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 cel l preparations of patients with various kinds of cancer and their pres ence in bone marrow is of prognostic significance. Chacterization of t hese 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 anticytokerat in antibody. Expression of Ki67, p53, Her-2/neu (c-erbB2), and 17-1A c ould be detected on A45-B/B3-stained cells by immunofluorescence using a fluorescein-labeled anti-mouse immunoglobulin specific for the Fc p art of mouse immunoglobulins. Reactivity for all antigens except for K i67 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 abe rrations and amplifications of the Her-2/neu oncogene could be assesse d by fluorescence in situ hybridization in the same A45-B/B3-stained c ells. This combination of immunophenotyping and genotyping may help in establishing the role of epithelial cells in bone marrow or periphera l stem cell harvests for tumor relapse and formation of metastases. (C ) 1998 Wiley-Liss,Inc.