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1664
Molecular Cytogenetic Analysis of Cytokeratin
20-Labeled Cells in Primary Tumors and Bone
Marrow Aspirates from Colorectal Carcinoma Patients
Virginia R. Litle, M.D.1,2
Robert S. Warren, M.D.1,2
Dan Moore II, Ph.D.2
Maria G. Pallavicini, Ph.D.2
BACKGROUND. Low frequency epithelial cells in bone marrow from colorectal carcinoma patients are associated with an increased risk of recurrence and decreased
survival. Current immunohistochemical approaches to detect epithelial cells in
1
Department of Surgery, University of California, San Francisco, California.
2
Cancer Center, University of California, San
Francisco, California.
bone marrow aspirates rely on antibodies against cytokeratin 18 (CK18). The predictive value of CK18-based detection strategies is limited by false-positives that
occur in approximately 30% of cases. Cross-reactivity of anti-CK18 antibodies with
nontumor cells may contribute to the false-positive rate. Cytokeratin 20 (CK20)
shows more restricted expression than CK18 and labels cells in colorectal tumors.
METHODS. Immunofluorescence assays were used to quantify CK20-labeled cells
in bone marrow aspirates and tumors from 18 Dukes stage C and D colorectal
carcinoma patients to determine whether CK20 is useful in detecting micrometastases. Fluorescent in situ hybridization was used to determine whether CKlabeled subpopulations carried genomic aberrations associated with colorectal
carcinoma.
RESULTS. CK20-labeled cells occurred at frequencies Ç5 1 1005 in control bone
marrow aspirates from patients without colorectal carcinomas. Approximately
1004 CK20-labeled cells were present in 4 of 11 bone marrow aspirates (45%)
from patients with Dukes stage D colon carcinoma. The mean frequency (5 1
1005) of CK20-labeled cells in Dukes stage C and D rectal carcinoma patients
was statistically similar to control values.
CONCLUSIONS. A subset of CK20-labeled cells in primary tumors and hepatic
metastases are aneusomic. CK20-labeled cells in bone marrow aspirates are
cytogenetically normal. These data demonstrate that CK20 cells in solid tumors
may be cytogenetically aberrant, but suggest caution in the use of CK20 to
detect micrometastases in bone marrow aspirates. Cancer 1997; 79:1664 – 70.
q 1997 American Cancer Society.
KEYWORDS: micrometastases, immunocytochemistry, recurrent-disease, fluorescent in situ hybridization.
M
Address for reprints: Dr. Maria Pallavicini, Cancer Genetics Group, 2340 Sutter St., Box 0808,
University of California, San Francisco, CA
94143-0808.
Received August 27, 1996; revision received
January 13, 1997; accepted January 24, 1997.
etastases are the major cause of disease progression and treatment failure in patients with colorectal carcinoma.1 Approximately 30% of colorectal carcinoma patients undergoing potentially
curative surgery will develop recurrent disease; 70 – 80% of disease
recurrence occurs at distant sites, even in patients without clinical
evidence of tumor cell dissemination at the time of surgery. Conventionally, identification of patients at risk for disease progression is
based on clinical stage. However, clinical stage is insufficient to accurately classify patients into risk groups for development of recurrent
disease. Thus, effort continues to be directed toward identifying new
prognosticators of tumor dissemination. Early diagnosis of patients
q 1997 American Cancer Society
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W: Cancer
CK20 Cells and Colorectal Carcinoma/Litle et al.
at increased risk of developing metastatic disease is
important to identify those who would benefit from
aggressive adjuvant therapy.
