Oesophageal cancer is a disease with a high degree of malignancy, and its prognosis
is worse than that of other gastrointestinal tumours. The two-year survival rate of
patients who receive surgery for oesophageal cancer is unfavourable, being about 30%
(Krevsky, 1995; Medical Research Council Oesophageal Cancer Working Party, 2002).
For those with risk factors for oesophageal cancer, which include smoking and drinking,
high age (50 to 70 years), and head and neck carcinomas, a common form of screening
is oesophageal endoscopy with iodine solution for detection of oesophageal carcinoma
in the early stage. As a result, the detected number of superficial oesophageal carcinomas,
in which tumour invasion is limited to the mucosa or submucosa, with a favourable
prognosis has increased (Brodmerkel, 1971; Endo et al, 1986). With endoscopic ultrasonography
(EUS) and endoscopic mucosal resection (EMR) we positively staged and treated oesophageal
cancer (Yanai et al 1996; Yanai et al, 1997; Takeo et al, 2001).
Although methods to diagnose superficial oesophageal cancer have advanced, an important
reason for the poor prognosis of oesophageal cancer is lymph node metastasis, even
in superficial carcinoma. Thus, the existence of lymph node metastasis determines
the strategy for treatment and the estimated prognosis of oesophageal cancer. Oesophageal
cancer limited to the epithelium (EP) and lamina propria mucosae (LPM) has little
lymph node metastasis. But the likelihood of lymph node metastasis for cancer with
muscularis mucosae (MM) invasion is almost 10%. The possibility of lymph node metastasis
with invasion of the submucosa (SM) increases to as much as 30% to 50% (Bogomoletz
et al, 1989; Kodama et al, 1998). Thus, for tumours with MM and SM invasion, operation
is considered necessary even in the case of superficial oesophageal tumours. Thus,
new parameters for lymph node metastasis and prognosis are needed for tumours with
MM and SM invasion.
Recently, the matrix metalloproteinase (MMP) family has been reported to play important
roles in the invasion and metastasis of colon cancer and gastric carcinoma (McDonnell
et al, 1991; Wilson et al, 1997; Adachi et al, 1998; Yamashita et al, 1998). In particular,
MMP-7 is characterized by (1) broad and strong proteolytic activity against a variety
of extracellular matrix substrates such as collagens, proteoglycans, laminin, fibronectin,
and casein, (2) lack of a c-terminal domain, so it is not regulated easily by tissue
inhibitor of metalloproteinases (TIMP), and (3) production by tumour cells themselves
(Miyazaki et al, 1990; Matrisian, 1992; Baragi et al, 1994; Adachi et al, 1998). It
has been reported that MMP-7 is present in gastric, colorectal, head and neck, and
hepatocellular cancers (Yoshimoto et al, 1993; Yamamoto et al, 1997; Adachi et al,
2001; Liu et al, 2002). MMP-7 has attracted much attention because it is reported
to be present in the tumour invasive front (Adachi et al, 1998; Adachi et al, 2001;
Lin et al, 2002). It has also been reported to have a correlation with invasion and
metastasis in oesophageal cancer (Adachi et al, 1998; Yamamoto, 1999; Ohashi, 2000;
Yamashita, 2000).
On the other hand, the members of the MMP family form a cascade and activate each
other. MMP-9, which works directly to degrade the extracellular matrix (Parsons et
al, 1997), is the last stage in the cascade. It decomposes type IV collagen, which
is the chief component of basement membrane, and also promotes tumour invasion and
metastasis. MMP-9 expression in tumours was reported in gastric, colon, pancreatic,
and cervical cancers (Torii et al, 1998; Baker et al, 2002; Kato et al, 2002; Nagakawa
et al, 2002).
To determine the appropriate treatment for superficial oesophageal carcinoma, we retrospectively
studied expression of MMP-7 and MMP-9 and investigated clinical pathological factors
and outcomes in superficial oesophageal cancer treated by endoscopic and surgical
resection.
