ONCOLOGY LETTERS 3: 699-703, 2012
Abstract. In the present study, we investigated the role of
matrix metalloproteinase (MMP)-2 and -9 as novel biomarkers
in the body fluid of patients with metastatic breast cancer. We
measured the expression of MMPs in 37 samples of body fluid
(10 peritoneal and 27 pleural fluids) from metastatic breast
cancer patients between 2000 and 2009. Zymography and
ELISA assays were used to determine the cut-off level and to
quantify MMP expression from a positive control, HT-1080
conditioned media. MMP expression in patient samples was
measured with ELISA and compared with other clinical
parameters. Ascitic carcinoembryonic antigen (CEA) and
pleural CEA were measured in patient samples with a chemi-
luminescent enzyme immunoassay. Body fluid cytology had a
positivity of 45% (9/20) for pleural fluid and 28.6% (2/7) for
ascites. However, MMP-2 had a positivity of 85.2% (23/27)
in 27 pleural fluid samples and 100% (10/10) in ascitic fluid
with cut-off levels of 8.6 and 0.14 ng/ml for MMP-2 and
-9, respectively. When body fluid CEA and MMP-2 were
combined, the positivity improved to 96% in pleural fluid and
100% in ascites. MMP-2 expression in body fluid did not show
any significant differences, but MMP-9 expression was lower
in ascites than in pleural fluids (p<0.005). Our results suggest
that MMP-2 expression in body fluid be used as an additive
diagnostic marker for metastatic breast cancer patients.
Breast cancer is one of the most common cancers in the world.
There are 2.5 million women diagnosed with breast cancer
in the United States, and in Europe. Additionally, 350,000
new cases are diagnosed each year, with a mortality rate of
130,000 patients, accounting for 17.5% of all cancer-related
mortality in Europe (1,2). In Korea, the incidence rate for
breast cancer has increased by 2.6% each year (3). In advanced
adenocarcinoma, progressed or stage IV cancer, malignant
peritoneal and pleural fluid may develop as the tumor
progresses, and this occurs in 10% of all cases (4).
Clinically, cancer antigen (CA) 15-3 is widely used as a
tumor marker for breast cancer, but it is mostly used with
plasma samples. Among the diagnostic methods using body
fluid, cytology is thought to be the most reliable, but it is limited
by low sensitivity (5). To compensate for the low sensitivity,
other diagnostic markers, such as carcinoembryonic antigen
(CEA), which has been reported to have a diagnostic value for
determining malignancy in pleural fluid, are being used clini-
cally (6). Ascitic CEA has recently been reported to have an
increased specificity in peritoneal fluid for diagnosing gastric
malignancy (7). However, the markers that are being used to
diagnose malignancy still pose problems of low sensitivity
with a wide variability, which is a limitation in routine clinical
use, particularly for predicting prognosis (8,9).
Matrix metalloproteinases (MMPs) are known to promote
cancer progression through extracellular matrix (ECM) and
basement membrane degradation, resulting in the exposure
of cryptic locations linked to invasion, metastasis and angio-
genesis (10-12). It has been reported that active MMPs are
indicators for metastasis in breast cancer (13). Additionally,
the overexpression of MMP-2 and -9 is reportedly correlated
with poor overall survival, suggesting that MMP-2 and -9 are
possible prognostic markers (11,14). Therefore, the improved
ability to detect malignancy in body fluids of breast cancer
patients using biomarkers such as MMPs may be helpful for
determining the proper treatment and predicting prognosis. In
this study, we evaluated the possibility of using MMP-2 and -9
expressed in body fluids as diagnostic markers for metastatic
Materials and methods
Patients. We collected the body fluids of 36 patients, who were
clinically diagnosed with metastatic stage IV breast carci-
noma with malignant ascites or pleural effusion (10 ascites,
Body fluid MMP-2 as a putative biomarker
in metastatic breast cancer
SEWON NOH1, JAE-JOON JUNG1,4,5, MINKYU JUNG1-3, KI-HYANG KIM1-3, HA-YOUNG LEE1-3,
BRANDON WANG1, JOANNA CHO1, TAE SOO KIM1,2, HEI-CHEUL JEUNG1-3 and SUN YOUNG RHA1-5
1Cancer Metastasis Research Center; 2Yonsei Cancer Center, Yonsei Cancer Research Institute;
3Department of Internal Medicine; 4National Biochip Research Center; 5Brain Korea 21 Project for Medical Science,
Yonsei University College of Medicine, Seoul, Republic of Korea
