Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates

Aim-The presence of malignant cells in the blood and bone marrow of patient s with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells mig ht be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and character isation of malignant cells present in bone marrow. Methods-In spiking experiments, SKBR3 cells were mixed with mononuclear cel ls in known proportions to mimic bone marrow samples with micrometastatic c ells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the M OC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and i nt2 copy numbers were also determined in 15 primary breast cancers, and bon e marrow samples from patients with amplification were analysed for microme tastatic cells by immunomagnetic enrichment and FISH. Results-In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed incr eased copy numbers of erbB2 and/or int2, and three of these patients had de tectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tu mour cells/10(7) mononuclear cells, respectively. The patient with 540 tumo ur cells/10(7) mononuclear cells showed high level amplification of erbB2 a nd suffered from a particularly aggressive disease, whereas the patient wit h 4000 tumour cells/10(7) mononuclear cells had favourable disease progress ion. Conclusion-These results demonstrate the feasibility and advantage of combi ning immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.