Download Cathepsin B Expression and Lamimn Degradation as Factors

[CANCER RESEARCH 54, 6133—6136, December 1, 1994)
Cathepsin B Expression and Lamimn Degradation as Factors Influencing Prognosis
of Surgically Treated Patients with Lung Adenocarcinoma'
Takashi Inoue,2 Teruyoshi Ishida, Kenji Suglo, and Keizo Sugimachi
Department ofSurgery
II, Faculty ofMedicine,
Kyushu University, Maidashi 3-1-1. Higashi-ku, Fukuoka 812, Japan
Department
ABSTRACT
We examined, immunohistochemically, tissues from primary lung ad
enocarcinomas. In 142 tumors, the mean overall labeling percentage of
cathepsin B was 263 ±22.3 (SD). The mean labeling percentage of
cathepsin B in cases with stage I disease was lower than that in cases with
stages ifiA, HIB, or IV disease (P < 0.05). Ofthe 115 tumors examined for
Inminin-positive
basement membranes, 54 (47%) had a continuous pat
tern and 61 (53%) had a discontinuous
pattern. The mean labeling
percentage ofcathepsin B was 35.0 ±24.2 in tumors with a discontinuous
pattern, compared with the 21.9 ±16.9 in those with a continuous pattern
(P < 0.01). The overall
5-year
survival
rates of patients
with high and low
cathepsin B expressions were 26% and 77%, respectively (P < 0.01),
including 45% and 94% for patients with stage I disease, respectively
(P < 0.01),
and 15%
and 60%
for those
with
stage
ifiB
disease,
respec
tively (P < 0.05). Multivarlate analysis using the Cox life table regressIon
model showed cathepsin B to be a significantly Independent factor asso
of Surgery
II, Faculty
of Medicine,
at Kyushu
University.
Data on
patients who died within the first postoperative month were excluded from the
present analysis. The stage of the disease was classified according to the size
of the tumor, nodal involvement, and the presence of distant metastasis (TNM
classification), using guidelines from the International Union Against the
Cancer (11). There were 68 patients with stage I, 9 with stage II, 34 with stage
lIlA, 16 with stage IIIB, and 15 patients with stage IV. There were 142
patients,
88 men and 54 women,
and their ages ranged
from 35 to 82 years
(mean, 63.1 years). Histology of the disease and histological degree of differ
entiation were determined according to the WHO classification system (12).
There were 75 patients with well-differentiated adenocarcinoma, 41 with
moderately differentiated adenocarcinoma, and 26 with poorly-differentiated
adenocarcinoma. Complete resection of the tumor by lobectomy or pneumo
nectomy combined with hilar and mediastinal lymphadenectomy was per
formed whenever it was medically feasible. All resected specimens were fixed
elated with death due to the disease We conclude from this study that
in 10% formalin, paraffin-embedded blocks were prepared, and each was cut
into 4 g.tm-thick slices. Each tumor was examined from a few sections that
tumors with a discontinuous
were divided from the cut surface with the greatest diameter. For routine
pattern
oflaminin
have a higher percentage
of cathepsin B, and the survival rate was poor for patients with a high
expressIon of cathepsin B. Thus, cathepsin B may be useM in assessing
histological
prognosis
tibody to cathepsin B was obtained from the Binding Site Ltd., Institute of
Researchand Development(Birmingham,EnglandPC 049, A 6576).An indirect
in lung adenocarcinoma.
studies
Immunostainlng
the sections
were stained
Procedure
of Cathepsin
with hematoxylin
and eosin.
B. The primary polyclonal an
INTRODUCTION
staining
Cathepsin B is a lysosomal cysteine proteinase which degrades the
extracellular matrix and the basement membrane; it includes proteo
(13). The deparaffinizedsectionswere treatedwith 0.03% hydrogenperoxidasein
100%methanolfor 25 miii at room temperatureto inhibitendogenousperoxidase.
Possiblebackgroundstainingwas also removedby applyingnormal rabbit serum,
glycan,
diluted 1:10, for 30 mm at room temperature. We then added sheep cathepsin B
collagen,
fibronectin,
and laminin
(1). Other
investigators
found that the enzyme activity of cathepsin B in malignant tissue was
higher than it was in normal tissue and that it was related to the
invasion and metastasis of the cancer (2, 3). Sloane et al. (4) used a
metastatic variant of B16 melanoma and found that the metastatic
capability correlated with cathepsin B activity in subcutaneous tumors
of mice. Laminin is a basement membrane adhesive glycoprotein of
about M1 1,000,000 (5) and it mediates the attachment of epithelial
and neoplastic cells. This glycoprotein facilitates the attachment of
metastatic tumor cells to other matrix components. There are data
showing that the destruction of laminin is related to a poor prognosis
(6, 7).