Received July 28, 2011; Accepted December 19, 2011
Correspondence to: Professor Sun Young Rha, Department of
Internal Medicine, Division of Medical Oncology, Yonsei University
College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul
120-752, Republic of Korea
Key words: ascites, biomarkers, metastatic breast cancer, matrix
metalloproteinase, pleural effusion
NOH et al: ADDITIVE ROLE OF BODY FLUID MMP-2 AS A DIAGNOSTIC BIOMARKER IN METASTATIC BREAST CANCER
27 pleural fluids; one patient had malignant ascites and pleural
effusion) at Yonsei Cancer Center, Yonsei University College
of Medicine, Yonsei University Health System between
October 2000 and September 2009. Medical records were
retrospectively reviewed for patient demographic and clinical
information including serum CEA and CA 15-3. The patients
had systemic metastasis with more than 2 sites of metastasis,
including at least one site of visceral metastasis. The patients
were heavily pretreated with systemic chemotherapy, with
the median chemotherapy regimen consisting of 3 chemo-
therapeutic agents (range, 1-7). Clinical and radiological
results confirmed that the body fluids originated from the
carcinomatosis of breast cancer, with no evidence of other
malignancies (15). When the body fluid was detected for the
first time in each patient, it was collected through paracentesis
or thoracentesis. Body fluid cytology based on cell block and
routine body fluid examinations were performed and samples
were kept at -70˚C until they were used for experimentation.
Body fluid cytology results were available in 7 peritoneal
and 20 pleural fluids. In addition, CEA expression from body
fluids (aCEA for peritoneal and pCEA for pleural fluids) was
evaluated with a chemiluminescent enzyme immunoassay
(Beckman Coulter Inc., Minnesota, MN, USA). Following
the previous result, a positive cut-off level of 5 ng/ml CEA for
body fluid was used (7). Patient survival was calculated from
the date body fluid was collected until the date of mortality due
to any cause. Signed consent was obtained from all patients.
Positivity of body fluid MMP-2 and -9. The cut-off level for
positivity of body fluid MMPs determined from a comparison
of zymography and ELISA was used based on previous
results (16). Briefly, conditioned media (CM) of HT-1080 and
human fibrosarcoma cells, were used as a positive control for
MMP-2 and -9. To overcome the difficulties of zymography,
which shows the qualitative biological activity of MMPs, and
also to quantify their activities, an ELISA assay was utilized.
Enzymatic activity and the quantitative expression level of MMP
were compared with the protein concentration of HT-1080 CM.
After confirming the positive correlation between zymography
and ELISA results (p<0.05), we determined the diagnostic
cut-off for MMP-9 as 0.14 ng/ml and 8.6 ng/ml for MMP-2 (16).
Patient samples were then quantified with ELISA assay.
Statistical analysis. To compare the expression levels of
body fluid MMPs with body fluid CEA and cytology, the
Mann-Whitney U-Test was used. In analyzing the overall
survival, we performed a log-rank test using the Kaplan-Meier
method. SPSS 13.0 was used to perform all statistical analyses.
P<0.05 was considered to indicate a statistically significant
Biomarker expression in the body fluid of breast cancer
patients. Our patient sample included only females (n=36),
and the total sample number was 37, as one patient had both
malignant ascitic and pleural effusion. The median age of the
patients was 54 years (range, 36-77). Body fluid cytology had
a 40.7% (11/27) positivity, serum CA15-3 had a mean value
of 159.3±298.4 µg/ml, serum CEA 22.9±67.9 ng/ml, and body
fluid CEA had a mean value of 60.9±124.1 ng/ml in all patients
(Table I). Median overall survival of all patients was 37 days
(range, 5-1463), suggesting that the patients had far advanced
disease, and patients were heavily pretreated when they
developed the malignant ascites of pleural effusion. Notably,
our results showed that patients with peritoneal fluid had a
significantly shorter survival, with a median of 16 days (range,
5-792), compared to patients with pleural fluid, who had a
median survival of 291 days (range, 10-1463), p<0.05 (Fig. 1).
Since body fluid CEA has been reported to have a role
as a biomarker (7), we evaluated CEA in body fluids. CEA
Table I. Patient characteristics.