MATERIALS AND METHODS
Patients and tissue samples
Fifty-five patients (average age 65. 2 years old, male/female ratio 41/14), consisting
of 34 with surgical resection for superficial oesophageal cancers between 1995 and
1999, and 21 with EMR between 1995 and 2001 at Yamaguchi University Hospital were
studied. No patient had received irradiation treatment or chemotherapy before the
resection. Forty-four oesophageal squamous cell carcinomas (SCC) and 1 adenocarcinoma
were included. The age range for the 55 patients was 44–85 years (mean, 65.2 years).
Lymph node metastases were detected in twelve patients but none of the patients had
distant metastasis. The depth of superficial oesophageal tumour invasion was EP in
9 cases, LPM in 6 cases, MM in 16 cases and SM in 24 cases. All 55 patients were followed
up. The median follow-up period after the first treatment was 34.2 months (1–81 months),
at which time 15 patients had already died. The reasons for death were as follows:
oesophageal carcinoma for 5, (survival time 9–47 months, average 22.8 months), other
carcinomas for 3, and cerebral infarction for 3.
One of the deepest sections from each tumour was selected for evaluation. It was pathologically
classified by standard methods using hematoxylin and eosin (HE) staining as follows
(Japanese Society for Esophageal Disease, 2001). According to the depth of invasion,
we classified the tumours into 4 groups: EP, LPM, MM, and SM. The tumours were classified
as well differentiated SCC, moderately differentiated SCC, poorly differentiated SCC,
and others. We also examined whether lymphatic permeation, venous invasion, and nodal
metastasis were present. Gross features of the superficial oesophageal cancer were
classified as elevated, flat and depressed. Infiltrative growth patterns (inf) were
classified into three types: α (expansile type: expansile growth of tumour nests forming
a distinct border with surrounding tissue), β (intermediate type: intermediate pattern,
between inf-α and inf-γ) and γ (infiltrative type: infiltrative growth of tumour nests
forming an indistinct border with surrounding tissue).
Immunohistochemistry for MMP-7 and MMP-9 was performed for each case. The patient's
prognosis after treatment was investigated by examining the medical record. Written
informed consent was obtained from all the patients or their relatives.
Immunohistochemistry
Four-micrometer sections of paraffin-embedded specimens from the deepest parts of
the tumours were dewaxed in xylene, and rehydrated through a series of graded ethanol.
After heating in a microwave oven (500 watts) for 6 min, the sections were allowed
to cool to room temperature in cold water. Endogenous peroxidase was blocked with
3% hydrogen peroxide in methanol for 30 min. Sections were washed twice for 3 min
in phosphate–buffered saline (PBS). Primary mouse monoclonal anti-human MMP-7, purified
IgG (141-7B2; Fuji Chemical, Toyama, Japan, diluted 1 : 100) and anti-human MMP-9,
and purified IgG (56-2A4; First Fine Chemical, Toyama, Japan, diluted 1 : 100) were
added and incubated in a moist chamber overnight at 4°C. After three washes in PBS
for 5 min each time, samples were incubated with Histofine simple stain MAX-PO (M)(Nichirei,
Tokyo) for 30 min at room temperature, according to the manufacturer's instructions.
The reactions were stopped by washing the sections three times for 5 min in PBS. Positive
reactions were visualized with hydrogen peroxide containing 0.05% 3,3′-diaminobenzine
(DAB) in PBS. The slides were counterstained with Mayer's hematoxylin solution for
nuclei. Negative control sections were made by omitting the primary antibody.
The score was calculated as the percentage of cells stained positively among tumour
cells counted. Four high-power fields (× 400) were selected. Cases were considered
positive when over 10% of carcinoma cells were stained. Immunostaining signals at
the invasive front were also calculated and the cases were considered positive when
not less than 10% of cells at the invasive front were stained (Kawano et al, 1997;
Yamamoto et al, 1999). All specimens were evaluated by two investigators (Y.T., T.Y.)
who were blinded to the patients’ clinical information. Disagreements were reviewed
and a conclusive judgment was made.
