The detection and elimination of minimal disseminated disease in patients with solid tumours is one of the main current topics in clinical oncology (Pantel, 1996).A variety of assays of widely varying sensitivity have been utilized for the detection of circulating tumour cells such as light microscopy, cytogenetic analyses, fluorescence in situ hybridization (FISH), Southern blot analysis, immunocytochemistry, polymerase chain reaction (PCR) and flow cytometry (Kvalheim, 1996;Pantel, 1996;Vrendenburgh et al, 1996;Schoenfeld et al, 1997;Traystman et al, 1997).Because of the fact that breast and ovarian cancers do not appear to have tumour-specific chromosomal aberrations, tumour cell detection by molecular methods is based on the amplification of tissue-specific transcripts Bostick et al, 1998). In immunocytochemical assays, epithelial specific antibodies have been used to detect isolated tumour cells in bone marrow (BM) and blood (Cote et al, 1991(Cote et al, , 1995(Cote et al, , 1996Diel et al, 1996;Franklin et al, 1996).In an effort to obtain greater sensitivity, several investigators have developed techniques for the enrichment of tumour cells before their identification by immunocytochemistry, PCR or FISH (Hardingham et al, 1993(Hardingham et al, , 1995Berois et al, 1997;Eaton et al, 1997;Hildebrandt et al, 1997;Naume et al, 1997;Martin et al, 1998).Therefore, the aim of our study was to analyse the presence and frequency of circulating tumour cells in the peripheral blood of patients with breast or ovarian cancer by using a combination of magnetic activated cell sorting (MACS) and interphase FISH.
MATERIALS AND METHODS
PatientsIn this study we included 48 adult patients with a median age of 60.6 ± 13.3 years (range 30-86). Thirty-five had the diagnosis of breast cancer, 12 ovarian cancer and one uterine sarcoma. Twentyfive patients had primary disease (6/25 without involvement of axillary lymph nodes, 8/25 with solid metastases, 11/25 with lymph node metastases) and 23 had relapsed (20/23 with solid metastases). PB-samples were obtained following informed consent at the time of diagnosis, during or after therapy. MACSsorted tumour cells were analysed by interphase FISH, using α-satellite probes specific for the centromeric regions of chromosomes 7, 12, 17 and the region 17 q11.2-q12 (HER-2/neu). Controls were PB-samples from five normal volunteers to determine the background for each probe.
Magnetic cell separationMononuclear cells were isolated from fresh blood by FicollHypaque gradient separation. After washing in PBS per 5 mm EDTA, 300 µl PBS buffer per 10 8 cells, 20 µl of CK-8 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were added. After gentle mixing and incubation for 15 min at 4°C, the cells were washed once in 5 ml buffer per 10 8 cells. The buffer was completely removed, and the cells were resuspended again in 400 µl buffer. The cell suspension was then applied to a prefilled Summary Detection of micrometastases in patients with solid tumours may aid the establishment of prognosi...
