J.
Nippon
•\O
Med.
riginal
Sch.,
Vol.
59,
No.
1
(1992)
(51) •\51•\
Articles•\
The
immunohistochemical
expressions
growth factors, epidermal receptors and c-erbB-2 in human
growth factor oncoprotein
pancreatic
Yoichiro The First Department
of epidermal
cancer
Yamanaka
of Surgery,
Nippon Medical
School
Summary
The expressions of epidermal growth factors (EGF), epidermal growth factor receptors (EGFR), and the c-erbB-2 oncoprotein were immunohistochemically examined in 25 cases of human pancreatic carcinoma and epineoplastic pancreatitis and in 10 non-cancerous/noninflammatory pancreatic tissues. The positive rates of EGF, EGFR, and the c-erbB-2oncoprotein in cancer tissues were 72%,36%,and 28%,respectively. EGF was stained mainly in the cytoplasm and partly on the surfaces of the cancer cells. EGFR and the c-erbB-2oncoprotein were stained mainly on the surfaces of the cancer cells and partly in the cytoplasm. The expressions of these 3 products correlated significantly with tumor invasion into the anterior and posterior areas surrounding the pancreas. In the EGF, EGFR, and c-erbB-2positive cancer tissues, some stromal cells, that is fibroblasts and endothelial cells, were also positive. In the adjacent pancreatic tissues with inflammation, these products were noted in some ductal cells, acinar cells, fibroblasts and endothelial cells. No distinct staining was detected in non-cancerous/non-inflammatory tissues. The survival period for patients who tested positive for these three proteins was statistically shorter than for those who tested negative. These results suggest that the coexpression of EGF and EGFR and the expression of the c-erbB-2 oncoprotein are related to the existence of the invasion of human pancreatic cancer. Furthermore, an immunohistochemical examination of these three products is useful in forming a prognosis for pancreatic cancer patients. Key words: human pancreatic cancer, epidermal growth factor, epidermal growth factor receptor, c-erbB-2oncoprotein, immunohistochemistry Introduction Among
the gastrointestinal
carcinomas,
human
pancreatic
cancer is well known for its poor
prognosis. Recently, many studies have suggested that expressions of growth factors and oncoproteins might be associated with lymph node metastasis, tumor size, infiltration, histologic grade and prognosis1) . The epidermal growth factor, stimulates proliferation of not only normal epidermal the epidermal Correspondence
growth to Yoichiro
Tokyo 113, Japan
factor
receptor2, 3). This receptor,
Yamanaka,
The First Department
a polypeptide consisting of 53 amino acids, cells but also some carcinoma cells through a glycoprotein
of Surgery,
Nippon
Medical
with a molecular School, 1-1-5 Sendagi,
weight of Bunkyo-ku,
•\ 52•\
(52)
170 kDa, is encoded associated
by the c-erbB-1
with regional
gene.
EGF positive
and EGFR
positive
cases
lymph node metastasis
and poor prognosis
in breast,
The c-erbB-2 gene has the same structure
as the HER-2/neu
gene9, 10), whose
have been
gastric
and colonic
carcinomas5-8) .
glycoprotein of 185 kDa existing on cell surfaces. The protein that of the EGFR/c-erbB-1 oncoprotein11) . In breast, gastric staining
of the c-erbB-2 oncoprotein
has a sequence homology and colonic carcinomas,
has been shown to correlate
the expressions
of EGF, EGFR and the c-erbB-2 oncoprotein
characteristics
of human
pancreatic
cancer.
was observed
in several
pancreatic
cancer
EGFR biochemically
existed
in human
in tumor growth.
Hall et al.18) reported
noted in human
pancreatic
and the c-erbB-2 cancerous
cancer.
oncoprotein
in human
and non-inflammatory
relationship
between
and
examined
epineoplastic
Furthermore,
and the cancer's
as well as the patients' Materials
1.
cancer,
tissues.
invasion,
mean survival
the
EGFR
EGF and role
was immunohistochemically
immunohistochemically
pancreatic
pancreatic
those expressions
and lymph node metastasis
study
that
that
tissues and might play an important
that the c-erbB-2 oncoprotein This
very important
Gamou et al. 15)and Korc et al. 16)reported
cancer
close to positive
in order to clarify
cell lines. Chen et al.17) demonstrated
pancreatic
is a
with poor prognosis12) . Moreover,
the erbB-2 mRNA level rises earlier than the EGFR mRNA level13, 14). It is therefore to investigate
product
EGF, EGFR,
pancreatitis
in the cases histologic
and non-
of cancer,
pattern,
tumor
the size
period were also studied.
