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Most Early Disseminated Cancer Cells Detected in Bone
Marrow of Breast Cancer Patients Have a Putative
Breast Cancer Stem Cell Phenotype
Marija Balic,
1
Henr y Lin,
1
LillianYoung,
1
Debra Hawes,
1
Armando Giuliano,
2
George McNamara,
3,4
Ram H. Datar,
1
and Richard J. C ote
1
Abstract Purpose: The presence of disseminated tumor cells (DTC) in the bone marrow of breast cancer
patients is an acknowledged independent prognostic factor.The biological metastatic potential of
these cells has not yet been shown. The presence of putative breast cancer stem cells is shown
both in primary tumors and distant metastases. These cells with a CD44
+
CD24
/low
phenotype
represent a minor population in primary breast cancer and are associated with self-renewal and
tumorigenic potential. Recognizing the potential effect of prevalence of putative stem cells among
DTC, we evaluated the bone marrow DTC.
Experimental Design: We employed the double/triple-staining immunohistochemistry
protocol and modified the established bone marrow cytokeratin (CK) staining protocol by
adding steps for additional antigens, CD44 and/or CD24.We evaluated 50 bone marrow speci-
mens, previously categorized as CK
+
from early breast cancer patients. CK
+
cells were examined
for CD44 and CD24 expression by light microscopy, fluorescence microscopy, and spectral
imaging.
Result s: We detected the putative stem cell ^ like phenotype in all CK
+
specimens. The mean
prevalence of putative stem/progenitor cells was 72% and median prevalence was 65 % (range,
33-100%) among the overall DTC per patient, compared with primary tumors where this pheno-
type is reported in <10 % o f c el ls .
Conclusions: This is the first evidence of the existence of the putative stem-like phenotype
within the DTC in bone marrow in early breast cancer patients. All patients had a putative
stem cell phenotype among the DTC and most individual DTC showed such phenotype. Future
molecular characterization of these cells is warranted.
The most important factor influencing the outcome of
patients with an invasive cancer is whether the tumor has
spread regionally or systemically (1). Metastases start out as
single cells that detach from the primary tumor and travel to
distal locations; these cells in transit, however, are not
detectable by routine histologic or radiologic methods. Tumor
cell dissemination to the bone marrow is an early event in
progression of breast cancer (1) and is associated with poor
prognosis (2–4). Disseminated tumor cells (DTC) are
commonly detected by immunohistochemistry for epithelial
antigens, such as cytokeratins (CK). The biology of these cells
is not well understood and is critical for therapeutic improve-
ments. Recent evidence suggests that only a certain type of
tumor cells, cancer stem cells or cancer initiating cells, harbor
tumorigenic potential (5). The hypothesis that the initiation
of malignancy has to take place in cancer stem cells derives
from the observation that it often takes many months or
years for the promotion stage of carcinogenesis to occur,
suggesting that the cancer stem cells must stay viable over a
long period (6). Such cells have been identified for hema-
tologic malignancies (7), brain tumors (8), and breast cancers.
For breast cancers, such cells have been identified to have a
CD44
+
CD24
/low
phenotype (9) and represent a minor
population (10-20%) within primary tumors (10). Cells with
this phenotype have been shown to be tumorigenic and
multipotent, capable of generating cells with all the different
lineages. One recently published study further characterized
these cells with additional neoangiogenic and cytoprotective
markers (11). These cells may be responsible for therapeutic
failure (12).
CD44 is a cell adhesion molecule known to be expressed in
most cell types (13, 14) and has been associated with stem cells
Human Cancer Biology
Authors’ Affiliations:
1
Depart ment of Pathology, Keck School of Medicine,
University of Southern California;
2
Joyce Eisenberg-Keefer Breast Center, John
Wayne Cancer Institute, Santa Monica, California;
3
Congressman Julian Dixon
Cellular Image Core, Childrens Hospital Los Angeles Saban Research Institute, Los
Angeles, C alifornia; and
4
Division of Cancer Immunothe rapeutics a nd Tumor
Immunology, City of Hope National Medical Center, Duarte, California
Received 1/25/06; revised 4/27/06; accepted 5/12/06.
Grant support: National Cancer Institute grants 5 R011 CA 585 40 and U10
CA76001and Doheny Eye InstituteSpecialized Imaging Core grant EY03040.