Ascites Pleural effusion Total
10 27 36
10 27 37
53 (40-64) 54 (36-77) 54 (36-77)
mean ± SD
mean ± SD
aOne patient who has body fluids of ascites and pleural effusion is
involved in each analysis independently. bSurvival after body fluid for-
mation. CEA, carcinoembyonic antigen; CA15-3, cancer antigen 15-3.
Figure 1. Overall survival compared between body fluids.
ONCOLOGY LETTERS 3: 699-703, 2012
expression differed in body fluids; ascites had a higher CEA
expression of 124.5±213 ng/ml than the CEA expression in
pleural fluids, which was 37.3±58.8 ng/ml. We then compared
MMP-9 and -2 expression in body fluids. Peritoneal fluid had
a lower MMP-9 expression level of 0.09±0.26 ng/ml compared
to 0.25±0.64 ng/ml from the pleural fluids. However, MMP-2
expression in ascites (34.1±20 ng/ml) was higher than that in
pleural fluids (29.9±24.5 ng/ml) (Table II). When we compared
the expression levels of various biomarkers (CEA, MMP-2,
MMP-9) in ascites and pleural fluids, CEA and MMP-2 were
not significantly different (data not shown). In comparison,
MMP-9 expression in pleural fluid was higher than that in
peritoneal fluid (p<0.05).
Improved malignancy detection using body fluid MMP-2.
Cytology information from 20 pleural and 7 peritoneal fluids
showed positive rates of 45 (9/20) and 28.6% (2/7), respectively,
demonstrating that cytology has an overall low sensitivity in
our samples, as reported in previous studies (5,9). Following
evaluation of other biomarkers in 27 pleural fluids, MMP-2
was found to have the highest positivity with 85.2% (23/27),
followed by CEA, with a 74.1% (20/27) positivity. MMP-9
showed the lowest positivity with 29.6% (8/27) (Table III).
Notably, in five patients with ascites and negative cytology from
peritoneal fluid, four patients were positive for CEA expression
(80%), one patient was positive for MMP-9 expression (20%),
and all five patients were positive for MMP-2 expression
(100%). By contrast, in 11 pleural fluids with negative cytology,
the positivity for CEA, MMP-2 and MMP-9 were 63.6 (7/11),
72.7 (8/11) and 27.3% (3/11), respectively. These results suggest
that body fluid MMPs, especially MMP-2, could be used as
diagnostic biomarkers in metastatic breast cancer.
When the biomarkers were combined, an increase in sensi-
tivity was observed. In pleural fluids, combining CEA and
MMP-2 increased the positivity to 96.3% (26/27). Combining
MMP-9 and MMP-2 showed a positive rate of 88.9% (24/27),
combining CEA and MMP-9 improved the positivity to 85.2%
(23/27), and combining all three markers had the same posi-
tive rate as combining just CEA and MMP-2, 96.3% (26/27)
(Table III). In the 10 peritoneal fluid samples, MMP-2 had
the highest positive rate with 100% (10/10), followed by CEA
which had a positivity of 80% (8/10), and MMP-9 had the
lowest positive rate with 10% (1/10). The combination of body
fluid CEA and body fluid MMP-2 had a positive rate of 100%
(10/10), which was equal to combining body fluid MMP-9
and body fluid MMP-2 or combining all three biomarkers.
The combination of body fluid CEA and body fluid MMP-9,
however, increased positivity to 80% (8/10) (Table III). As a
result, MMP-2 was more sensitive to detecting malignancy
in body fluids and had an additional diagnostic role when
combined with body fluid CEA.
Previous reports have suggested that body fluid CEA is a
marker with a relatively high sensitivity of approximately 80%
in the body fluid of various types of cancer, confirming results
of this study, obtained from the body fluid of metastatic breast
cancer patients (6,7,16). However, our results showed that
body fluid MMP-2 had an even higher sensitivity than body
fluid CEA for detecting malignancy in breast carcinoma. In
pleural fluids, the combination of body fluid MMP-2 and body
fluid CEA improved sensitivity to almost 100%, indicating
that MMP-2 alone, or in addition to CEA, may be used as a
diagnostic biomarker. In peritoneal fluid, MMP-2 had a posi-
tivity of 100%, suggesting that body fluid MMP-2 is a useful
diagnostic biomarker in metastatic breast cancer patients, in
addition to cytology and body fluid CEA.
