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NEOPLASIA
Brief report
Bmi-1 is useful as a novel molecular marker for predicting progression
of myelodysplastic syndrome and patient prognosis
Keichiro Mihara, Moniruddin Chowdhury, Nanae Nakaju, Sachiko Hidani, Akihiro Ihara, Hideo Hyodo,
Shin’ichiro Yasunaga, Yoshihiro Takihara, and Akiro Kimura
The International Prognostic Scoring System (IPSS) has been widely used to predict the prognosis of patients with myelodysplastic syndrome (MDS). However,
IPSS does not always provide a sufficiently precise evaluation of patients to
allow the appropriate choice of clinical
interventions. Here, we analyzed the expression of Bmi-1, which is required to
regulate the self-renewal in CD34ⴙ cells
from 51 patients with cases of MDS and
acute myeloid leukemia preceded by MDS
(MDS-AML). Higher positivity rate of Bmi-1
was preferentially seen in refractory anemia with excess blasts (RAEB), RAEB in
transformation (RAEB-T), and MDS-AML
compared with refractory anemia (RA)
and RA with ringed sideroblasts (RARS).
IPSS score was positively correlated with
the percentage of Bmi-1 expression. Pa-
tients with RA and RARS with a higher
percentage of Bmi-1ⴙ cells showed disease progression to RAEB. Here, we propose Bmi-1 as a novel molecular marker
to predict the progression and prognosis
of MDS. (Blood. 2006;107:305-308)
© 2006 by The American Society of Hematology
Introduction
Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic disorders characterized by aberrant hematopoiesis and dysplasia.1,2 Because the disease entity includes quite heterogeneous
pathogeneses, the clinical course and prognosis are highly variable.
The International Prognostic Scoring System (IPSS) has achieved
international acceptance to estimate prognosis in patients with
MDS.3,4 However, IPSS score is not necessarily enough to evaluate
the patient for the purpose of choosing intervention therapies. It
would be helpful for the clinical treatment of patients with MDS to
establish molecular markers that clearly reflect the disease
progression.
Because the most critical event for the progression of MDS and
prognosis of patients with MDS is susceptibility to acute leukemia,
it is necessary to monitor the appearance and increase of leukemic
stem cells (LSCs) in patients with MDS. Thus, we focused on
molecular mechanisms supporting LSCs. Bmi1 is a member of the
Polycomb group of transcriptional repressor genes, which may be
expressed restrictedly in stem cells and progenitors.5,6 Some studies
have demonstrated that Bmi-1 is required to regulate the adult
self-renewing hematopoietic and LSCs.7-12 Others have shown that
overexpression of the Bmi-1 gene could cause neoplastic proliferation of cells.13-16 A number of reports on Bmi-1 provide perspectives on the close association of its expression with the progression
of hematopoietic malignancies.17-21
In this study, we therefore examined the positivity of Bmi-1
expression in CD34⫹ cells from patients with MDS by flow
cytometry to test whether Bmi-1 would be a novel biomarker
that is well correlated with the disease progression and prognosis of the patients.
From the Department of Hematology and Oncology, Research Institute for
Radiation Biology and Medicine, Hiroshima University, Hiroshima; the
Department of Internal Medicine, National Kure Medical Center, Kure; and the
Department of Stem Cell Biology, Research Institute for Radiation Biology and
Medicine, Hiroshima University, Hiroshima, Japan.
Reprints: Keichiro Mihara, Department of Hematology and Oncology,
Research Institute for Radiation Biology and Medicine, Hiroshima University,
1-2-3 Kasumi, Minami-ku, Hiroshima, Japan 734-8553; e-mail: kmmihara@
hiroshima-u.ac.jp.
Submitted June 15, 2005; accepted August 18, 2005. Prepublished online as
Blood First Edition Paper, September 20, 2005; DOI 10.1182/blood-2005-062393.
BLOOD, 1 JANUARY 2006 䡠 VOLUME 107, NUMBER 1
Study design
Cells
Four acute myeloid leukemia (AML) cell lines (KG1, HL60, HEL, and
U937) were obtained from American Type Culture Collection (ATCC;
Manassas, VA). Mono7 originated from MDS was available in our
laboratory. Normal T cells were obtained from healthy donors and
cultured in RPMI-1640 complete medium in the presence of interleukin-2
and/or PHA.
Patients
We studied bone marrow samples obtained from 50 patients with newly
diagnosed MDS or MDS-AML (case 19 occurred in the same patient as
case 27, which developed into refractory anemia with excess blasts
[RAEB]). We used 10 bone marrow samples from healthy donors and from
patients with renal anemia, iron deficiency anemia, and lymphoma without
bone marrow involvement as controls. The patients with refractory anemia
(RA) and RA with ringed sideroblasts (RARS) received supportive care
such as blood-cell transfusion and low-intensity therapy, including granulocyte colony-stimulating factor (G-CSF), erythropoietin, and cyclosporine.
