Techniques to identify low frequency (i.e., 10(-4)-10(-5)) tumor cells
in bone marrow and peripheral blood of cancer patients provide opport
unities for early detection of disseminated disease and characterizati
on of the properties of cells released from the tumor, Low frequency e
pithelial cells in marrow and blood can be detected using immunophenot
ypic markers directed against intracellular and/or cell surface antige
ns. However, nonspecific phenotypic labeling may compromise the abilit
y to discriminate tumor from nontumor cells, We describe optimization
of slide-based approaches that facilitate identification and subsequen
t molecular cytogenetic characterization of rare tumor cell population
s in hematopoietic tissues, Colon tumor cells seeded in a hematopoieti
c background provided a model system to optimize methodologies that ar
e applicable to detection and quantification of micrometastases in cli
nical specimens, Mixtures of cytogenetically aberrant epithelial cells
and hematopoietic cells on slides were labeled with an anti-cytokerat
in 20 (anti-CK20) antibody that recognizes >90% of colon adenocarcinom
as, Computerized image analysis was used to record the location of the
immunofluorescent cells on microscopic slides. Cells on slides were t
hen hybridized using fluorescence in situ hybridization (FISH) with re
peat-sequence DNA probes to detect aneusomies, Previously discriminate
d epithelial cells were relocated for molecular cytogenetic characteri
zation. Tumor cells present at frequencies approximating 5 x 10(-5) we
re discriminated on the basis of immunofluorescence and cell size, A l
ow frequency (4 x 10(-4)) population of normal hematopoietic cells als
o labeled with anti-CK20 (data not shown). This strategy of sequential
immunophenotyping and molecular cytogenetic analyses may be useful to
discriminate tumor from nontumor cells in cancer patients with microm
etastatic disease. (C) 1996 Wiley-Liss, Inc.