Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.
OBJECTIVE
The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow.


METHODS
In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic 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 increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression.


CONCLUSIONS
These results demonstrate the feasibility and advantage of combining 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.
Forus
A
A
Department of Tumour Biology, Norwegian Radium Hospital, Oslo, Norway.
Høifødt
H K
HK
Overli
G E
GE
Myklebost
O
O
Fodstad
O
O
D001707:Biopsy, Needle; D019046:Bone Marrow Neoplasms / Q000175:diagnosis* / Q000556:secondary*; D001943:Breast Neoplasms / Q000473:pathology*; D005260:Female; D051518:Fibroblast Growth Factor 3; D005346:Fibroblast Growth Factors / Q000235:genetics; D018734:Genes, erbB-2; D006801:Humans; D018189:Immunomagnetic Separation; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009363:Neoplasm Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012680:Sensitivity and Specificity
C496444:FGF3 protein, human; D051518:Fibroblast Growth Factor 3; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D005346:Fibroblast Growth Factors