The costs of publication of this article were defrayed in part by the paymentof page
charges. This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Richard J. Cote, Department of Pathology, University of
Southern California, and Norris Comprehensive Cancer Center, Keck School of
Medicine, 2011Zonal Avenue, Los Angeles, CA 90033. Phone: 323-865 -0212;
Fax: 323-865-0077; E-mail: cote_r
@
ccnt.hsc.usc.edu.
F2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-06-0169
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on July 3, 2021. © 2006 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
in normal breast tissue (15). CD24 is expressed in the early
stages of B-cell development and is highly expressed on neutro-
phils, but is absent in normal T cells or monocytes (16).
Whereas CD24 is not present in adult human tissues, it has
been shown to be expressed in human carcinomas (17, 18).
Although considered in one study as a prognostic marker in
breast cancer, where higher CD24 expression as seen by
immunohistochemistry was considered to favor the worse
prognosis (19), recent data suggest a different role for CD24 in
the carcinogenesis of breast cancer, such that decreased
expression or loss of CD24 seems to be the characteristic of
the stemness of a tumor cell (9, 11). CD24 reduces stromal
cell–derived factor-1–mediated migration and signaling via
CXCR4 in breast cancer cell lines with enhanced CD24
expression, suppressing their metastatic potential. On the
contrary, the metastatic potential of CD24
cells is increased
as evidenced by small interfering RNA inhibition (20). This role
of CD24 is also supported by in vivo analysis of nonobese
diabetic severe combined immunodeficient mouse models.
Taking into account the significance of DTC in breast cancer
(2–4), a reasonable next step is that we analyze the com-
partments known to be customarily associated with such
cells—the bone marrow and the lymph nodes—to identify the
potential cancer stem cells and characterize them. The
underlying hypothesis, therefore, is that cancer stem cells
comprise a subpopulation of DTC, and that immunohisto-
chemical identification of this subpopulation may provide a
tool to assess the malignant potential of specific cells and thus
may help to present the clinical course of the malignancy. In
the present study, we show for the first time an application in
bone marrow of a newly developed immunohistochemical
protocol to stratify DTC into subpopulations, and further
show that, surprisingly, even in the early-stage breast cancer
bone marrow samples studied, a vast majority of the DTC
match the phenotype criterion of putative cancer stem/
progenitor cells.
Materials and Methods
Patients and specimen collection
Bone marrow specimens were obtained from early breast cancer
patients, participating in the American College of Surgeons Oncology
Group Z-0010 clinical trial for detection and molecular characteriza-
tion of DTC. Following patient informed consent, unilateral bone
marrow aspirates from upper iliac crest were processed as described
below. For the present analysis, frozen cytospin slides from early
breast cancer patients previously categorized as CK
+
were used.
Because the analysis of patient samples is still ongoing, the study
remains blinded for clinical details and follow-up data are not yet
available. The study was approved by local Institutional Review
Boards.
Preparation of cultured cell slides
Breast cancer cell lines MCF-7, MDA-MB-231, SK-BR-3, HCC-38,
HCC-1395, and HCC-70 were purchased from American Type Culture
Collection (Manassas, VA). Cells were grown in appropriate growth
medium as suggested by American Type Culture Collection and
harvested by trypsinization before reaching confluence. The cells were
subjected to cytocentrifugation (at a density of f1,000/mm
2
) onto
positively charged slides by centrifugation at 1,000 rpm for 10 minutes.
Slides were air-dried overnight, followed by acetone fixation for
10 minutes, and stored at 20jC until use for immunohistochemical
analysis.
Bone marrow preparation
The procedure for processing the bone marrow aspirates has
previously been published (21). Briefly, the mononuclear cell
fraction containing any DTC was enriched by Ficoll-Hypaque density
gradient centrifugation, using Beckman GS-6 centrifuge, at 400 g
for 35 minutes and washed twice with PBS before transferring
the cells onto microscope slides by cytocentrifugation at a density
of 5 10
5
/mm
2
. Subsequently, the cells were fixed in acetone for
10 minutes and stored at 20jC.
Immunohistochemistry
Antibodies . A cocktail of two different mouse monoclonal
antibodies against CK was used as primary immunohistochemistry
detection reagent for circulating tumor cells: AE-1 (Signet, Dedham,
MA) against low and intermediate type I acidic keratins and
CAM5.2 (Becton Dickinson, San Jose, CA) against CK8 and CK18.