The patients with RAEB, RAEB in transformation (RAEB-T), and MDSAML received high-intensity therapy, including chemotherapy and bone
The publication costs of this article were defrayed in part by page charge
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© 2006 by The American Society of Hematology
305
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306
BLOOD, 1 JANUARY 2006 䡠 VOLUME 107, NUMBER 1
MIHARA et al
marrow transplantation. Informed consent was obtained from these patients
and donors. An independent pathologist confirmed the diagnosis.
Immunoblot analysis and flow cytometric assessment
Immunoblot analysis using anti–Bmi-1 monoclonal antibody (Upstate Cell
Signaling Solutions, Lake Placid, NY) was performed. The immunoblot results
demonstrated that Bmi-1 was highly expressed in 5 AML cell lines. For flow
cytometric analysis, cells were stained with anti-CD34 antibody-PE (BD
Biosciences, San Jose, CA). After fixation with 3% paraformaldehyde in PBS
supplemented with 5% bovine serum albumin, the cells were incubated with
anti–Bmi-1 monoclonal antibody. They were incubated with goat anti–mouse
IgG antibody-FITC (BD Biosciences). We confirmed that the Bmi-1 expression
of all cell lines was detected by flow cytometric analysis.
Table 1. Characteristics of patients with MDS and MDS-AML
Case
FAB
Sex
Age (y)
Outcome
Follow-up (mo)
Blasts (%)
Karyotype
IPSS
Risk
Bmi-1 (%)
1
RA
M
62
Alive
8
1.0
Normal
0
Low
1.53
2
RA
F
60
Alive
8
3.5
Normal
0
Low
4.80
3
RA
F
56
Alive
8
0.5
Normal
0
Low
0.45
4
RA
F
38
Alive
7
1.5
Normal
0
Low
7.58
5
RA
F
63
Alive
8
3.5
Normal
0
Low
8.88
6
RA*
M
75
Alive
21
1.5
Normal
0
Low
23.00
8.39
7
RA
F
66
Alive
22
2.0
46, XX, add(5)(q13)
0.5
Int-1
8
RA
F
56
Alive
19
1.0
Normal
0.5
Int-1
7.50
9
RA
M
51
Alive
48
2.0
Normal
0.5
Int-1
1.36
10
RA*
M
61
Dead
10
2.5
46, XY, add(13)(q14)
0.5
Int-1
25.84
11
RA*
F
80
Alive
8
1.0
Normal
0.5
Int-1
16.04
12
RA
M
63
Alive
27
0.5
46, XY, ⫹1, der(1;7)(q10;p10)
1
Int-1
8.33
13
RA
M
81
Dead
12
3.5
47, XY, ⫹8
1
Int-1
0.70
14
RA
M
75
Alive
5
2.5
47, XY, ⫹14
1
Int-1
0.31
15
RARS
F
47
Alive
22
1.5
Normal
0
Low
5.18
16
RARS
F
67
Alive
108
1.5
Normal
0.5
Int-1
0.10
17
RARS
M
96
Alive
4
2.5
Normal
0.5
Int-1
1.24
18
RARS*
F
75
Alive
22
3.5
Normal
0.5
Int-1
13.15
19
RARS*
M
72
Dead
10
2.0
46, XY, del(5)(q15q33),
1
Int-1
63.44
del(11)(q21)
20
RAEB
M
78
Alive
7
8.5
Normal
1
Int-1
48.12
21
RAEB
M
80
Dead
7
5.0
Complex
1.5
Int-2
59.25
22
RAEB
M
73
Dead
4
7.5
Complex
2
Int-2
60.80
23
RAEB
M
66
Dead
2
7.5
Complex
2
Int-2
62.86
24
RAEB
F
66
Dead
6
8.5
Complex
2
Int-2
78.51
25
RAEB
F
49
Alive
28
18.5
Normal
2
Int-2
61.88
26
RAEB
M
81
Dead
24
13.0
Normal
2
Int-2
53.00
27
RAEB
M
72
Dead
6
18.5
Complex
2.5
High
69.06
28
RAEB
M
69
Dead
2
11.0
Complex
3
High
60.28
29
RAEB
F
93
Dead
6
14.0
Complex
3
High
36.60
30
RAEB
M
63
Dead
2
11.0
46, XY, ⫺7
3
High
31.52
31
RAEB
M
55
Dead
1
11.5
Complex
3
High
40.34
32
RAEB-T
M
78
Alive
18
20.5
Normal
2
Int-2
7.58
33
RAEB-T
M
70
Dead
10
23.0
46, XY, add(21)(q22)
2.5
High
73.96
34
RAEB-T
M
81
Dead
2
29.5
45, X, ⫺Y, t(8;21)(q22;q22)
3
High
71.94
35
RAEB-T
M
74
Dead
1
20.0
Complex
3
High
83.80
36
RAEB-T
M
74
Dead
11
21.0
45, XY, add(3)(q13.2), ⫺7
3.5
High
88.81
37
RAEB-T
M
74
Dead
5
25.0
Complex
3.5
High
73.98
38
RAEB-T
F
81
Dead
5
20.5
Complex
