Download Expression Pattern and Clinical Prognostic

Jpn J Clin Oncol 2009;39(10)625 – 631
doi:10.1093/jjco/hyp094
Original Articles
Expression Pattern and Clinical Prognostic Relevance of Bone
Morphogenetic Protein-2 in Human Gliomas
Ce Liu1,†, Gang Tian2,†, Yanyang Tu3, Jianfang Fu4, Chuan Lan2 and Nan Wu2
1
Department of Neurosurgery, The Second Affiliated Hospital of the General Hospital of the People’s Liberation
Army, Beijing, 2Department of Neurosurgery, Southwest Hospital, The Third Military Medical University, Chongqing,
3
Department of Neurosurgery, 63870 Hospital of the People’s Liberation Army and 4Department of Endocrine
Secretion, Xijing Hospital of the Fourth Military Medical University, Shanxi, PR China
Received April 27, 2009; accepted July 13, 2009
Objective: Bone morphogenetic protein-2 (BMP-2) is normally expressed in the embryo promoting the development of several organs. Aberrant expression of BMP-2 occurs in various
tumors. However, a correlation between BMP-2 expression in human gliomas and patients’
prognosis has not been reported. To address this question, this study was to investigate the
BMP-2 expression pattern in human gliomas and to evaluate its prognostic relevance.
Methods: We analyzed the expression of the BMP-2 antigen in a series of 98 gliomas of
various grade and histology by immunohistochemistry on paraffin-embedded sections. Then,
the correlation of BMP-2 expression pattern with clinical–pathological features of patients and
its prognostic relevance were determined.
Results: Immunohistochemical analysis with anti-BMP-2 antibody revealed dense and spotty
staining in the tumor cells and its expression levels became significantly higher as the
gliomas’ grade advanced (P , 0.001). The median survival of patients with intensively positive BMP-2 expression was significantly shorter than that with negative expression (318 vs.
1197 days, P , 0.0001). The Kaplan – Meier survival curves showed that the BMP-2
expression was not only a significant predictor of survival in high-grade gliomas (grade IV,
P ¼ 0.02), but also in lower-grade gliomas (grades II and III, P , 0.001).
Conclusions: These results indicate that BMP-2 is a highly sensitive marker for gliomas
prognosis and suggest that the expression level of BMP-2 may be a potent tool for the clinical
prognosis of gliomas patients.
Key words: bone morphogenetic protein-2 – glioma – prognosis
INTRODUCTION
Gliomas, the most common primary malignant brain tumors,
comprise approximately one-third of intrinsic neoplasms of
the central nervous system (CNS) in both adults and children
(1). Gliomas are divided into four stages, or grades, in
increasing level of malignancy according to the World
Health Organization (WHO) classification scheme (2).
For reprints and all correspondence: Nan Wu, Department of Neurosurgery,
Southwest Hospital, The Third Military Medical University, Chongqing
400038, PR China. E-mail: [email protected]
†
Contribute equally to this work.
Grades I and II are the least malignant phenotypes, whereas
grades III (anaplastic astrocytoma, anaplastic oligoastrocytoma and anaplastic oligodendroglioma) and IV (glioblastoma multiforme, GBM) constitute the most malignant as
well as the most reported glioma histologies. The survival of
patients with gliomas is closely related to WHO tumor
grade. GBM is the most aggressive gliomas in adults (3).
Unfortunately, advances in surgery, radiotherapy and chemotherapy do only have a minor effect on the natural course
of this tumor with a mean overall survival time of only 9 –
15 months from the time of diagnosis (4). Therefore, there is
an urgent need for more effective therapeutic approaches
# The Author (2009). Published by Oxford University Press. All rights reserved.
626
Over-expression of BMP-2 in human gliomas
based on a better understanding of the pathophysiologic and
molecular properties of gliomas.
Bone morphogenetic proteins (BMPs), members of the
transforming growth factor-b (TGF-b) superfamily, play an
important role in embryonic development events, such as
neurogenesis, apoptosis, gastrulation and hematopoiesis (5).