Further, mouse monoclonal primary antibodies anti-CD44 (clone
156-3C11, LabVision, Fremont, CA), binding to standard isoform
of CD44, and anti-CD24 (clone SN3b, LabVision) were used to
detect the putative breast cancer stem/progenitor cell markers
(10, 19).
Single-marker immunohistochemistry
Antibodies were titered until optimal concentration was deter-
mined by two independent trained personnel (M.B. and D.H.),
viewing under 40and 100objective lenses. The following
procedure was used for evaluating the expressions of the three
markers (CK, CD44, and CD24) on different cell lines and bone
marrow specimens. Slides were brought to room temperature before
blocking with normal horse serum for 30 minutes and incubated
with primary antibody diluted in blocking buffer for 1 hour.
Subsequently, the slides were washed thrice with PBS and incubated
with biotinylated secondary antibody (antimouse). After washing in
PBS thrice to remove the unbound secondary antibody, the immune
complexes were made visible by 30-minute incubation with avidin-
biotin-horseradish peroxidase complexes (Vector Laboratories, Bur-
lingame, CA) and 3,3¶-diaminobenzidine (DAB; Biogenex, San
Ramon, CA) as substrate. Cultured MCF7 and MDA-MB-231 were
used as positive controls for each run in single-, double-, and triple-
marker immunohistochemistry.
Double-marker immunohistochemistry
We optimized the double marker immunohistochemistry proce-
dure using slides with MDA-MB-231 cell line following the
completion of the single marker immunohistochemistry. The
optimized protocol is briefly described below. Slides were brought
to room temperature and incubated with normal horse serum
blocking solution for 30 minutes, with Universal block solution for
10 minutes, and with Tris-HCl for 5 minutes. Subsequently, the
slides were incubated with a cocktail of first primary antibodies for
2 hours (AE1 at 1:200 dilution and CAM5.2 at 1:100 dilution from
stock solution) following similar detection system with DAB as
described above in single-marker immunohistochemistry. The
second primary antibody was incubated either overnight (CD24
antibody at 1:100 dilution from stock solution) or with similar
procedure with a different detection system, alkaline phosphatase
with Vector Red as substrate (Vector Laboratories) and the addition
of a 30-minute blocking step of endogenous alkaline phosphatase
activity with 0.1 mol/L levamisole along with PBS with 5 mmol/L
levamisole for all the washes before the application of the avidin-
biotin alkaline phosphatase complexes. When the slides were
assessed for CK and CD44 staining, the procedure was slightly
modified. The slides were first incubated with anti-CD44 antibody
(at 1:300 dilution from stock solution) for 1 hour, followed by
detection using horseradish peroxidase/DAB system, and then with
the cocktail for anti-CK antibodies as described above, followed by
detection with alkaline phosphatase-Vector Red system.
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Triple-marker immunohistochemistry with immunoflu-
orescence
Additional CD44 assessment was done on the slides where the
immunodetection was already completed for CK and CD24. The slides
were first soaked in warm water for a few minutes to remove the
coverslips. After washing with PBS thrice, additional blocking was done
with normal horse serum for 30 minutes and incubated with primary
antibody for 2 hours (CD44 at dilution 1:200 from stock solution).
After washing in PBS thrice, Alexa Fluor 488 goat anti-mouse
immunoglobulin G (Molecular Probes, Invitrogen, Carlsbad, CA) was
added at 1:100 dilution from the stock solution and incubated for
1 hour. Following nuclear staining with 4¶,6-diamidino-2-phenylindole
(Vecatshield mounting medium containing 4¶,6-diamidino-2-phenyl-
indole), the slides were coverslipped and sealed with nail polish. The
protocol optimized for triple staining in cultured cell line slides was
used to analyze the clinical bone marrow samples.
DTC identification
All preparations were analyzed by two independent trained
personnel (M.B. and D.H.) using 40objective lens and 100
objective lens with oil immersion.
When slides were analyzed for CK and CD44 staining, CK was
represented by red cytoplasmic staining and CD44 by brown
membranous staining. When slides were assessed for CK and CD24,
cells stained for CK, which was brown cytoplasmic staining, and
CD24, which was membranous red staining, were classified as
CK
+
CD24
+
cells (see Fig. 1A), and cells dominant with only brown
cytoplasmic staining were classified as CK
+
CD24
(putative breast
cancer stem/progenitor cells; Fig. 1B). By changing the focal plane of
the objective, some cells previously considered negative for membrane
CD24 staining did show some membrane staining, and were
characterized as CK
+
CD24
+
.