Rarely occurring epithelial cells in bone marrow
aspirates from patients with solid tumors are presumed to be micrometastases (i.e., cells associated
with disease progression). Epithelial cells in bone marrow aspirates, detected immunohistochemically using
antibodies directed against members of the cytokeratin (CK) family, are present in bone marrow aspirates
of approximately 12 – 35% of patients with colorectal
tumors, as well as other cancers.2 – 5 CK-labeled cells
are also detected, albeit at lower frequencies, in patients without cancer.6 Epithelial cells in bone marrow
aspirates from colorectal carcinoma patients at the
time of initial surgery are associated with increased
risk of recurrence and decreased disease free survival.6,7 The predictive value of micrometastases assays
to stratify patients into groups at increased risk of developing overt metastases is nonuniform. Approximately 40% of patients without detectable epithelial
cells in bone marrow aspirates will develop recurrent
disease at distant sites (false-negatives), and 30% of
patients with CK/ cells may not recur (false-positives).7,8 Typically, 105 – 106 bone marrow cells are
screened for the presence of CK/ cells. The underlying
basis for false-negatives and false-positives is poorly
understood. Nonspecific cell labeling with monoclonal
antibodies against CK epitopes that are not tumor specific may contribute to false-positive results.6 The majority of prognostic studies of epithelial cells in bone
marrow aspirates have utilized antibodies against CK8
and/or 18. However, it has been reported that CK8/18
epithelial cells may be present, albeit at low and variable frequencies (approximately 2 – 5 1 1006), in the
bone marrow of some noncancer patients.5,9,10 Identification of markers with increased specificity for colorectal tumor cells may improve the prognostic value
of micrometastases assays.
CK20, a member of the CK family, is expressed in
95% of primary and metastatic colorectal adenocarcinomas.11,12 The liver and lymph nodes do not contain
immunohistochemically-labeled CK20 cells12 nor is
CK20 mRNA detected in hematopoietic cells.13 Therefore, it was postulated that CK20 may be useful in
discriminating colorectal tumor cells that disseminate
to hematopoietic tissues, such as bone marrow. The
authors applied a combined immunophenotype and
genotype approach to detect and quantify low frequency CK20/ populations in bone marrow aspirates
and tumor specimens from colorectal carcinoma patients and to assess the molecular cytogenetic features
of discriminated subpopulations.14 Fluorescent in situ
hybridization (FISH; 15,16) was used to measure aneuso-
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1665
mies characteristic of colorectal tumors in bone marrow aspirates and resected tumors from discriminated
CK-20 labeled populations. Thus, CK20-labeled cells
that carry molecular cytogenetic aberrations are considered to be tumor cells.
MATERIALS AND METHODS
Tissues
Bone marrow aspirates and biopsies of primary colorectal tumor and/or gross hepatic metastasis were
taken after informed consent was obtained from 18
patients undergoing surgery for Dukes Stage C or D
colorectal carcinoma. Bone marrow (4 – 10 mL) was
aspirated from the posterior iliac crest after induction of anesthesia. Bone marrow mononuclear cells
were isolated using a Hypaque density gradient
(Sigma, St. Louis, MO). Approximately 104 low density cells were deposited onto ethanol-cleaned glass
slides using a Shandon cytocentrifuge (Shandon,
Pittsburgh, PA) and stored at 020 7C prior to immunostaining and FISH. Tumor biopsies (1 – 2 gm) were
obtained during surgery and placed in phosphatebuffered saline (PBS) (0.14 M NaCl, 2.7 mM KCl, 5.4
mM Na2HPO4 , and 1.8 mM KH2PO4 ; pH 7.0) on ice
prior to processing. Biopsies were sliced with a razor
blade and lightly touched (‘‘touch preps’’) onto ethanol-cleaned slides that were dried overnight at
room temperature (RT) and stored at 020 7C.