Methods
Materials
Thirty-five human pancreatic tissues were examined, of which 25, including 13 regional metastatic lymph nodes were tumorous and 10 were non-tumorous and non-inflammatory. The tissues were obtained through surgery at the First Department
of Surgery, Nippon Medical
School Hospital, from 1984 to 1989. 2. Immunohistochemical assay All of the specimens were fixed in neutralized 10% formalin solution, embedded in paraffin, and cut into 2.0 micrometer sections. The deparaffinized hydrogen peroxide (11202)/methanol temperature. For EGF staining
the sections
sections were pretreated
with 0.3%
(1: 100 dilution) and with 10% normal goat serum at room were incubated
with rabbit antiserum
to human EGF
(Wakunage Pharmaceutical Co., Ltd., Hiroshima, Japan) at a dilution of 1: 10 at 4°C for 24 hours and incubated with anti-rabbit IgG biotinylatd antibody (Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1: 50 at room temperature
for 1 hour.
For EGFR staining the sections were incubated with mouse antiserum
to human EGFR
(Tansformation Research, Inc., Framingham, MA) at a dilution of 1: 10 at 4°C for 48 hours and reacted with anti-mouse IgG biotinylated antibody (Vector Laboratories, Inc.) at a dilution of 1: 10 at 4°C for 24 hours. For c-erbB-2 oncoprotein staining the sections were incubated
with antiserum
to human
c-erbB-2 protein product (Nichirei Co., Ltd., Tokyo, Japan) at a dilution of 1: 50 at 4°C for 24 hours and reacted with anti-mouse IgG biotinylated antibody (Vector Laboratories, Inc.) at a dilution of 1: 100 at room temperature for 1 hour.
(53) •\53•\
Table
1
Histopathological and clinical factors in the general rules for cancer of the pancreas laid down by the Japan Pancreas Society
All the slices were then reacted with avidin-biotin-peroxidase an ABC kit (Vector Laboratories, submandibular
complex (ABC) for 1 hour with
Inc.). As positive controls, normal tissue from the human
gland was used for EGF, A431 cells for EGFR, and human breast cancer (tubular
adenocarcinoma) tissue for the c-erbB-2 oncoprotein. As negative controls, the sections were incubated with non-immunized rabbit IgG for EGF and with non-immunized mouse IgG for EGFR and the c-erbB-2 protein product instead of each immunized primary antiserum. After treatment with 0.03% diaminobenzidine solution containing 0.01% hydrogen peroxide, the sections were counterstained
with methylgreen for light microscopic examination.
The level of immunoreactivity of EGF, EGFR and the c-erbB-2 oncoprotein correlated as follows with the percentage of positively stained cancer cells: more than 50%,+++; 25 to 50%,++;less than 25%, +; 0%, -. 3. Histopathological All the tissues
and clinical evaluations
were stained
with hematoxylin
and eosin for light microscopic study.
Histological and clinical findings of carcinomas were classified according to the General Rules for Cancer of the Pancreas laid down by the Japan Pancreas Society19)(Table 1). 4.
Statistics
The data obtained in this study were analyzed for statistical significance using the chi-square test with Yates' correction or the Wilcoxon test. Results 1. The
Histopathological 25 primary
differentiated
tubular
lesions
findings were
classified
adenocarcinomas,
as follows:
6 moderately
1 papillary
differentiated
adenocarcinoma, tubular
14 well
adenocarcinomas,
1
•\ 54•\
(54)
Table
2
Characteristics
of the cancer
patients
poorly differentiated tubular adenocarcinoma, 1 mucinous adenocarcinoma, and 2 undifferentiated carcinomas. Carcinomas whose histological pattern was papillary adenocarcinoma, well or moderately differentiated tubular adenocarcinoma, or mucinous adenocarcinoma were defined here as differentiated types. Carcinomas whose histologic pattern was poorly differentiated tubular adenocarcinoma or undifferentiated carcinoma were defined as undifferentiated types. Adjacent to each cancerous lesion, there were fibrotic changes, atrophy of acini and hyperplasia of ductal epithelium, which are close to the findings of chronic pancreatitis.