3.5
High
63.96
39
RAEB-T
M
71
Dead
8
20.5
Complex
3.5
High
99.59
40
MDS-AML
M
79
Dead
18
35.0
Normal
NA
NA
74.31
41
MDS-AML
F
65
Dead
13
47.0
45, XX, add(5)(q22),
NA
NA
99.99
add(7)(p11.2), ⫺22
42
MDS-AML
F
76
Dead
17
34.0
Complex
NA
NA
86.11
43
MDS-AML
M
68
Dead
4
39.0
Complex
NA
NA
97.04
44
MDS-AML
F
70
Dead
5
61.0
46, XY, add(8)(q22), ⫺21, ⫹mar
NA
NA
90.44
45
MDS-AML
M
77
Dead
17
74.5
47XY, ⫹8
NA
NA
82.52
46
MDS-AML
M
81
Dead
6
35.0
Complex
NA
NA
99.00
47
MDS-AML
M
75
Dead
4
45.0
Complex
NA
NA
82.13
48
MDS-AML
F
60
Dead
20
30.5
46, XX, del(6)(q?)
NA
NA
100.00
49
MDS-AML
M
64
Dead
5
83.0
46, XY
NA
NA
76.17
50
MDS-AML
M
59
Dead
1
85.5
Complex
NA
NA
93.84
51
MDS-AML
M
65
Dead
3
49.0
Complex
NA
NA
82.55
Bmi-1 (%) denotes the positivity of Bmi-1 in
which developed into RAEB.
NA indicates not applicable.
*Progression to RAEB from RA or RARS.
CD34⫹
cells; Complex denotes more than 3 chromosomal abnormalities. Case 19 occurs in the same individual as case 27,
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BLOOD, 1 JANUARY 2006 䡠 VOLUME 107, NUMBER 1
BMI-1 PREDICTS PROGRESSION AND PROGNOSIS OF MDS
307
Statistical analysis
The correlation between the positivity of Bmi-1 expression of CD34⫹ cells
and IPSS was evaluated by the Spearman correlation coefficient. We used
the Fisher protected least significant difference (Fisher PLSD) to evaluate
differences in Bmi-1 expression ratio of CD34⫹ cells according to
French-American-British (FAB) classification. We then used the Cox
proportional hazards model to evaluate the correlation of prognosis with the
percentage of Bmi-1 expression. P values less than .05 were considered
statistically significant. These studies were approved by the Hiroshima
University institutional review board for these studies.
Results and discussion
Bmi-1 protein is related to cell proliferation
Bmi-1 is known to play a role for sustaining self-renewing cell
activity by repressing the INK4A locus encoding p16INK4A and
p19ARF, which are capable of inducing growth arrest, cellular
senescence, and apoptosis.22-24 In this context, to determine whether
cells expressing Bmi-1 proliferate, a BrdU-labeling assay was
performed. Normal proliferating T cells, which incorporated BrdU,
were predominantly stained with anti–Bmi-1 antibody. Furthermore, Bmi-1 positivity was also higher in CD34⫹ cells mobilized
with G-CSF than for controls (data not shown). Thus, it appears
that Bmi-1 protein not only sustains self-renewing stem cells, but it
also plays an important role in providing cells the potential for
proliferation.
Bmi-1 expression in CD34ⴙ cells is high in RAEB, RAEB-T, and
MDS-AML, and is correlated with IPSS score
Because CD34⫹ cells, in which LSCs are supposed to be enriched,
have an important role in leukemogenesis, by applying flow
cytometric analysis, we examined the percentage of Bmi-1 expression in CD34⫹ bone marrow cells from 51 patients with cases of
MDS and MDS-AML, whose profiles are summarized in Table 1.
Representative results of flow cytometric patterns are shown in
Figure 1A. Higher positivity rate of Bmi-1 was preferentially seen
in RAEB, RAEB-T, and MDS-AML compared with RA and
RARS. The percentages of Bmi-1⫹ cells in CD34⫹ cells in RA and
RARS were almost the same as that in controls (Figure 1B).