Slides assessed for all the three antigens, CK, CD24, and CD44,
were first analyzed with light microscopy. CK
+
cells were marked and
subsequently examined for CD44 expression using a Leica DM LB2
microscope equipped with Diagnostic Instruments 7.3 3 shot color
camera viewed with a Chroma filter set consisting of an excitation
filter of 480/40 nm, dichroic filter of 505-nm long pass, and an
emission filter of 5,350/50 nm (Diagnostic Instruments, Sterling
Heights, MI).
Fig. 1. Aand B, double staining immunohistochemistry results for breast cancer
bone marrow samples. Thebone marrow samples show presence of distinct
stem-like and non-stem tumor cells (magnification, 40). A, two tumor cells with
brown cytoplasmic staining for CK (DAB) and red membranous staining for CD24
(Vector Red). B, an example of a CK
+
CD2 4
cell (putative breast cancer stem cell).
Cto F, the applicability of spectral imaging analysis for immunohistochemistry
double-stained samples, where the chromogens are not easily distinguishable with
the naked eye. Here, the cytospun bone marrowaspirates were assessed for CK
(red; stained with Fast Red) and CD44 (brown ; stained with DAB).The cells were
counterstained with hematoxylin. C, transmittedlight image, showing overall cell
population subjected to double-staining for CK and CD44. Arrow, two CK positive
cells that are difficult to assess for their CD44 positivity. Dand E, spectrally
unmixed images with pseudocolors assigned to hematoxylin (D)andDAB(CD44;
E) using SpectraView software, showing that all the cells in the field show nuclear
hema toxylin as well as CD4 4 staining, including the t wo cells d enoted by a rrow.
F, fluorescence image obtained for CK with Cy3 filter for Fast Red. Gto J,
applicability of spectral imaging analysis for immunohistochemistry double-stained
samples for CK and CD24. In this case, the expression of CK was assessed using
DAB (brown) and that of CD24 with Fast red (red ). The cells were counterstained
with hematoxylin. G, transmitted light image, showing overall cell population
subjected to double-staining for CK and CD24. Arrow, a CK-positive cell that
appears as CD24
/low
. To confirm that this c ell is indeed CD24
/low
,wedidfurther
spectralimaging analysis. Spectrally unmixed images with pseudocolors assigned
to hematoxylin (H)andDAB(CK;I). These images show that, whereas all cells in
the field show nuclear hematoxylin, only the cell denoted is positive for CK (DAB).
J, fluorescence image obtained for CD24 staining with Cy3 filter for Fast Red.
Spectral imaging analysis of the cell denoted in (G) shows that the cell at this
location expresses no/low CD24 (red). K, another CK-positive cell; L, the same c ell
with specific membranous fluorescence demarking the CD44 positivity; note the
surrounding CD44
+
lymphocy tes.
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Spectral imaging
When it was not possible to distinguish with the naked eye if the cell
is single or double stained, spectral imaging was done. Images were
acquired at the CHLARI Congressman Julian Dixon Cellular Image Core
with a Leica DM RXA microscope using a HC Plan 20/0.70 numerical
aperture Ph2 circular differential interference contrast microscopy
objective lens, 1 optovar, and Koehler illumination (Leica Micro-
systems, Inc., Bannockburn, IL). A SKY/SD-300/VDS-1300 spectral
imager with Spectral Imaging 2.5 and SpectraView 1.1 software
(Applied Spectral Imaging, Inc., Carlsbad, CA) was used. Transmitted
light spectral images were acquired at maximum tungsten-halogen lamp
power (12 V) through a CB11 color balancing filter and ND4 neutral
density filter. Spectral image exposure time was 20 ms per frame to
avoid saturating the charge-coupled device detector. Spectral image
wavelength range was 400 to 750 nm. Vector Red fluorescence was
viewed through a Chroma Technology HQ Cy3 filter set (Rockingham,
VT) with a Sutter Instruments LS300/LLG380 Xenon arc lamp/1-m
liquid light guide (Novato, CA). DAB is not fluorescent and did not
attenuate the red fluorescence from these dyes. Fluorescence images
were acquired in SD-300/VDS-1300 direct view mode as 12-bit
monochrome images and saved as 16-bit TIFF images. Spectral color
images were saved from SpectraView and merged with monochrome
images using Adobe Photoshop 6.0 (San Jose, CA). Figure 1C to F
shows the applicability of spectral imaging analysis for immunohisto-
chemistry double-stained samples for analysis of CK
+
cell for CD44
expression, and Fig. 1Gto Jfor analysis of CK
+
cell for CD24
expression.