Thirteen
lymph nodes with metastatic carcinomas were also histologically confirmed. In the 10 non-cancerous and non-inflammatory tissues, neither fibrosis, atrophy of acini, nor hyperplasia of ductal epithelium were found. 2.
Immunohistochemical
expressions
of EGF, EGFR
and
the c-erbB-2
onco-
protein in pancreatic carcinomas The characteristics of the cancer patients are shown in Table 2. EGF was stained mainly in the cytoplasm and on the surfaces of cancer cells in 18 of the 25 (72%) cases (Fig. 1) . EGFR was stained mainly on the surfaces and in the cytoplasm of the cells in 9 cases (36%) (Fig. 2). The c-erbB-2 oncoprotein was seen mainly on the surfaces and in the cytoplasm of the cells in 7 cases
(55) •\55•\
Fig.
Fig.
1
EGF
3
is found
both
in the
surface
of
(original
magnification, •~100)
The
some
c-erbB-2
surface
in the cells
and
the
Fig.
2
EGFR
cells
exists
mainly
of some
on
on
cytoplasm
the
Fig.
4
EGF
pancreatic
the
of some
(original
is seen
cytoplasm
(original
on
carcinoma .
oncoprotein
and
carcinoma
cytoplasm
pancreatic
.
exists
tissue
in some
of
(Fig.
shown
in
EGFR
was
3.
3) . The Table noted
only
in
the
inflammatory EGF the
aci•¬r
of
where
the
when
the
and
EGF
was
of
also
detected
expressions
tissues
of
EGF,
expression (Table
of
epineoplastic
EGFR
cells
(D) , acinar cells
pancreatitis
cells (E)
in
(original
.
the
product
cells
endothelial
epineolastic
and
of each
in the
c-erbB-2 is
oncoprotein
apparent.
In
all
is cases
4) .
EGF,
EGFR
pancreatitis
and and
the
in
c-erbB-2
onco-
non-cancerous/non-
tissues EGFR
were
of
epineoplastic
tissues cells
expressions
heterogenous
Immunohistochemical
protein
in
intensity
3,
partially carcinoma
.
ductal
(F) and
magnification, •~100)
(28%)
and
pancreatic
magnification, •~100)
(A) , fibroblasts
magnification, •~100)
surface
and
in
small
found
in some
ductal
pancreatitis numbers
of ductal
cells,
acinar
(Fig.
4,
5).
cells,
fibroblasts
cells, The
fibroblasts, c-erbB-2
and
and
endothelial
oncoprotein
endothelial
cells
was (Fig.
cells seen
in
6) . The
•\ 56•\
Table
(56)
3
Intensity of the expressions of EGF, EGFR and the c-erbB-2 oncoprotein in primary lesions of human pancreatic
Fig.
5
EGFR cells
is detected (A) , and
epineoplastic tion, •~100)
in some endothelial pancreatitis
ductal cells
Table
4
Relationship between the expressions of EGF and EGFR in primary lesions of pancreatic carcinoma
carcinoma
cells (E)
(original
(D) , acinar in
tissue
Fig. of
magnifica-
6
The
c-erbB-2
cells
(A) and
weakly
.
cells
oncoprotein in small
stained (E)
(original
in
exists
numbers
fibroblasts
tissue
(F)
and
of epineoplastic
magnification, •~100)
tissues in which these reaction products were noted in pancreatitis
in
some
of ductal
acinar cells
(D) ,
endothelial pancreatitis
.
coincided with those in
carcinoma. Regardless of whether positive cells were found or not in pancreatitis
and cancer,
positive cells existed only very rarely in all the surrounding non-inflammatory regions. Neither EGF, EGFR or the c-erbB-2 oncoprotein was detected in the islet cells. In non-cancerous/non-inflammatory tissues, EGF, EGFR and the c-erbB-2 oncoprotein were only very rarely found in ductal cells, acinar cells, fibroblasts or endothelial cells. 4.
The relationship
erbB-2
between
histological
findings
and EGF, EGFR
and
the c-
oncoprotein
As shown in Table 5, a significant correlation was demonstrated between the invasion into the anterior area (s) and EGF, EGFR and the c-erbB-2 oncoprotein (p
Nippon
•\O
Med.
riginal
Sch.,
Vol.