Immunostaining and Microscopy
Immunolabeling with anti-CK antibodies was performed after cell fixation. Briefly, bone marrow cells
were fixed in 0.5% formaldehyde in PBS for 60 minutes
at RT followed by fixation in Carnoy’s (methanol/acetic acid, 3:1 volume/volume; 020 7C). Tumor touch
preps were fixed only in methanol/acetone. Normal
goat serum (diluted 1:50 in PN buffer; 0.1 M monobasic sodium phosphate, 0.1 M dibasic sodium phosphate, and 0.1% Nonidet P-40, pH 8.0) was applied to
the fixed cells in a humidified chamber for 20 minutes
at RT to reduce nonspecific antibody binding. The
cells were then labeled with anti-CK20 (Ks 20.8; Dakopatts, Glosstrup, Denmark) (25 mg/104 cells) for 20
minutes at RT. After 2 5-minute washes with PN buffer,
cells were incubated with fluorescein isothiocyanate
(FITC)-conjugated goat antimouse immunoglobulin
(Ig)G (Caltag, South San Francisco, CA) (0.1 mg/104
cells) for 20 minutes at RT. After 2 5-minute washes
with PN buffer, cells were counterstained with 4*,6diamidino-2-phenylindole (DAPI) (Sigma) in antifade
solution (0.4 mg/mL).17 Some specimens were also labeled with antibodies that recognize CK8/18 (FITCCAM 5.2, Becton Dickinson, San Jose, CA) (0.0125 mg/
104 cells). A colon carcinoma cell line, Colo 205 (Ameri-
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CANCER May 1, 1997 / Volume 79 / Number 9
can Type Culture Collection # CCL 222, Rockville, MD),
cultured in RPMI media, was used as a positive control
for CK immunostaining.
CK positive (CK/) cells in bone marrow aspirates
and tumor touch preps were visualized using fluorescence microscopy. Approximately 105 cells (contained
on approximately 10 slides) per bone marrow aspirate
were screened for the presence of CK/ cells. The total
number of hematopoietic cells on each slide was estimated by averaging the number of cells in each of ten
fields and then tracking the number of fields needed
to scan the area of deposited cells. Between 280 – 2885
cells in the primary tumor were screened to quantify
CK20/ cells in primary tumors and hepatic metastases.
After the frequency of CK20/ cells was determined by
manual fluorescence microscopy, slides containing
CK20/ cells were subsequently analyzed using computerized image microscopy as previously described.14
The coordinates of the CK20/ cells, as well as an image
of the surrounding field (s), were recorded to facilitate
relocation of the CK/ cell after hybridization, which
often abrogated the CK-linked FITC signal.
FISH
FISH was performed as described by Pinkel et al.15
with modifications. Cells previously labeled with antiCK20 antibody were refixed onto the slide with Carnoy’s (10 minutes at RT) to reduce cell loss during
hybridization.14 The cells were treated with pepsin
(0.01 mg/mL) (Sigma) in 0.01 N HCl for 2 minutes at
37 7C to improve accessibility of DNA probes. After
cell dehydration in a 2-minute ethanol series (70%,
85%, and 100%), 10 mL of hybridization mixture (final
concentration of 55% formamide, 21 standard saline
citrate [SSC]; 0.3 M NaCl, and 0.03 M sodium citrate
[pH 7.0], and 10% dextran sulfate [pH 7.0], 1 ml H2O,
2 mg/mL of Texas Red-labeled chromosome 7 or 8,
and fluorescein-labeled chromosome 8 or 17 alpha
satellite probes [Oncor, Inc., Gaithersburg, MD]) was
applied to the cells on slides. Twenty ml of hybridization mixture was applied to tumor touch preps to ensure coverage of the specimen. The DNA probes and
interphase cells were denatured simultaneously by
placing the slides on the bottom of a metal box in a
73 7C water bath for 2 minutes. After overnight incubation at 37 7C, the slides were washed in 11 SSC at 73
7C, 21 SSC at 37 7C, 21 SSC at RT, and distilled water at
RT and then dehydrated in a 2-minute ethanol series.
Nuclei were counterstained with DAPI in antifade solution. Hybridization domains were enumerated18 in
the relocated cells that were labeled previously with
anti-CK antibodies.
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RESULTS
Patient/Specimen Characteristics
Bone marrow aspirates and tumor specimens were
collected from 11 patients with colon carcinoma and
7 patients with rectal carcinoma. The median age at
the time of surgery was 57 years (Table 1). Liver metastases were present in all patients except Patient 101
and Patient 115. None of the patients showed clinical
evidence of bone metastases. Carcinoembryonic antigen (CEA) levels at the time of diagnosis/surgery
ranged from 1 – 9100 ng/mL. Control bone marrow
specimens were obtained from 12 patients without colorectal carcinoma (data not shown).