It has been demonstrated that some BMPs are implicated in
the development of several cancers (6 – 8). BMP-2 was originally described because it induces the entire cascade of
endochondral bone formation (9). It is required for normal
embryonic development. BMP-2 function as morphogens
required for the development of the lung, heart, digits, limbs,
CNS and epidermis (10). It enhances cell proliferation,
stimulates cell survival pathways and induces self-renewal of
stem cells during the development. Interestingly, BMP-2 has
been demonstrated to play different roles on cancer cells
dependent on the tissue and environment where it is
expressed. For example (11), BMP-2 has been shown to
stimulate the growth of pancreatic carcinoma cells and prostate cancer cells in the absence of androgen. On the other
hand, BMP-2 clearly inhibits the growth of tumor cells of
many origins including cancers arising from thyroid,
androgen-dependent prostate in the presence of androgen,
myeloma, gastric and pancreatic cells. It has also been
reported that BMP-2 is over-expressed in 98% of lung carcinomas (12). However, the biological role of BMP-2 in
human gliomas has not been elucidated. To address this
question, this study was to investigate the BMP-2 expression
pattern in human gliomas and to evaluate its prognostic
relevance.
PATIENTS AND METHODS
PATIENTS
AND
TISSUE SAMPLES
Our study was approved by the Ethics Committee of the
second affiliated hospital of the General Hospital of the
People’s Liberation Army, Beijing, PR China. Ninety-eight
Chinese patients with glioma, whose detailed clinical and
pathological information was available, were selected from
the second affiliated hospital of the General Hospital of the
People’s Liberation Army from 11th January 1996 to 7th
July 2005. There were 68 males and 30 females (2.267:1),
with a mean age of 48.17 years (range from 9 to 76 years).
The clinical characteristics of these patients are listed in
Table 1. All lesions were classified as primary glioma
because the onset of disease was ,3 months before diagnosis and there was no prior history of malignant astrocytoma.
Patient characteristics, including the Karnofsky performance
scale (KPS) score, were collected before initial surgery.
Surgical resection, chemotherapy and radiotherapy were
attempted in all patients. Surgically resected tissues were
paraffin-embedded, sectioned at 4 mm thickness and used for
histochemical and immunohistochemical staining. Two or
more pathologists and neurosurgeons classified their grades
according to the WHO guidelines. Ten normal brain tissues
were used as control samples for the immunohistochemical
analysis.
All patients were given a follow-up investigation. Total
survival time was defined as the time interval between initial
craniotomy and the day of the patient’s death. All the
patients died of other diseases but not gliomas or unexpected
events were excluded from the case collection.
IMMUNOHISTOCHEMICAL STAINING
The expression levels of BMP-2 in glioma tissues were analyzed by immunohistochemical staining. Tissues were fixed
in 10% buffered formalin and embedded in paraffin.
Commercially available monoclonal antibody to BMP-2
(Santa CruzTM , USA) was used. Immunohistochemical staining was carried out using the avidin – biotin method and a
commercially available kit (Vectastain Elite ABC kit, Vector
Laboratories, Burlingame, CA, USA). Deparaffinized
Table 1. Clinical characteristics of 98 patients with gliomas included in the population-based immunohistochemical staining
WHO grades No. of patients Gender
Histology No. of patients Mean age (years) Tumor location
Male Female
Grade II
17
10
7
Frontal Non-frontal
Grade IV
Total
35
46
98
20
38
68
15
8
30
Median survival (days)
70
,70
A
4
45.21
3
1
3
1
1292 (240– 2002)
OA
7
47.98
4
3
6
1
1287 (241– 1998)
O
Grade III
KPS
6
46.19
3
3
6
0
1298 (240– 2003)
AA
13
49.75
6
7
11
2
372 (33–1538)
AOA
11
50.99
4
7
9
2
370 (35–1539)
AO
11
50.02
5
6
10
1
375 (33–1531)
pGBM
40
48.86
16
24
15
25
330 (32–1890)
sGBM
6
49.08
2
4
4
2
327 (29–1886)
98
48.98
43
55
64
34
396 (39–1927)
KPS, Karnofsky performance scale; A, astrocytoma; OA, oligoastrocytoma; O, oligodendroglioma; AA, anaplastic astrocytoma; AOA, anaplastic
oligoastrocytoma; AO, anaplastic oligodendroglioma; pGBM, primary glioblastoma; sGBM, secondary glioblastoma.