59,
No.
1
(1992)
(51) •\51•\
Articles•\
The
immunohistochemical
expressions
growth factors, epidermal receptors and c-erbB-2 in human
growth factor oncoprotein
pancreatic
Yoichiro The First Department
of epidermal
cancer
Yamanaka
of Surgery,
Nippon Medical
School
Summary
The expressions of epidermal growth factors (EGF), epidermal growth factor receptors (EGFR), and the c-erbB-2 oncoprotein were immunohistochemically examined in 25 cases of human pancreatic carcinoma and epineoplastic pancreatitis and in 10 non-cancerous/noninflammatory pancreatic tissues. The positive rates of EGF, EGFR, and the c-erbB-2oncoprotein in cancer tissues were 72%,36%,and 28%,respectively. EGF was stained mainly in the cytoplasm and partly on the surfaces of the cancer cells. EGFR and the c-erbB-2oncoprotein were stained mainly on the surfaces of the cancer cells and partly in the cytoplasm. The expressions of these 3 products correlated significantly with tumor invasion into the anterior and posterior areas surrounding the pancreas. In the EGF, EGFR, and c-erbB-2positive cancer tissues, some stromal cells, that is fibroblasts and endothelial cells, were also positive. In the adjacent pancreatic tissues with inflammation, these products were noted in some ductal cells, acinar cells, fibroblasts and endothelial cells. No distinct staining was detected in non-cancerous/non-inflammatory tissues. The survival period for patients who tested positive for these three proteins was statistically shorter than for those who tested negative. These results suggest that the coexpression of EGF and EGFR and the expression of the c-erbB-2 oncoprotein are related to the existence of the invasion of human pancreatic cancer. Furthermore, an immunohistochemical examination of these three products is useful in forming a prognosis for pancreatic cancer patients. Key words: human pancreatic cancer, epidermal growth factor, epidermal growth factor receptor, c-erbB-2oncoprotein, immunohistochemistry Introduction Among
the gastrointestinal
carcinomas,
human
pancreatic
cancer is well known for its poor
prognosis. Recently, many studies have suggested that expressions of growth factors and oncoproteins might be associated with lymph node metastasis, tumor size, infiltration, histologic grade and prognosis1) . The epidermal growth factor, stimulates proliferation of not only normal epidermal the epidermal Correspondence
growth to Yoichiro
Tokyo 113, Japan
factor
receptor2, 3). This receptor,
Yamanaka,
The First Department
a polypeptide consisting of 53 amino acids, cells but also some carcinoma cells through a glycoprotein
of Surgery,
Nippon
Medical
with a molecular School, 1-1-5 Sendagi,
weight of Bunkyo-ku,
•\ 52•\
(52)
170 kDa, is encoded associated
by the c-erbB-1
with regional
gene.
EGF positive
and EGFR
positive
cases
lymph node metastasis
and poor prognosis
in breast,
The c-erbB-2 gene has the same structure
as the HER-2/neu
gene9, 10), whose
have been
gastric
and colonic
carcinomas5-8) .
glycoprotein of 185 kDa existing on cell surfaces. The protein that of the EGFR/c-erbB-1 oncoprotein11) . In breast, gastric staining
of the c-erbB-2 oncoprotein
has a sequence homology and colonic carcinomas,
has been shown to correlate
the expressions
of EGF, EGFR and the c-erbB-2 oncoprotein
characteristics
of human
pancreatic
cancer.
was observed
in several
pancreatic
cancer
EGFR biochemically
existed
in human
in tumor growth.
Hall et al.18) reported
noted in human
pancreatic
and the c-erbB-2 cancerous
cancer.
oncoprotein
in human
and non-inflammatory
relationship
between
and
examined
epineoplastic
Furthermore,
and the cancer's
as well as the patients' Materials
1.
cancer,
tissues.
invasion,
mean survival
the
EGFR
EGF and role
was immunohistochemically
immunohistochemically
pancreatic
pancreatic
those expressions
and lymph node metastasis
study
that
that
tissues and might play an important
that the c-erbB-2 oncoprotein This
very important
Gamou et al. 15)and Korc et al. 16)reported
cancer
close to positive
in order to clarify
cell lines. Chen et al.17) demonstrated
pancreatic
is a
with poor prognosis12) . Moreover,
the erbB-2 mRNA level rises earlier than the EGFR mRNA level13, 14). It is therefore to investigate
product
EGF, EGFR,
pancreatitis
in the cases histologic
and non-
of cancer,
pattern,
tumor
the size
period were also studied.
