Sequential immunotyping and genotyping of tumor cells in bone marrow of cancer patients: a model study

Epithelial cells can be detected in bone marrow or peripheral stem cell preparations of patients with various kinds of cancer and their presence in bone marrow is of prognostic significance. Characterization of these cells has been hampered by their low frequency. Here we present a method that may allow sequential immunophenotyping and genotyping of epithelial cells in bone marrow. To simulate in vivo situations, cells from the colon cancer cell line HT29 were seeded into bone marrow and were first detected by the Fab fragment of the A45-B/B3 anticytokeratin antibody. Expression of Ki67, p53, Her-2/ neu (c-erbB2), and 17-1A could be detected on A45-B/B3-stained cells by immunofluorescence using a fluorescein-labeled anti-mouse immunoglobulin specific for the Fc part of mouse immunoglobulins. Reactivity for all antigens except for Ki67 persisted after A45-B/B3 labeling even when a scoring step for the presence of epithelial cells was performed before proceeding with the immunophenotyping. After immunophenotyping, numerical chromosomal aberrations and amplifications of the Her-2/neu oncogene could be assessed by fluorescence in situ hybridization in the same A45-B/B3-stained cells. This combination of immunophenotyping and genotyping may help in establishing the role of epithelial cells in bone marrow or peripheral stem cell harvests for tumor relapse and formation of metastases.