Jpn J Clin Oncol 2009;39(10)
sections were treated with methanol containing 3% hydrogen
peroxide for 10 min before conducting antigen retrieval
using a microwave oven at 958C for 5 min and cooling at
258C for 2 h. After washing with phosphate-buffered saline
(PBS), blocking serum was applied for 10 min. The sections
were incubated with an anti-BMP-2 monoclonal antibody
(1:500) overnight at 48C. Negative control sections were
incubated with PBS instead of the primary antibody. Then,
the section was incubated with a secondary biotinylated antibody (DakoCytomation Inc.) followed by incubation with a
streptavidin – peroxidase complex (DakoCytomation Inc.) for
10 min at room temperature. Reaction products were developed using diaminobenzidine containing 0.3% H 2 O 2 as a
substrate for peroxidase and nuclei were counterstained with
diluted hematoxylin. Positive and negative immunohistochemistry controls were routinely used. Reproducibility of
staining was confirmed by reimmunostaining via the same
method in multiple, randomly selected specimens.
The score of BMP-2 immunoreactivitiy in tissue sections
was evaluated according to the intensity of staining (value of
0 for absence, 1 for weak, 2 for moderate, 3 for high and 4
for very high intensity). Each section was independently analyzed in a blind study by two independent observers.
STATISTICAL ANALYSIS
The software of SPSS version 13.0 for Windows (SPSS Inc.,
IL, USA) and SAS 9.1 (SAS Institute, Cary, NC, USA) was
used for statistical analysis. Continuous variables were
expressed as X̄ + s. Statistical analysis were performed with
Fisher’s exact test for any 2 2 tables’ Pearson x 2 test for
non-2 2 tables, x 2 trend test for ordinal datum, Kaplan –
Meier and the Cox regression analysis for the question of
survival analysis. A difference between means was considered significant if the P value was ,0.05.
RESULTS
EXPRESSION
OF
BMP-2
IN
HUMAN GLIOMA TISSUE
The expression and location of BMP-2 in 98 patients of
primary gliomas were examined using immunostaining
analysis. The positive expression rate of BMP-2 (83/98,
84.7%) in patients with gliomas were higher than those in
normal brain tissues (both 1/10, 10%) significantly (P ,
0.001, Table 2). BMP-2 expression occurred mainly on the
cytoplasm, which is similar to the results from previous
Table 2. Expression of bone morphogenetic protein-2 (BMP-2) in human
gliomas and normal brain tissues
Groups
No. of patients 0 (n, %) 1þ to 2þ (%) 3þ to 4þ (%) P value
Gliomas 98
Normal 10
15 (15.3) 56 (57.1)
9 (90.0)
1 (10%)
27 (27.6)
0 (0)
,0.001
627
studies (13,14). Representative pictures of immunohistochemistry staining of BMP-2 are shown in Fig. 1.
CORRELATION OF BMP-2 EXPRESSION WITH
GRADING OF HUMAN GLIOMAS
THE
CLINICAL
Table 3 summarizes the substantial differences in BMP-2
expression between tumors of different histologic classifications. In the majority of low-grade tumors (WHO grade
II), BMP-2 was either not detectable (5 of 17, 33.3%) or
expressed only in 1þ to 2þ intensity (12 of 17, 66.7%).
With progression to anaplastic gliomas (WHO grade III) and
GBM (WHO grade IV), the percentage of BMP-2-negative
tumors were only 22.9% (8 of 35) and 4.4% (2 of 46),
whereas the proportion of tumors in which BMP-2 was
detectable in 3þ to 4þ intensity were 14.3% (5 of 35,
WHO grade III) and 47.8% (22 of 46, WHO grade IV).
However, there was no significant difference in BMP-2
expression between tumors of different histologic classifications in the same WHO grade (Grade II – IV: P ¼ 0.32,
0.39 and 0.16, respectively).
Fisher’s exact test or x 2 test (Table 4) showed no significant statistical association of BMP-2 expression pattern with
age, gender and tumor location (all P . 0.05), indicating
that these features might not affect the expression of BMP-2.
A significant association of BMP-2 expression pattern with
histologic grade and KPS score was observed (P , 0.001
and P ¼ 0.006, respectively). This indicated that patients
with GBM (grade IV) and a lower KPS score tend to express
a high level of BMP-2.