Methods
Materials
Thirty-five human pancreatic tissues were examined, of which 25, including 13 regional metastatic lymph nodes were tumorous and 10 were non-tumorous and non-inflammatory. The tissues were obtained through surgery at the First Department
of Surgery, Nippon Medical
School Hospital, from 1984 to 1989. 2. Immunohistochemical assay All of the specimens were fixed in neutralized 10% formalin solution, embedded in paraffin, and cut into 2.0 micrometer sections. The deparaffinized hydrogen peroxide (11202)/methanol temperature. For EGF staining
the sections
sections were pretreated
with 0.3%
(1: 100 dilution) and with 10% normal goat serum at room were incubated
with rabbit antiserum
to human EGF
(Wakunage Pharmaceutical Co., Ltd., Hiroshima, Japan) at a dilution of 1: 10 at 4°C for 24 hours and incubated with anti-rabbit IgG biotinylatd antibody (Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1: 50 at room temperature
for 1 hour.
For EGFR staining the sections were incubated with mouse antiserum
to human EGFR
(Tansformation Research, Inc., Framingham, MA) at a dilution of 1: 10 at 4°C for 48 hours and reacted with anti-mouse IgG biotinylated antibody (Vector Laboratories, Inc.) at a dilution of 1: 10 at 4°C for 24 hours. For c-erbB-2 oncoprotein staining the sections were incubated
with antiserum
to human
c-erbB-2 protein product (Nichirei Co., Ltd., Tokyo, Japan) at a dilution of 1: 50 at 4°C for 24 hours and reacted with anti-mouse IgG biotinylated antibody (Vector Laboratories, Inc.) at a dilution of 1: 100 at room temperature for 1 hour.
(53) •\53•\
Table
1
Histopathological and clinical factors in the general rules for cancer of the pancreas laid down by the Japan Pancreas Society
All the slices were then reacted with avidin-biotin-peroxidase an ABC kit (Vector Laboratories, submandibular
complex (ABC) for 1 hour with
Inc.). As positive controls, normal tissue from the human
gland was used for EGF, A431 cells for EGFR, and human breast cancer (tubular
adenocarcinoma) tissue for the c-erbB-2 oncoprotein. As negative controls, the sections were incubated with non-immunized rabbit IgG for EGF and with non-immunized mouse IgG for EGFR and the c-erbB-2 protein product instead of each immunized primary antiserum. After treatment with 0.03% diaminobenzidine solution containing 0.01% hydrogen peroxide, the sections were counterstained
with methylgreen for light microscopic examination.
The level of immunoreactivity of EGF, EGFR and the c-erbB-2 oncoprotein correlated as follows with the percentage of positively stained cancer cells: more than 50%,+++; 25 to 50%,++;less than 25%, +; 0%, -. 3. Histopathological All the tissues
and clinical evaluations
were stained
with hematoxylin
and eosin for light microscopic study.
Histological and clinical findings of carcinomas were classified according to the General Rules for Cancer of the Pancreas laid down by the Japan Pancreas Society19)(Table 1). 4.
Statistics
The data obtained in this study were analyzed for statistical significance using the chi-square test with Yates' correction or the Wilcoxon test. Results 1. The
Histopathological 25 primary
differentiated
tubular
lesions
findings were
classified
adenocarcinomas,
as follows:
6 moderately
1 papillary
differentiated
adenocarcinoma, tubular
14 well
adenocarcinomas,
1
•\ 54•\
(54)
Table
2
Characteristics
of the cancer
patients
poorly differentiated tubular adenocarcinoma, 1 mucinous adenocarcinoma, and 2 undifferentiated carcinomas. Carcinomas whose histological pattern was papillary adenocarcinoma, well or moderately differentiated tubular adenocarcinoma, or mucinous adenocarcinoma were defined here as differentiated types. Carcinomas whose histologic pattern was poorly differentiated tubular adenocarcinoma or undifferentiated carcinoma were defined as undifferentiated types. Adjacent to each cancerous lesion, there were fibrotic changes, atrophy of acini and hyperplasia of ductal epithelium, which are close to the findings of chronic pancreatitis.