Statistical analysis
Statistically significant differences were determined by Fisher's exact probability
test for gender, lymphatic permeation, venous invasion, and nodal metastasis. Pearson's
chi-squared test was performed for gross feature classification, histological differentiation
grade, and infiltrative growth pattern. Other factors such as age and depth of invasion
were subjected to the Cochran-Armitage trend test. The P-value was calculated using
exact methods.
Probability distributions of overall survival were calculated using the Kaplan-Meier
method and compared by the log-rank test. To determine independent factors that were
significantly related to the prognosis for patients with oesophageal cancer, multivariate
analysis was performed using Cox's proportional hazards model with a stepwise procedure.
The variables included in the multivariate analysis were the following categories:
gender, age (under 59, 60–64, 65–69, and over 70), gross features (elevated, flat,
depression), depth of invasion (EP, LPM, MM, SM), histological differentiation grade
(well differentiated SCC, moderately differentiated SCC, poorly differentiated SCC,
and others), lymphatic permeation (positive, negative), venous invasion (positive,
negative), infiltrative growth patterns (α,β,γ), nodal metastasis (positive, negative),
MMP-7 (positive, negative), MMP-7 on the invasive front (positive, negative), MMP-9
(positive, negative), and MMP-9 on the invasive front (positive, negative). For all
statistical tests, a P value of less than 0.05 was defined as statistically significant.
All analyses were performed using SAS version 6.11 (SAS Institute Inc., Cray, NC,
USA).
RESULTS
Expression of MMP-7
The cytoplasms and cell membranes of carcinoma cells were stained brown for MMP-7,
but stromal cells, except for some monocytes, were not stained (Figure 1A, B
Figure 1
Immunohistochemical staining of oesophageal cancer for MMP-7 and MMP-9. (A) MMP-7-positive
SCC; MMP-7 was stained in cancer cell cytoplasm, (B) MMP-7-negative SCC, (C) MMP-9-positive
SCC; MMP-9 was stained in tumour cytoplasm, (D) MMP-9-negative SCC (× 400).
). Expression of MMP-7 was demonstrated in 13 of the 55 cases (23.6%) (Table 1
Table 1
Association of MMP-7 expression and clinicopathologic findings
MMP-7
MMP-7 at invasive front
Positive
Negative
P
Positive
Negative
P
Variables
(n=13)
(n=42)
value
(n=7)
(n=48)
value
Gender
Male
11
30
0.447
6
35
0.664
Female
2
12
1
13
Age
≦59
2
14
0.461
1
15
0.851
60–64
4
5
3
6
65–69
3
7
2
8
70≦
4
16
1
19
Gross features
Elevated
4
7
0.291
3
8
0.057
Flat
1
1
1
1
Depressed
8
34
3
39
Depth of invasion
EP
0
9
0.06
0
9
0.013*
LPM
2
4
0
6
MM
2
14
0
16
SM
9
15
7
17
Differentiation grade
SCC (well)
2
8
0.569
0
10
0.334
SCC (moderately)
9
22
6
25
SCC (poorly)
1
10
1
10
Others
1
2
0
3
Lymphatic permeation
Positive
8
13
0.059
6
15
0.01*
Negative
5
29
1
33
Venous invasion
Positive
1
7
0.664
1
7
1
Negative
12
35
6
41
Infiltrative growth pattern
α
4
24
0.186
1
27
0.043*
β
8
17
5
20
γ
1
1
1
1
Nodal metastasis
Positive
7
5
0.004*
5
7
0.004*
Negative
6
37
2
41
*
Significant.
). MMP-7 was immunostained at the invasive front in 7 cases (12.7%). MMP-7 expression
was significantly correlated with positive nodal metastasis (P=0.004) only. In the
invasive front, MMP-7 expression was significantly correlated with deep tumour invasion
(P=0.013), positive lymphatic permeation (P=0.010), positive nodal metastasis (P=0.004)
and unclear infiltrative growth pattern (P=0.043).