Result s
We evaluated bone marrow samples for the presence and
prevalence of putative cancer stem/progenitor cells. We
accomplished this by using pan-CK expression to first identify
tumor cells, followed by characterization of these DTC into
CD44
+
CD24
(cancer stem/progenitor cell subpopulation) and
CD44
+
CD24
+
(non-stem population). We first assessed six
different breast cancer cell lines for CD44, which were all found
to be immunohistochemistry positive. Furthermore, as proof of
principle, we stained 10 slides, selected from the 50 breast
cancer cases, for CK and CD44. In these 10 cases, we detected 22
CK
+
cells, and all of them were CD44
+
. In three cases where
CD44
+
reactivity was difficult to distinguish with the naked
eye, we did spectral imaging analysis to evaluate the cells for
the expression of CD44 (as shown in an example in Fig. 1C-F).
These observations, coupled with the evidence that most cells
(breast as well as hematopoietic; refs. 13, 14) and breast cancer
cell lines are CD44
+
, led us to reason that most of the breast
cancer DTC are both CK and CD44 positive. We employed the
double immunohistochemistry staining protocol optimized for
cell lines on cytospin preparations from bone marrow, which
were previously characterized as CK
+
.
Of 100 slides (from 50 clinical samples, 2 slides each), 2 were
excluded from the analysis due to poor sample quality. The
remaining 98 slides were evaluated for the presence and
prevalence of CK
+
CD24
+
or CK
+
CD24
cells.
Only 3 of 50 (6%) clinical bone marrow specimens have not
been confirmed for being positive on repeat immunohisto-
chemistry because no CK
+
cells could be detected in either slide.
This finding is attributable to sampling variability during
cytospin preparations, an acknowledged clinical reality. In all
47 (100%) clinical bone marrow specimens with CK
+
cells,
CK
+
CD24
phenotype was identified (Fig. 1B, G-J). The mean
percentage of cells with CK
+
CD24
phenotype detected in DTC
per clinical sample was 74.87% (range, 33-100%). The median
percentage of such cells per clinical sample was 71%. Table 1
provides the numbers of detected cells with either of the two
phenotypes and percentages of CK
+
CD24
cells in each bone
marrow specimen. On analyzing a total of 332 CK
+
cells, 232
(70.59%) cells showed CK
+
CD24
phenotype.
To further ascertain the prevalence of CD44 expression across
CK
+
cells, we have additionally done reevaluation of the bone
marrow specimens where possible. Of the 100 slides that were
assessed for CK and CD24, 71 slides underwent the additional
analysis. We used the cultured cell line MDA-MB-231 as
positive control, and the lymphocytes as internal positive
control, where the majority are positive for CD44. When
specific membranous fluorescence was undetectable in a slide,
it was regarded as insufficient immunohistochemical reaction.
Six of 71 slides showed insufficient staining for CD44. Two
hundred-eighteen cells could be identified in the remaining 65
slides as CK
+
. Of these, 19 (9%) were categorized as CD44
;the
rest of the cells (91%) showed specific positive membranous
reaction (see Fig. 1L). The CD44
cells were evenly distributed
between the CK
+
CD24
(4%) and CK
+
CD24
+
(5%) cell
populations. In none of the specimens could we identify only
CK
+
CD44
cells. From 199 CD44
+
cells, 141 (71%) were
CD24
and 58 (29%) were CD24
+
.CK
+
CD44
+
CD24
cells
represented 65% of the total pool of 218 cells, whereas
CK
+
CD44
+
CD24
+
cells represented 26%.
Discussion
Whereas identifying the rare tumor cells in a background
of numerous hematopoietic cells from bone marrow is itself
a labor-intensive task requiring much expertise, distinguishing
the tumor cells further between different subpopulations
(stem/non-stem) poses a new challenge. Using an optimized
immunohistochemistry procedure, we show here that the
majority of CK
+
cells (DTC) have the putative stem cell
phenotype (CD44
+
CD24
+
; refs. 9, 11). Further, we show here
for the first time the usefulness of spectral imaging for
analysis of single DTC and expression of additional markers.