We evaluated the expression of MMP-2 and -9 in the body fluid
of metastatic breast cancer patients to determine the possi-
bility of using MMPs as biomarkers. MMPs are reportedly
involved in prognosis and are used as prognostic markers in
tissue and plasma samples (17,18). Our study focused on body
fluid samples, which have an advantage over tissue and plasma
samples in terms of their availability and representation of the
direct effect from cancer. Body fluids may be obtained from
patients as soon as the fluids accumulate, but tissue samples
are limited in their availability. Moreover, plasma samples are
not directly in contact with the cancer, and thus may contain
numerous non-specific target molecules, whereas body fluids
form directly at the site of cancer and are capable of reflecting
cancer status as well as tumor burden. Therefore, body
Table II. Comparison of body fluid biomarker expression
between ascites and pleural effusion.
N MMP-9 MMP-2 CEA
Pleural effusion 27 0.25±0.64 29.9±24.5
10 0.09±0.26 34.1±20 124.5±213
Total 37 0.20±0.56 31.1±23.19 60.9±124.1
MMP-9, matrix metalloproteinase-9; MMP-2, matrix metallopro-
teinase-2; CEA, carcinoembyonic antigen.
Table III. Positivity of single and multiple biomarkers in body
n % %
CEA, carcinoembryonic antigen; MMP-9, matrix metallopro-
teinase-9; MMP-2, matrix metalloproteinase-2.
NOH et al: ADDITIVE ROLE OF BODY FLUID MMP-2 AS A DIAGNOSTIC BIOMARKER IN METASTATIC BREAST CANCER
fluids provide advantages over tissue and plasma samples for
understanding pathogenesis and diagnosis, and for predicting
clinical outcomes in breast cancer.
Cytology from body fluid is known to be the most reliable
marker for diagnosis, but has an extremely low sensitivity (5).
Due to the low sensitivity, other cancer-related biomarkers
such as CEA and telomerase activity are being used in patient
samples, but still yield unsatisfactory sensitivities and speci-
ficities, and are not applicable due to difficulties in detection
methods. We aimed to identify diagnostic biomarkers with
increased sensitivity by evaluating MMP-2 and -9 expression
in body fluids to be used alone or in addition to body fluid
CEA, for detecting malignancy within body fluids from meta-
static breast cancer.
Since our study focused on assessing body fluid MMP-2
and -9 as diagnostic markers in breast cancer, the choice of
method for evaluation was important, as the assay directly
affects accuracy and practicality. While zymography allows
visualization of the enzymatic activity of MMPs qualitatively,
ELISA provides a quantitative amount of MMP protein
expression. The combination of methods allows us to create an
accurate assessment of MMP expression (19). Using the two
assays with HT-1080 CM, we determined a cut-off level for
MMP-9 of 0.14 and 8.6 ng/ml for MMP-2 based on the minimal
level of expression that could be sufficient for diagnosis.
In our experiment, body fluid MMP-2 had a positivity of
85.2% in pleural fluid and 100% in peritoneal fluid. When body
fluid MMP-2 was combined with body fluid CEA, positivity
was increased to 96.3% in pleural fluid. Compared to body fluid
MMP-9 with limited diagnostic features, body fluid MMP-2
alone or in combination with body fluid CEA was useful as an
additive diagnostic marker in the body fluid of breast cancer
patients. Although body fluid MMPs did not seem to have
any prognostic role in our study (data not shown), the type of
body fluid had a prognostic role. Breast cancer patients with
peritoneal fluid had a significantly shorter survival compared
to patients with pleural fluid. This observation may be corre-
lated with disease burden considering the site of metastasis
from the original breast tumor. Previously, it was reported
that MMP-2 and MMP-9 are involved in breast cancer inva-
sion (20,21). Our results have shown that MMP-2 has a higher
expression and positivity than MMP-9. Findings of a previous
report that evaluated 23 malignant body fluids showed that
MMP-2 (87%) had a higher positivity than MMP-9 (78.3%) in
different cancer origins, corresponding with our result (22).