CK20-Labeled Cells in Tumor Specimens
The frequency of CK20/ cells in primary and metastatic tumors was measured to confirm that the antiCK20 antibody labeled epithelial cells in colorectal adenocarcinomas. CK20-expressing cells were detected
in four of four primary tumors and six of six resected
hepatic metastases. CK20/-labeled cells showed weblike cytoplasmic structures that resembled the staining
patterns typically associated with the cytoskeleton matrix. The mean frequency (24%) of CK20/ cells varied
among primary tumors (range, 18 – 36%) (Table 2).
Similar levels (35% mean frequency) of CK20/ cells
and variation (range, 10 – 59%) were present in hepatic
metastases. CK20/ cells in the solid tumors also labeled with antibodies against CK8/18, indicating that
the labeled cells coexpressed multiple members of the
CK family.
Molecular cytogenetic analysis of epithelial cells
in the tumor specimens was used to determine
whether the CK20-expressing cells were cytogenetically aberrant. Six patients (four with colon carcinoma
and two with rectal carcinoma) were selected for combined immunophenotype/genotype analysis. CK20/
cells within individual tumor touch preps were identified microscopically and their locations recorded using
the image acquisition system. The tumor touch preps
were then hybridized with pairs of alpha satellite DNA
probes specific for chromosomes 1, 7, 8, and 17. After
relocalization, hybridization domains were enumerated in the CK20/ subpopulation. The frequency of
CK20/ cells with 1, 2, and ú3 hybridization domains
for chromosomes 7 and 17 are shown in Figures 1a
and 1b, respectively. Between 40 – 70% of the CK20labeled cells in the primary tumors and/or hepatic
metastases carried trisomies involving chromosomes
7 and 17. Heterogeneity in the molecular cytogenetic
profile of CK20-expressing cells is evident in CR21,
CR133, and CR115, in which approximately 50% of
the discriminated subpopulation carried normal copy
numbers of chromosomes 7 and 17. Trisomies involv-
W: Cancer
CK20 Cells and Colorectal Carcinoma/Litle et al.
1667
TABLE 1
Patient Characteristics
Patient
ID no.
23
108
150
153
151
155
146
133
116
103
21
20
123
124
144
101
127
115
Gender
Age
(yrs)
Dukes stage and
location of primary
tumor
CEA level
(ng/mL)
M
F
M
M
F
M
M
M
M
M
F
M
F
M
F
M
M
M
40
53
64
37
69
54
46
74
76
62
73
64
68
44
57
47
68
34
D sigmoid colona
D left colonb
D sigmoid colonb
D left colonb
D sigmoid colonb
D sigmoid colonb
D right colonb
D colonb
D rectosigmoidb
D right colona
D sigmoid colonb
D rectalb
D rectalb
D rectalb
D rectalb
D rectala
D rectala
C rectal
6
29
117
38
351
57
1
72
NA
268
9100
313
59
18
NA
1
NA
2
CEA: carcinoembryonic antigen; M: male; F: female; NA: not available.
a
Carcinomatosis.
b
Hepatic metastases only.
TABLE 2
CK20-Labeled Cells (%) in Solid Tumors
TABLE 3
Chromosome Specific Molecular Cytogenetic Analysis of CK20/ Cells
in Bone Marrow Aspirates
Patient ID no.
No. of cells
scored
Frequency (%)
CK20/ cells
115a
101a
21a
108a
108b
133b
23b
127b
21b
116b
2885
654
935
2809
737
908
1620
430
579
559
18
21
23
36
10
11
31
50
53
59
ing chromosomes 1 and 8 occurred in greater than
90% of the CK20/ cells in these same tumor specimens
(data not shown).
CK-20-Labeled Cells in Bone Marrow Aspirates
Detection and quantitation of CK20-labeled cells in
bone marrow aspirates were performed on aspirates
from patients with and without colorectal carcinoma.
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2 copies N
õ2 copies N
ú2 copies N
1
7
8
17
9
39
30
71
5
12
5
16
0
0
0
2
CK: cytokeratin; N: number of cytokeratin 20 positive cells with 2, õ2, or ú2 copies of each chromosome
in bone marrow aspirates from eight patients.
CK: cytokeratin.
a
Primary tumor.
b
Hepatic metastases.
