analysis of unseparated MNC (n = 11). Enrichment of TC by LMS techniques enables us to examine large numbers of MNC from BM or PB, which can result in the detection and characterization of minimal residual disease with increased sensitivity and specificity.