Thirteen
lymph nodes with metastatic carcinomas were also histologically confirmed. In the 10 non-cancerous and non-inflammatory tissues, neither fibrosis, atrophy of acini, nor hyperplasia of ductal epithelium were found. 2.
Immunohistochemical
expressions
of EGF, EGFR
and
the c-erbB-2
onco-
protein in pancreatic carcinomas The characteristics of the cancer patients are shown in Table 2. EGF was stained mainly in the cytoplasm and on the surfaces of cancer cells in 18 of the 25 (72%) cases (Fig. 1) . EGFR was stained mainly on the surfaces and in the cytoplasm of the cells in 9 cases (36%) (Fig. 2). The c-erbB-2 oncoprotein was seen mainly on the surfaces and in the cytoplasm of the cells in 7 cases
(55) •\55•\
Fig.
Fig.
1
EGF
3
is found
both
in the
surface
of
(original
magnification, •~100)
The
some
c-erbB-2
surface
in the cells
and
the
Fig.
2
EGFR
cells
exists
mainly
of some
on
on
cytoplasm
the
Fig.
4
EGF
pancreatic
the
of some
(original
is seen
cytoplasm
(original
on
carcinoma .
oncoprotein
and
carcinoma
cytoplasm
pancreatic
.
exists
tissue
in some
of
(Fig.
shown
in
EGFR
was
3.
3) . The Table noted
only
in
the
inflammatory EGF the
aci•¬r
of
where
the
when
the
and
EGF
was
of
also
detected
expressions
tissues
of
EGF,
expression (Table
of
epineoplastic
EGFR
cells
(D) , acinar cells
pancreatitis
cells (E)
in
(original
.
the
product
cells
endothelial
epineolastic
and
of each
in the
c-erbB-2 is
oncoprotein
apparent.
In
all
is cases
4) .
EGF,
EGFR
pancreatitis
and and
the
in
c-erbB-2
onco-
non-cancerous/non-
tissues EGFR
were
of
epineoplastic
tissues cells
expressions
heterogenous
Immunohistochemical
protein
in
intensity
3,
partially carcinoma
.
ductal
(F) and
magnification, •~100)
(28%)
and
pancreatic
magnification, •~100)
(A) , fibroblasts
magnification, •~100)
surface
and
in
small
found
in some
ductal
pancreatitis numbers
of ductal
cells,
acinar
(Fig.
4,
5).
cells,
fibroblasts
cells, The
fibroblasts, c-erbB-2
and
and
endothelial
oncoprotein
endothelial
cells
was (Fig.
cells seen
in
6) . The
•\ 56•\
Table
(56)
3
Intensity of the expressions of EGF, EGFR and the c-erbB-2 oncoprotein in primary lesions of human pancreatic
Fig.
5
EGFR cells
is detected (A) , and
epineoplastic tion, •~100)
in some endothelial pancreatitis
ductal cells
Table
4
Relationship between the expressions of EGF and EGFR in primary lesions of pancreatic carcinoma
carcinoma
cells (E)
(original
(D) , acinar in
tissue
Fig. of
magnifica-
6
The
c-erbB-2
cells
(A) and
weakly
.
cells
oncoprotein in small
stained (E)
(original
in
exists
numbers
fibroblasts
tissue
(F)
and
of epineoplastic
magnification, •~100)
tissues in which these reaction products were noted in pancreatitis
in
some
of ductal
acinar cells
(D) ,
endothelial pancreatitis
.
coincided with those in
carcinoma. Regardless of whether positive cells were found or not in pancreatitis
and cancer,
positive cells existed only very rarely in all the surrounding non-inflammatory regions. Neither EGF, EGFR or the c-erbB-2 oncoprotein was detected in the islet cells. In non-cancerous/non-inflammatory tissues, EGF, EGFR and the c-erbB-2 oncoprotein were only very rarely found in ductal cells, acinar cells, fibroblasts or endothelial cells. 4.
The relationship
erbB-2
between
histological
findings
and EGF, EGFR
and
the c-
oncoprotein
As shown in Table 5, a significant correlation was demonstrated between the invasion into the anterior area (s) and EGF, EGFR and the c-erbB-2 oncoprotein (p