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CK20/ cells were detected in bone marrow aspirates
of approximately 60% of patients (7 of 12) without
colorectal carcinoma at frequencies between 0.2 – 1.1
1 1004 (Fig. 2). CK20/ cells were detected in bone
marrow aspirates from 11 of 11 colon carcinoma patients and 5 of 7 rectal carcinoma patients. The CK20/
frequency (with the upper bound for the 95% confidence interval based on the number of cells scored)
in individual specimens is shown in Figure 2. Four of
11 patients with colon carcinoma carried bone marrow
CK20/ cells at levels that were significantly higher than
those detected in patients without colorectal carcinoma. As a group, bone marrow aspirates from patients with colon carcinoma contained significantly
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CANCER May 1, 1997 / Volume 79 / Number 9
FIGURE 1. Aneusomy analysis of cytokeratin 20-labeled subpopulations
in colorectal tumors. The number of hybridization domains for chromosomes 7 and 17 are shown in panels a and b, respectively. medium grey:
õ2 copies; light grey: 2 copies; black: ú2 copies.
more CK20/ cells (mean, 1.6 1 1004) than patients with
rectal carcinoma or those without colorectal carcinoma (Mann-Whitney U test, P õ 0.05). The CK20/
mean frequency (5 1 1005) in the rectal carcinoma
patients was similar to patients without colorectal carcinoma. The frequency of bone marrow CK20/ cells
was unrelated to patient gender or age, location or
grade of the primary tumor, or the presence of carcinomatosis. However, the frequency of CK20/ cells in
specimens that contained levels of CK20/ cells that
significantly exceeded those in control bone marrow
correlated with preoperative CEA levels (correlation
coefficient, 0.918). Selected bone marrow specimens
containing CK20/ cells were subsequently labeled with
antibodies specific for CK8 and CK18. Approximately
1 – 2 1 105 cells from each of 12 patients with colorectal
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carcinoma and 4 without cancer were analyzed. CK8/
18/ cells were not detected in these 16 bone marrow
specimens.
Morphologic evaluation of the CK20/ cells in bone
marrow aspirates from patients with and without colorectal carcinoma indicates a morphologically heterogeneous subpopulation with respect to size and nuclear to
cytoplasmic ratio. The FITC-linked fluorescence pattern
in individual cells was also variable, ranging from diffuse
cytoplasmic fluorescence to fluorescently labeled filamentous structures that appeared to resemble the cytoplasmic web often associated with CK labeling. There
were no discriminating morphologic or immunofluorescent features between CK20 labeled cells in bone marrow aspirates from patients with and without colorectal
carcinoma (data not shown).
Molecular cytogenetic analysis was used to determine whether the discriminated CK20-labeled cells in
bone marrow aspirates carried numerical chromosome abnormalities suggesting their derivation from
the primary tumor or hepatic metastases. Selection of
hybridization probes for chromosomes 1, 7, 8, and 17
was based on aneusomies involving these chromosomes in the CK20/ population in solid tumor specimens. Approximately 93% (101 of 109) of the CK20/
cells in the bone marrow carried 2 copies of chromosomes 1, 7, 8, or 17 (Table 3). One hybridization domain of these chromosomes was present in approximately 26% (26 of 101) of the CK20/ cells, suggestive
either of monosomy or inefficient hybridization. In
contrast to trisomies observed in CK20/ cells in the
tumor specimens (Fig. 1), only 2 of 109 CK20/ cells in
the bone marrow showed ú2 (e.g., 3 and 4) chromosome 17 hybridization domains. Approximately five to
ten CK20 negative cells that were present in the same
field as the CK20/ cells carried two copies of chromosome 7 or 17.
DISCUSSION
Although associations between the presence of epithelial cells in bone marrow aspirates and risk of recurrence and/or disease free survival have been established for colorectal and breast carcinoma in some
studies,6 – 8 false-positives and false-negatives continue
to limit the accuracy of micrometastases assays to predict recurrence. False-negatives are often attributed to
sampling errors, whereas the involvement of multiple
factors is postulated to contribute to false-positives.