Potential enhancement of the above assay can come through
application of newer techniques such as quantum dot
labeling, which would allow the labeling of cells with up
to six different markers and simultaneous analysis of their
expression.
To our surprise, all the patients with CK
+
DTC had cells with
putative breast cancer stem cell phenotype, and in fact, even
when the assessment for CD44 was completed on the sub-
stantial portion of the slides, the majority (71%) of DTC were
found to have a putative breast cancer stem cell – like pheno-
type. The majority of DTC isolated from bone marrow have
previously been shown to be viable tumor cells with
proliferative potential under specific culturing conditions
(22); our results may be attributable to this finding because
the vast majority of the detected cells have a stem cell – like
phenotype. Somewhat unexpected, the high percentage of CK
+
cells having CD44 expression in bone marrow may be
attributable to the previous findings in cell lines that, as an
adhesion factor, CD44 also represents a homing factor of breast
cancer cells to settle down in bone marrow (23).
Although DTC are regarded as the prerequisite for relapse
and metastasis (24), no studies have yet examined these cells
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for the existence of putative stem cell phenotype. Abraham
et al. (10) have recently analyzed primary breast tumors for
prevalence of CD44
+
CD24
/low
cells and their effect on
clinical outcome. They found that 70% of patients had
<10% of cells with CD44
+
CD24
/low
phenotype and that
higher prevalence of these cells correlated with distant
metastases, in particular, bone metastases. However, in their
study, the finding had no effect on overall survival of breast
cancer patients. Kristiansen et al. (19) considered higher CD24
expression as a prognostic marker in breast cancer to favor the
worse prognosis. Their immunohistochemistry data, however,
show differential expression of CD24 in tumor cells, with a
high number of CD24
+
cells, but cell nests can be recognized
with low or no expression of CD24
. These data can be
explained given the recent evidence that CD24
stem cells
give rise to both CD24
+
and CD24
cells (9) and that the
Table 1. Presence and distribution of putative stem cells across all the bone marrow specimens analyzed
Patient no. No. CK
+
CD24
cells No. CK
+
CD24
+
cells Total no. CK
+
cells % CK
+
CD24
phenotype
1 10 6 16 62.5
2 9 5 14 64.3
32 24 50
40 00
5 4 2 6 66.7
6 5 3 8 62.5
7628 75
87 29 77.8
96 28 75
10 3 0 3 100
11 11 1 12 91.6
12 5 6 11 45.5
13 5 1 6 83.3
14 7 4 11 63.6
15 9 3 12 75
16 9 5 14 64.2
17 3 2 5 60
18 6 3 9 66.7
19 11 5 16 68.8
20 6 2 8 75
21 2 0 2 100
22 1 2 3 33
23 7 2 9 77.8
24 1 0 1 100
25 7 5 11 58.3
26 14 6 20 70
27 1 0 1 100
28 2 0 2 100
29 2 0 2 100
30 7 2 9 77.8
31 2 0 2 100
32 4 3 7 57.1
33 5 3 8 62.5
34 0 0 0
35 5 3 8 62.5
36 6 0 6 100
37 8 3 11 72.7
38 7 3 10 70
39 1 0 1 100
40 4 0 4 100
41 3 0 3 100
42 4 2 6 66.7
43 5 2 7 71.4
44 3 0 3 100
45 4 1 5 80
46 3 2 5 60
47 5 2 7 71.4
48 0 0 0
49 1 1 3 50
50 2 2 4 50
Average of CK
+
CD24
phenotype/total DTC per patient 74.87
%CK
+
CD24
phenotype in overall total DTC 70.59 (240 of 340)
NOTE: The table provides the total number of CK
+
CD24
,CK
+
CD24
+
,andCK
+
cells, analyzed from two cytospun slides from bone marrow of
each patient. For each patient the percentage of CK
+
cells with CK
+
CD24
phenotype is presented along with the mean percentage per patient
and the overall percentage of all CK
+
cells with CK
+
CD24
phenotype.
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larger proportion of cells in tumors is represented by CD24
+
cells. In addition, it is still not clear if the number of cancer
stem cells—or simply their presence—in the primary tumor is
critical for further disease development.