Expression of MMP-9
Expression of MMP-9 was observed in the cytoplasm of carcinoma cells, stromal fibroblasts
and vascular endothelial cells (Figure 1C, D). It was detected in 26 cases (47.3%)
(Table 2
Table 2
Association of MMP-9 expression and clinicopathologic findings
MMP-9
MMP-9 at invasive front
Positive
Negative
P
Positive
Negative
P
Variables
(n=26)
(n=29)
value
(n=22)
(n=33)
value
Gender
Male
22
19
0.13
18
23
0.361
Female
4
10
4
10
Age
≦59
7
9
0.562
5
11
0.95
60–64
8
1
7
2
65–69
5
5
4
6
70≦
6
14
6
14
Gross features
Elevated
6
5
0.867
5
6
0.871
Flat
1
1
1
1
Depressed
19
23
16
26
Depth of invasion
EP
1
8
0.004*
0
9
0.001*
LPM
2
4
2
4
MM
7
9
5
11
SM
16
8
15
9
Differentiation grade
SCC (well)
2
8
0.003*
2
8
0.32
SCC (moderately)
12
19
12
19
SCC (poorly)
10
1
6
5
Others
2
1
2
1
Lymphatic permeation
Positive
16
5
0.001*
13
8
0.012*
Negative
10
24
9
25
Venous invasion
Positive
5
3
0.455
5
3
0.244
Negative
21
26
17
30
Infiltrative growth pattern
α
11
17
0.633
8
20
0.298
β
14
11
13
12
γ
1
1
1
1
Nodal metastasis
Positive
9
3
0.049*
8
4
0.047*
Negative
17
26
14
29
*
Significant.
). At the invasive front MMP-9 was observed in 22 cases (40%). Tumours with MM and
SM invasion had significantly more MMP-9 expression (P=0.004). Poorly differentiated
SCC had more MMP-9 expression than moderately and well differentiated carcinomas (P=0.003).
MMP-9 expression was significantly correlated with positive lymphatic permeation (P=0.001),
and positive nodal metastasis (P=0.049). On the other hand, expression of MMP-9 at
the invasive front was significantly correlated with deep tumour invasion (P=0.001),
positive lymphatic permeation (P=0.012), and positive nodal metastasis (P=0.047).
Coexpression of MMP-7 and MMP-9
We divided subjects into two groups with coexpression of MMP-7 and MMP-9, and the
others. Coexpression was detected in 11 cases (20%) (Table 3
Table 3
Association of MMP-7+MMP-9 coexpression and clinicopathologic findings
MMP-7+MMP-9
Invasive front MMP-7+MMP-9
Positive
Others
P
Positive
Others
P
Variables
(n=11)
(n=44)
value
(n=6)
(n=49)
value
Gender
Male
10
31
0.255
5
36
1
Female
1
13
1
13
Age
≦59
1
15
0.341
1
15
0.863
60–64
4
5
2
7
65–69
3
7
2
8
70≦
3
17
1
19
Gross features
Elevated
4
7
0.158
2
9
0.134
Flat
1
1
1
1
Depressed
6
36
3
39
Depth of invasion
EP
0
9
0.008*
0
9
0.025*
LPM
0
6
0
6
MM
2
14
0
16
SM
9
15
6
18
Differentiation grade
SCC (well)
1
9
0.452
0
10
0.132
SCC (moderately)
8
23
6
25
SCC (poorly)
1
10
0
11
Others
1
2
0
3
Lymphatic permeation
Positive
7
14
0.082
5
16
0.026*
Negative
4
30
1
33
Venous invasion
Positive
1
7
1
1
7
1
Negative
10
37
5
42
Infiltrative growth pattern
α
2
26
0.058
1
27
0.064
β
8
17
4
21
γ
1
1
1
1
Nodal metastasis
Positive
6
6
0.008*
4
8
0.017*
Negative
5
38
2
41
*
significant.