Interestingly, the positivity of Bmi-1 expression at diagnosis was
significantly higher in the patients with progression to RAEB
(cases 6, 10, 11, 18, and 19) (28.29% ⫾ 20.30%) compared with
those without progression (4.03% ⫾ 3.53%) (P ⬍ .001). Especially, even though the patient with case 19, whose percentage of
Bmi-1⫹ cells was as high as 63.44% at diagnosis, had IPSS score of
1.0, the disease developed into RAEB in 3 months. However, the
patient with case 32 with RAEB-T, had IPSS score of 2.0 and
Bmi-1 positivity of about 7.58%, and has remained alive for more
than 18 months after diagnosis without chemotherapy. Individuals
with cases 20 to 31 with RAEB have died, except for the patient
with case 25, who received successful allogeneic bone marrow
transplantation, and the patient with case 20, who has been
observed for just 7 months. Furthermore, Bmi-1 positivity in
CD34⫹ cells was positively correlated with IPSS score (Figure 1C)
(P ⬍ .001). Within a given IPSS group, cases of RA and RARS that
progressed to RAEB showed the highest percentage of Bmi-1.
Bmi-1 positivity in CD34⫹ cells independently seemed to predict
the risk of mortality (hazard ratio, 1.025; P ⬍ .001). We then
Figure 1. Expression of Bmi-1 in CD34ⴙ cells from MDS and MDS-AML.
(A) Representative expression of Bmi-1 in bone marrow mononuclear cells from
patients with RA, RAEB, and MDS-AML by flow cytometry is shown. The positivity of
Bmi-1 in CD34⫹ cells was highest in the patient with case 44 with MDS-AML and also
high in the patient with case 23 with RAEB. It was lowest in the patient with case 7
with RA. (B) The percentage of Bmi-1 expression in CD34⫹ cells according to MDS
subtype is as follows: RA, 8.19% ⫾ 8.20%; RARS, 16.62% ⫾ 26.67%; RAEB,
55.27% ⫾ 13.79%; RAEB-T, 70.45% ⫾ 27.74%; MDS-AML, 88.68% ⫾ 9.29%; and
controls, 4.02% ⫾ 3.89%. Data are expressed as percentage of mean ⫾ SD of
results obtained. *Progression to RAEB from RA or RARS. (C) Bmi-1 expression ratio
in CD34⫹ cells was correlated with IPSS score in patients with MDS. The R
value ⫽ 0.809 and P ⬍ .001 obtained indicates a good correlation between the 2
parameters (Bmi-1 [%] and IPSS score) considered. *Progression to RAEB from RA
or RARS.
analyzed whether Bmi-1 is expressed more strongly in the stemcell subpopulations among CD34⫹ cells, which are supposed to be
heterogeneous. However, there was no significant difference in the
positivity of Bmi-1 expression between hematopoietic stem cells
(CD34⫹CD38⫺ cells)25 and progenitors (CD34⫹CD38⫹ cells) in
MDS and MDS-AML. Further phenotypic analyses did not reveal
any correlation of Bmi-1 positivity in CD34⫹ cells in MDS and
MDS-AML with other maturation markers, including CD13,
CD33, or CD7 (data not shown). These results suggest that
increased Bmi-1 expression could reflect qualitative difference of
CD34⫹ cells. Although heterogeneity in study population of
patients with MDS has prevented us from predicting the disease
progression and prognosis, it might be possible to stratify patients
to estimate them according to Bmi-1 expression in CD34⫹ cells.
The detection and follow-up of this marker might also tell us when
to initiate chemotherapy and/or stem-cell transplantation. Because
our study suggested that Bmi-1 expression is useful for a molecular
marker to predict progression and prognosis of MDS, the prospective study with a large group of patients is required to further
evaluate Bmi-1 as a molecular maker for MDS.
Acknowledgments
We thank M. Kuroda and R. Matsumoto for their technical support.
We also thank Dr H Harada for his help in collecting patient
samples.
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308
BLOOD, 1 JANUARY 2006 䡠 VOLUME 107, NUMBER 1
MIHARA et al
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2006 107: 305-308
doi:10.1182/blood-2005-06-2393 originally published online
September 13, 2005
Bmi-1 is useful as a novel molecular marker for predicting progression
of myelodysplastic syndrome and patient prognosis
Keichiro Mihara, Moniruddin Chowdhury, Nanae Nakaju, Sachiko Hidani, Akihiro Ihara, Hideo Hyodo,
Shin'ichiro Yasunaga, Yoshihiro Takihara and Akiro Kimura
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