In addition, MMP-9 expression was significantly higher in
pleural than in peritoneal fluid. Breast cancer patients initially
form pleural fluid, which may invade to cause the formation
of peritoneal fluid in the abdomen. Since MMP-2 and -9 are
involved in cancer invasion, MMP expression may be higher
in pleural fluid in preparation for cancer invasion, whereas
MMP expression may be lower in peritoneal fluid, suggesting
that invasion has already occurred. Studies have shown that
MMP-9 is capable of being downregulated after invasion and
body fluid formation has occurred, suggesting that MMP-9
expression is tightly controlled, which may contribute to the
low level of MMP-9 in peritoneal fluid (23,24). In one patient
who had both ascitic fluid and pleural effusion, MMP-9
expression in the pleural fluid (0.55 ng/ml) was much greater
than that in the ascitic fluid (0.02 ng/ml).
Previous reports determined that cancer invasion is corre-
lated with poor survival (25). We observed that the patients
with malignant ascites showed a shorter survival compared to
the patients with pleural effusion. As pleural and peritoneal
effusions occur in various parts of the body with different
biology, we consider that the body fluid itself may differ in
the expression and biological role of each molecule, which
may also predict prognosis. Previous reports have suggested
that MMP-9 and MMP-2 overexpression correlates with
poor overall survival in tissue and plasma samples of various
cancers (17,18). However, results from our experiment did not
concur with these reports, except for the prognostic potential
of the site of malignant effusion. This may be related to the
small sample size in our study. Moreover, since our study used
body fluid, rather than tissue and plasma, the expression of
MMPs may differ, since MMPs are under tight control in the
body and in cells (23,24).
Although we focused on the role of body fluid biomarkers,
if we could use the serum biomarkers in addition to the body
fluid biomarkers, more reliable information would be obtained
in order to understand the status of the patients, including
tumor burdens and prognosis. Among the numerous tumor
markers, serum CEA and CA 15-3 are mostly used for breast
cancer patients. However, in our patient set, the level of those
serum markers were not correlated with each other or with
body fluid biomarkers. In addition, pleural CEA has been used
for the detection of malignancy from pleural fluid. However,
ascitic CEA has recently been suggested as a detection factor
for malignancy by our previous report in gastric cancer.
Therefore, in this study we compared ascitic and pleural
CEA with body fluid MMPs. Our study was unique in that it
analyzed MMP expression in body fluids of metastatic breast
cancer patients, allowing us to compare the differences that
potentially exist between each patient. Moreover, the use of an
ELISA assay has several benefits for use in clinical practice;
it is easy, quantitative and a small amount of the body fluid is
required. Previous studies have suggested that MMP-2 and -9
be used as diagnostic markers in tissue and plasma samples.
However, this is the first study to use malignant body fluids
from breast cancer to evaluate MMPs as possible diagnostic
markers. In particular, our results suggest that MMP-2 is a
highly sensitive diagnostic marker for metastatic breast cancer
patients. The limitations of the current study are: i) the patient
heterogeneity, ii) the study is retrospective with a small sample
size, and iii) the determination of the assay cut-off level is
arbitrary. Patient heterogeneity with tumor heterogeneity is
the essential problem of translational research. In our study,
patients with relatively homogeneous clinical features were
selected. The patients were required to have clinically evident
malignant ascites or pleural effusion regardless of cytology
results, considering the false negativity of body fluid cytology.
Since systemic chemotherapy after body fluid formation
may affect prognosis, we selected patients who had received
active systemic chemotherapy prior to body fluid formation.
Therefore, patients who developed ascites or pleural effusion
at the time of breast cancer diagnosis were excluded. Multiple
sites of metastasis were also observed, including a minimum
of 1 visceral metastasis from the breast cancer. All the patients
were previously heavily pretreated with active systemic treat-
ment. In addition, this study is the first study to evaluate the
ONCOLOGY LETTERS 3: 699-703, 2012
proof-of-concept of whether the biomarker in the body fluid
may work in clinical practice and also the feasibility of ELISA
for stratifying the patients. In almost all the patients who
develop body fluids, the fluid examination is easily performed
in clinical practice, and the collection of body fluid is feasible.
Gathering the fluid provides biological information, which may
be of clinical use, and therefore this practice is worthwhile.
However, validation with large numbers of prospectively
collected samples is required for the further clinical develop-
ment of these novel body fluid biomarkers.
This manuscript was supported by the Public Welfare and
Safety research program through the National Research
Foundation of Korea (NRF) funded by the Ministry of
Education, Science and Technology (2010-0020841).
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