For example, inability of the discriminated epithelial
cells to form metastases due to lack of viability or absence of genetic/cellular characteristics conducive to
their proliferation in a bone marrow microenvironment is of particular concern in colorectal carcinoma
patients, in whom clinical bony metastases are infre-
W: Cancer
CK20 Cells and Colorectal Carcinoma/Litle et al.
1669
FIGURE 2. Frequency of cytokeratin 20/ cells in bone marrow aspirates from patients with and
without colorectal carcinoma. The 95% confidence intervals are shown for each specimen. A sample
frequency is considered to differ significantly from baseline when the 95% lower bound is above
the upper bound (dashed line) for patients without colorectal carcinoma.
quent. Labeling of nontumor epithelial cells and/or
nonspecific labeling of hematopoietic cells are particularly problematic in rare event analyses,14 and in
some cases it has been suggested that CK18-labeled
cells may not derive from the tumor.6 CK20 shows
more restricted expression than CK8/18, and thus in
principle provides opportunities to improve the specificity of micrometastases detection.
Phenotype and genotype analyses of CK20/ cells
in primary tumors and hepatic metastases were performed to establish whether CK20 expressing cells in
colorectal tumors represent tumor cells. Immunophenotypic analyses of CK20 labeling of cells in specimens
of colorectal tumors indicate that all primary and metastatic tumors contain CK20/ cells at frequencies between 10 – 59%. Similar frequencies of CK20/ cells in
primary and metastatic colon adenocarcinomas were
estimated using immunohistochemical labeling.12 Aneusomies occur in greater than 70% of colorectal tumors19 – 21 and thus are useful molecular markers to
determine whether discriminated subpopulations are
likely to contain tumor cells. Combined immunophenotype/genotype analyses14 of CK20 cells hybridized
with chromosome specific DNA probes demonstrate
that 40 – 60% of CK20/ cells in 6 of 6 tumors are aneusomic for chromosomes 1, 7, 8, or 17, thereby confirming that they are tumor cells. CK20 cells that carry
normal copy numbers of the measured chromosomes
may either be normal epithelial cells or, alternatively,
tumor cells with molecular cytogenetic aberrations
undetected using the selected DNA probes.
Bone marrow aspirates from colorectal carcinoma
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patients contain CK20-labeled cells. The average level
of CK20/ cells in bone marrow aspirates from colorectal carcinoma patients is 1.2 1 1004 , which is approximately an order of magnitude higher than bone
marrow CK8/18-labeled cells in previous studies.7 Although CK20 and CK8/18 colabel subpopulations in
the solid tumor specimens, CK20 cells in bone marrow
aspirates did not coexpress CK8 or CK18. Furthermore,
CK20/ cells were observed in bone marrow aspirates
from patients without colorectal carcinoma. CK20/
cells occurred at levels above those found in patients
without colorectal carcinoma in only 4 of 18 patients
with colon carcinoma. CK20/ cells were not elevated in
bone marrow aspirates from rectal carcinoma patients
(Fig. 2). These data contrast with CK18-based detection of epithelial cells, which suggest that CK18/ bone
marrows are more prevalent among patients with rectal carcinoma than those with colon carcinoma.7
CK20-labeled cells are morphologically heterogeneous
and indistinguishable from surrounding hematopoietic cells. Similarly, CK18/ cells may resemble hematopoietic cells in some cases and malignant epithelial
cells in others.2,6 Although CK20-labeled cells were cytogenetically aberrant in primary tumors and hepatic
metastases, genotypic analyses of CK20 cells in bone
marrow aspirates indicate a normal complement of
chromosomes 1, 7, 8, and 18. Thus, it is unclear
whether CK20-labeled cells in bone marrow aspirates
derive from the primary tumor and/or hepatic metastases. Similarly, it is unknown whether CK18/ cells
in bone marrow aspirates carry molecular cytogenetic
abnormalities. Thus, although the frequency of CK20/
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CANCER May 1, 1997 / Volume 79 / Number 9
cells is elevated in bone marrow aspirates from 45%
of colon carcinoma patients, the likelihood that these
cells derive from the tumor is uncertain and suggests
caution is necessary in the use of CK20 to discriminate
micrometastases in bone marrow.
10.
11.
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