We are well aware of the fact that our present study as well as
previous studies has analyzed these cells for a predefined
phenotype, and that this is intended only as a preliminary
morphologic characterization of these cells. Whereas the
finding of DTC with putative stem/progenitor cell – like
phenotype in majority of breast cancer patient bone marrow
is somewhat unexpected, it must be noted that this phenotypic
characterization needs to be supplemented with functional
analysis of these cells, which would inform on their capability
of self-renewal. Therefore, further molecular characterization
and biological analysis of the putative stem cells in prospec-
tively collected patient samples must be done. Nonetheless,
considering the present evidence, which suggests the effect of
such cells on the progression of breast cancer (9, 11, 25) and
other solid tumors (8, 24), we strongly believe that our assay
permits an initial identification of putative stem-like cell
subpopulation among the DTC, which can then permit further
molecular and functional characterization.
Using the assay we present here as a basic morphologic
assay, it should also be possible to better characterize DTC
for additional stem cell–like markers. Some of the molecular
features of putative stem cells are emerging in the recent
literature (11, 26). Their data suggest that only a small
proportion of the CD44
+
CD24
/low
cells may have the self-
renewal capability. However, even if only a proportion of the
putative stem-like cells detected in our study have an ability
to perpetuate indefinitely, this finding indicates that at least
a proportion of the cancer cells detected in the bone marrow
of patients with early-stage breast cancer are capable of
forming metastases. Further, the majority of the DTC
detected in the bone marrow have the putative stem cell
phenotype, as compared with what has been reported in the
primary and metastatic tumor formations (where only a
minority of cells have this phenotype; refs. 9, 10), clearly
suggesting that there is a selection for these stem-like cells
and that DTC detected in the bone marrow of patients with
early-stage breast cancer are not merely shed from the
primary tumor.
Breast cancer patient population has a lifetime enhanced
risk of death from cancer when compared with similar normal
population group (27). This may be attributable to the
potential effect of the existence of cancer stem cells among the
DTC in patients. Recent pooled analysis by Braun et al. (3) in
a large number of patients analyzed shows that although the
presence of DTC is associated with a poor prognosis, a
significant proportion of patients still do well even after
10 years. The reason for this variability could be that a
proportion of these cells may be capable of remaining
‘‘dormant,’’ settled in bone marrow and/or distant organs,
which are driven towards the recurrent tumorigenesis only
after unknown initiation events occur in these cells. In
addition, besides their presence, the environment of DTC
may be as crucial in determining the destiny of the cells and
final patient outcome (24).
This is the first evidence of the existence of the putative
stem/progenitor cell (CD44
+
CD24
) subpopulation within the
DTC component in bone marrow. Because we could identify
the putative breast cancer stem cell phenotype in all patients
where we detected CK
+
cells, our data suggest that the majority
of the patients with DTC may have a lifetime risk for relapse,
an idea that has been suggested previously (28). Existence of
putative stem cells will be a clinically relevant issue that we
must address. Recognizing that it is desirable to correlate these
findings of existence of putative stem cells among the DTC
population with the actual clinical progression of breast cancer
patients, we will in the future examine the clinical correlates
for these patients once the clinical outcome data become
available from American College of Surgeons Oncology Group
clinical trial.
Whereas the possible presence of early tumor dissemination
is the rationale behind the use of systemic adjuvant chemo-
therapy in patients who have undergone definitive local
treatment of the primary tumor (29, 30), the presence of
cancer stem cells may explain the failure of adjuvant
chemotherapy in a proportion of early-stage breast cancer
patients. Further research aimed at enrichment and expression
profiling of CD44
+
CD24
population of DTC in a larger set of
prospectively collected bone marrow specimens can help
identify additional, novel potential therapeutic targets and
further define the biological potential of these cells.
Acknowledgments
We thankJustinTsai (LabVision, Fremont, CA) for the kind technical support.
Human Cancer Biology
www.aacrjournals.orgClin Cancer Res 20 06;12(19) October 1, 20 06 5620
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2006;12:5615-5621. Clin Cancer Res
Marija Balic, Henry Lin, Lillian Young, et al.
Cancer Stem Cell Phenotype
Marrow of Breast Cancer Patients Have a Putative Breast
Most Early Disseminated Cancer Cells Detected in Bone
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