PROGNOSTIC RELEVANCE
GLIOMAS
OF
BMP-2 EXPRESSION
IN
HUMAN
Univariate analyses of each factor with the Cox log-rank
analysis (Table 5) show that the histologic grade, KPS score
and BMP-2 expression were significantly associated with
prognosis. Among them, BMP-2 expression was the most
significant (P , 0.0001). The median survival of patients
with a high BMP-2 expression level was significantly shorter
than those with a low BMP-2 expression level (318 vs. 1197
days, P , 0.0001, Table 5 and Fig. 2A), whereas the BMP-2
expression pattern varied widely in each grade as shown in
Table 4. The Kaplan – Meier survival curves (Fig. 2B and C)
showed that the BMP-2 expression was not only a significant
predictor of survival in high-grade glioma (grade IV, P ¼
0.0002), but also in lower-grade glioma (grades II and III,
P ¼ 0.003). In multivariate analysis, the histologic grade,
KPS score and BMP-2 expression were significant predictors
of survival (Table 6). These results are consistent with the
previous research on other tumors and suggest that
the BMP-2 expression, which is inversely correlated with the
survival of patients with gliomas, will be a powerful tool for
clinical prognosis.
628
Over-expression of BMP-2 in human gliomas
Figure 1. Immunohistochemical analysis for anti-human bone morphogenetic protein-2 (BMP-2) antibody. Paraffin-embedded sections of representative
gliomas and normal brain tissues were stained with the antibody against human BMP-2. The photograph (A) is a normal brain tissue which showed negative
or weak staining for BMP-2. In contrast, the gliomas that have moderate and high expression levels of BMP-2 with dense and spotty staining are shown in (B)
and (C), respectively. The photograph (D) shows the negative staining for non-specific antibody.
Table 3. BMP-2 expression in human glioma tissues with histologic classification
WHO grades
No. of patients
Histology
No. of patients
BMP-2 (n, %)
0
Grade II
17
Grade IV
35
46
1þ to 2þ
3þ to 4þ
A
4
1 (25.0)
3 (75.0)
0 (0)
OA
7
2 (28.6)
5 (72.4)
0 (0)
O
Grade III
P value
6
2 (33.3)
5 (67.7)
0 (0)
AA
13
4 (30.8)
8 (61.5)
1 (7.7)
AOA
11
2 (18.2)
7 (63.6)
2 (18.2)
AO
11
2 (18.2)
7 (63.6)
2 (18.2)
pGBM
40
2 (5.0)
20 (50.0)
18 (45.0)
sGBM
6
2 (33.3)
4 (66.7)
0 (0)
0.32
0.39
0.16
BMP-2, bone morphogenetic protein-2.
DISCUSSION
Because patients with similar stages of glioma showed a
marked discrepancy in survival, the standard histological
methods do not precisely predict which tumors will undergo
rapid malignant progression and are difficult to give an accurate prognosis to patients. Several new molecular prognostic
factors are being evaluated in the hope that they may
contribute to better assessment of the survival probability
and, consequently, the tailoring of treatment for each individual patient. In this study, we attempted to clarify the role
of BMP-2 in human gliomas. We found that many cells in
all grades of glioma stained positive for BMP-2 (Table 2 and
Fig. 1). The immunohistological determination of BMP-2
expression alone was strongly associated with the prognosis
Jpn J Clin Oncol 2009;39(10)
629
Table 4. BMP-2 expression in human glioma tissues with different
clinical–pathological features
Clinical–
pathological
features
No. of
patients
BMP-2 (n, %)
0
P value
1þ to 2þ 3þ to 4þ
Histologic classification (WHO)
Grade II
17
5 (33.3) 12 (66.7)
0 (0)
Grade III
35
8 (22.9) 22 (62.9)
5 (14.3)
Grade IV
46
2 (4.4)
22 (47.8)
22 (47.8)
,55
40
6 (15.0) 23 (57.5)
11 (27.5)
55
58
9 (15.5) 33 (56.9)
16 (27.6)
Male
68
10 (14.7) 40 (58.8)
18 (26.5)
Female
30
5 (16.7) 16 (53.3)
9 (30.0)
70
64
10 (15.6) 42 (65.6)
12 (18.8)
,70
34
5 (14.7) 14 (41.2)
15 (44.1)
Frontal
43
6 (14.0) 23 (53.5)
14 (32.6)
Non-frontal
55
9 (16.4) 33 (60.0)
13 (23.6)
,0.001
Age
0.32
Gender
0.28
KPS
0.006
Tumor location
0.030
Table 5. Univariate analyses with the Cox log-rank test of the effect on
median survival
Factors
No. of patients
Median survival
P value
0
15
1197
,0.0001
1þ to 2þ
56
469
3þ to 4þ
27
318
BMP-2
Histologic classification (WHO)
Grade II
17
1269
Grade III
35
371
Grade IV
46
328
,55
40
728
55
58
307
Male
68
397
Female
30
418
70
64
895
,70
34
277
Frontal
43
438
Non-frontal
55
392
0.0012
Age
0.1601
Gender
0.2035
KPS
0.0092
Tumor location
0.3196
Figure 2. Kaplan – Meier survival curves defined by the BMP-2 expression
of patients with histologic grades II – IV (A) or with grades II and III (B),
and IV (C). In each grade, glioma patients with the negative (0) or low (1þ
to 2þ) BMP-2 expression survived for significantly longer than those with
the high (3þ to 4þ) BMP-2 expression.