). MMP-7 expression alone was found in 2 cases (4%), and MMP-9 expression alone in
15 cases (27%). Twenty-seven cases (49%) were negative for both MMP-7 and MMP-9. At
the invasive front, the numbers of cases with coexpression, only MMP-7, only MMP-9
and negative expression of MMP-7 and MMP–9 were 6 (11%), 1 (2%), 16 (29%), and 32
(58%), respectively. Coexpression of MMP-7 and MMP-9 was significantly correlated
with deep tumour invasion in MM and SM (P=0.008), and positive nodal metastasis (P=0.008).
Moreover, expression at the invasive front was significantly correlated with deep
tumour invasion (P=0.025), positive lymphatic permeation (P=0.026) and positive nodal
metastasis (P=0.017).
Survival analysis
Patients with MMP-7 expression-positive cancer and MMP-9 invasive front-positive cancer
had significantly shorter overall survival times than those with negative expression
(P=0.025, P=0.026, respectively) (Figures 2
Figure 2
Comparison of overall survival curves for patients with MMP-7-positive and MMP-7-negative
superficial oesophageal cancer. MMP-7-positive patients have a less favourable prognosis
than those who are MMP-7-negative.
, 3
Figure 3
Comparison of overall survival curves for patients with MMP-9-positive and MMP-9-negative
superficial oesophageal cancer. Patients who were MMP-9-positive at the invasive front
had a less favourable prognosis than those who were MMP-9-negative at the invasive
front.
). In the multivariate analysis, only expression of MMP-9 at the invasive front had
prognostic significance for overall survival (P=0.004, risk ratio 5.68). The other
factors were not statistically significant (Table 4
Table 4
Multivariate analysis of MMP-7, MMP-9 and pathologic parameters
Risk ratio
95% CI
P value
MMP-9 invasive front
Negative
1
Positive
5.68
(1.75∼18.444)
0.004*
Age (years)
≦59
1
60–64
0.758
(0.102∼5.62)
0.787
65–69
3.652
(0.659∼20.222)
0.138
70≦
4.645
(0.918∼23.491)
0.063
CI=Confidence Interval
*
Significant.
). However, older patients tended to have poorer prognoses at the ages of 60–64 (P=0.787),
65–69 (P=0.138), and over 70 (P=0.063).
DISCUSSION
MMP-7 plays important roles in tumour invasion and metastasis. There have been some
reports on MMP-7 in oesophageal cancer. Ohashi et al. (2000) examined 148 primary
oesophageal cancers immunohistochemically, and showed that cancers with SM invasion
had significantly more MMP-7 expression (44%) than those with MM invasion. They also
reported that 61.9% of positive venous invasion cases had expression of MMP-7, which
was significantly more than in negative cases. Yamamoto et al. (1999) used immunohistochemistry
to study 100 oesophageal cancers at the invasive front. They reported that the MMP-7
expression rate was 49% and that the expression was related to the depth of invasion
(P<0.0001), advanced tumour stage (P=0.0159), recurrence (P=0.0002), recurrence within
the first operative year (P=0.002), disease-free time (P=0.0007) and overall survival
(P=0.0004). Yamashita et al. (2000) reported that the MMP-7 m-RNA expression rate
detected by northern blot analysis was 65% in 48 cases, and that expression was related
to nodal metastasis (P<0.05) and prognosis (P<0.01). It was also determined to be
of prognostic significance for predicting overall survival (P=0.0005) in multivariate
analysis.
In our study, MMP-7 expression was significantly related to nodal metastasis (P=0.004)
and a less favourable prognosis (P=0.025). Thus, examination for the presence of MMP-7
in tumour biopsy specimens before treatment is expected to be useful for decision
making about the treatment course and to predict prognosis. There was no correlation
with age, gender, gross features, deep growth invasion, differentiation grade, lymphatic
permeation, venous invasion, or infiltrative growth pattern. The rate of MMP-7 expression
was a little low, 23.6% of 55 patients, because we examined only superficial oesophageal
cancer.