of glioma patients (Tables 3 – 5 and Fig. 2). The BMP-2
expression pattern was predictive, especially in higher-grade
gliomas. In our small series of cases, BMP-2 expression was
associated with a poor prognosis and correlated better with
clinical course than with the histological grading. Although
the prognostic significance of the histological diagnosis
strongly depends on the experience of the respective
630
Over-expression of BMP-2 in human gliomas
Table 6. Multivariate analyses with the Cox regression model
Factors
Type
P value Relative
risk
95% CI for
relative risk
BMP-2
3þ to 4þ
0.0138
1.887
1.069
3.398
Histologic classification
(WHO)
Grade IV
0.0302
1.633
1.052
2.823
Age
55
0.2156
1.189
0.733
1.954
Gender
Male
0.1933
0.906
0.717
1.851
KPS
,70
0.0208
0.298
0.123
0.592
Tumor location
Non-frontal 0.3827
1.372
0.791
2.108
Lower Upper
CI, confidence interval.
neuropathologist, analysis of both available data sets
revealed that BMP-2 expression was superior in predicting
the patient’s survival.
During normal brain development, stem cells are regulated by BMPs, which signal through the canonical proteins Smad1, Smad5 and Smad8 (15). BMPs are
well-characterized inducers of CNS stem cell differentiation, astroglial fate, mitotic arrest and apoptosis; in contrast, the endogenously secreted BMP antagonist Noggin
limits glial differentiation and redirects normal post-natal
stem cells to generate neurons (16,17). Consistent with this
role, BMPs promote the differentiation of glioma-derived
precursors; however, BMP application during tumor cell
engraftment slowed but did not stop the growth of brain
tumors or eventual animal death (18). This suggests that
the brain microenvironment counteracts the actions of
BMPs in ways not present under culture conditions.
Indeed, the continued widespread expression of BMPs in
the brain after birth begs the question of how newly
forming tumors escape their differentiating effects (19,20).
In the present study, we chose BMP-2 from among the
other BMPs as our research target because it plays important roles in glial development and it interacts with TGF-b
in human glioma to antagonize TGF-b-induced inhibition
of multidrug transport (21). The aberrant expression of
embryonic proteins in cancer has highlighted the importance of this class of genes in the development of cancer.
This is of particular interest since the activities of these
embryonic proteins have been shown to be associated with
those of BMP-2 during development (22). Recent studies
have suggested that tumor growth and metastasis originates
from a small subset of cancer cells, which can self-renew
to form daughter cells. These studies suggest that tumor
growth is dependent on ‘stem cell-like’ tumor cells. It will
be of interest to see if BMP-2 regulates stem cell-like
cancer cells (23 – 25).
We believe that BMP-2 may be a good target for diagnosis and therapy in human gliomas for several reasons.
(i) BMP-2 is expressed in the majority of human gliomas.
(ii) BMP-2 expression in gliomas is high and there is little to
no expression in normal brain tissue, implying that
anti-BMP-2 therapy would have minimal toxicity. (iii)
BMP-2 expression itself showed a significant correlation
with prognosis in patients with gliomas; furthermore, it is
not only a significant predictor of survival in high-grade
gliomas, but also in lower-grade gliomas.
In conclusion, BMP-2 is a highly sensitive marker for
gliomas prognosis, suggesting that the expression level of
BMP-2 may be a potent tool for the clinical prognosis of
gliomas patients. Our findings will not only be useful for
understanding glioma, but for effective clinical diagnosis
and therapy. Additional studies of in vivo molecular signaling and mechanism to induce a high expression of BMP-2 in
glioma are likely to further highlight the advantage of diagnosis with BMP-2.
Acknowledgement
C.L., G.T. and N.W. carried out the experiment of this
manuscript and drafted the manuscript; Y.T. and J.F. participated in the experiment and revised the manuscript, C.L.
participated in the design of the study and approved the final
manuscript.
Conflict of interest statement
None declared.
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