On the other hand MMP-7 expression at the invasive front was also correlated with
positive nodal metastasis. Cases with tumour invasion of the SM had significantly
more MMP-7 expression at the invasive front. Moreover, we newly showed that MMP-7
expression at the invasive front was significantly correlated with positive lymphatic
permeation and unclear infiltrative growth pattern. These results supported the possibility
of an effect of MMP-7 on the tumour invasive front. At the invasive front of oesophageal
cancer, MMP-7 may have an important role in invasion and metastasis.
There have been reports about the roles of MMP-9 expression in tumour invasion and
metastasis, but this is controversial in oesophageal cancer. Ohashi et al. (2000)
immunohistochemically studied 148 oesophageal cancers, and found that tumours with
SM invasion had higher MMP-9 expression (51.9%) than those with MM invasion. They
showed that the presence of venous invasion was related to MMP-9 expression, as it
occurred in 65.1% of cases. But Koyama et al. (2000) found 60% expression in immunohistochemistry
for 39 oesophageal tumours, and reported that there was no relation of MMP-9 expression
to any clinicopathologic factor. They found a relation of MMP-9 expression to venous
invasion (P=0.0022) by zymography, so active MMP-9 protein might lead to tumour invasion.
Sato et al. (1999) found no relation to depth of invasion, nodal metastasis, distant
metastasis, staging, sex, age or differentiation grade. Murray et al. (1998) also
reported that MMP-9 expression had no relation to staging.
In our study, MMP-9 expression at the invasive front was significantly related to
deep invasion, positive lymphatic permeation, and positive nodal metastasis, like
MMP-7 expression at the invasive front. In the MMP family activation cascade, MMP-9
is downstream of MMP-7 and destroys the extracellular matrix directly (Parsons et
al, 1997). Judging from the above, it is reasonable to consider that MMP-9 expression
behaves similarly to MMP-7 expression. Moreover, in both univariate and multivariate
analyses, MMP-9 expression at the invasive front was an independent prognostic factor.
Thus MMP-9 expression at the invasive front in the resected tumour may indicate the
necessity for additional treatment after operation.
MMP-9 expression for the whole tumour was also related to the oesophageal tumour differentiation
grade, along with depth of invasion, lymphatic permeation and nodal metastasis. MMP-9
may have a role in the destruction of the extracellular matrix of the tumour overall,
not only at the invasive front. Thus, MMP-9 expression examined by a pre-treatment
biopsy of the tumour surface may be a good marker for lymphatic permeation, nodal
metastasis and depth of invasion. The presence of MMP-9 expression may be important
to determine the strategy for treatment.
We examined the significance of MMP-7 and MMP-9 coexpression and classified patients
into two groups, those with both MMP-7-positive and MMP-9-positive expression and
the others. Coexpression was significantly correlated with deep invasion into the
MM and SM, and nodal metastasis. Although it had no relation with minute changes like
lymphatic permeation, coexpression of MMP-7 and MMP-9 may suggest nodal metastasis
in oesophageal cancer.
Even superficial oesophageal cancers with invasive depths of MM and SM have about
10–50% lymph node metastasis, so it is often difficult to choose between EMR and surgical
resection. Immunohistochemistry for MMP-7 and MMP-9 before treatment in these cases
will provide important information for treatment policy. MMP-7-negative and MMP-9-negative
biopsy findings might be useful as an indication for EMR without operation because
they could predict shallow depth of invasion and little lymphatic metastasis.
In conclusion, combined MMP-7 and MMP-9 expression may be a good marker for the malignancy
level of oesophageal cancer and for the presence of lymphatic metastasis, not venous
invasion. MMP-9 expression at the invasive front can be a significant prognostic variable
for predicting overall survival.