In an immunohistochemical study on breast cancer in which axil
lary nodes were negative, a high level cathepsin D, an aspartic
proteinase, was shown to be a prognostic factor (8). As for cathepsin
B, studies on enzyme activity in various human malignant tissue were
done (9, 10). Relationships between proteinase and basement mem
brane, with regard to clinical prognosis, apparently have not been
documented. We attempted to clarify correlations between the fmd
ings with cathepsin B and laminin concerning the clinical prognosis.
MATERIALS
technique
and the avidin-biotin-peroxidase
complex
method
were used
polyclonal antibody,diluted 1:200, for 2 h at room temperature . After washing
each section with phosphate-buffered saline, we applied a biotinylated secondary
antibody and avidin with biotinylatedhorseradishperoxidase (Vector Laborato
rica, Inc., Burlingame,
CA). Peroxidase
labeling was developed
with 3,3'-diami
nobenzidine and hydrogen pemxidase, and sections were evaluated as positive or
negative;
a reaction
was considered
positive
only when strong
brown
deposits
were visible. Omission of the primary antibody resulted in a negative staining in
all cases. The proportion
ofcathepsin
B-positive
cells was determined
by counting
500 cancer cells from 5 high-power fields at random. The overall mean labeling
percentagewas 26.5 ±22.3. Patientswere separatedinto two groups: for patients
with less than the mean labeling percentage of cathepsin B in the tumor cell, the
designationwas a low cathepsin B; and those with a mean labelingpercentageor
more of cathepsin B were considered to have a high percentage of cathepsin B.
Immunostainlng Procedure of Laminin. The primary monoclonal anti
body
to laminin
Temecula,
was
obtained
CA) and an indirect
from
Chemicon
staining
technique
International,
Inc.
(04—91;
and the strept-avidin-biotin
peroxidase complex method was used (14). After blocking endogenous
per
oxidase with H202, all deparaffinized sections were incubated with 0.4%
pepsin in 0.01 n HO for 3 h at 37°Cto reveal the antigenic site of the basement
membrane. Possible background staining was also removed by applying nor
mal goat serum, diluted 1:10, for 10 mm at room temperature. We applied
rabbit laminin monoclonal antibody, diluted 1:250, overnight at room temper
AND METHODS
Surgical Specimens. Between January 1985 and May 1991, 142 patients
with primary adenocarcinoma of the lung were surgically treated in the
ature, then biotinylated secondary antibody and strept-avidin with rabbitradish
peroxidase (Nichirei Corp., Tokyo, Japan) were applied for 10 and 5 mm,
respectively. Positive controls were those with a basement membrane under
lying the endothelium of vessels and the normal bronchial mucosa. Depending
Received 4/6/94; accepted 10/3/94.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 This
work
was
supported
in
part
by
Grant-in-Aid
06671350 from the Ministry of Education, Japan.
2 To
whom
requests
for
reprints
should
be addressed.
for
General
Scientific
Research
on the continuity of laminin-positive basement membrane in the tumor, we
separated patients into two groups; in cases when the basement membrane
resembled the normal respiratory mucosa and the vessels of a normal lung the
designation was a continuous pattern; those with a focally defective basement
membrane, a fragmentary basement membrane, or without a basement mem
brane were considered to have a discontinuous pattern.
6133
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@
•
CAThEPSIN B AND LAMININ IN LUNG CANCER
Statistical
Analysis.
The BMDP statistical package program (BMDP, Los
Angeles, CA) for the IBM (Armok, NY) 4381 mainframe computer was used
for the @2
test, or Fisher's exact test was used to analyze the statistical
Table 1 Mean percentage of cathepsin B intients adenocarcinomaLabeling
with lung
BVariablesNo.
of patients(mean
SD)SexMale8826.3±21.7Female5426.9
% of cathepsin
±
significance of differences between immunoreactivity for cathepsin B and
clinicopathological factors. Fisher's exact test was used where there were six
or fewer items in a group. The survival rates were calculated by the Kaplan
Meier method (15). Comparison among the survival curves was made using the
Log rank test (16). The BMDP P2L program was used for multivariate
adjustment of covariates by the Cox regression analysis (17). In all analysis,
the difference was considered significant when P < 0.05.
23.4StageI6820.8±
±
17.3II920.7
22W'lIlA3433.2
±
24.0IIIB16313@[email protected]
±
RESULTS
Cathepsin B in Lung Adenocarcinoma. The immunoreactivity of
cathepsin B was diffuse and strong in the cytoplasm of some cancer
cells (Fig. 1A). The overall mean labeling percentage of cathepsin B
was 26.5 ± 22.3. The mean labeling percentage of cathepsin B
assessed according to various factors is given in Table 1. The mean
labeling percentage of cathepsin B in cases with stage I disease was
statistically lower than that in cases with stage LIlA, IIIB, or IV
disease (P < 0.05), and the mean-labeling percentage of cathepsin B
in cases with complete resection was statistically lower than that in
cases with incomplete resection (P < 0.01).
A
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-—i..
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.-@
.
22.4Moderate4126.3
22.5Poor2624.7
22.5CurabilityComplete10922.9
±
20.6Incomplete3338.4
±
23W'LamininContinuous5421.9
±
16.9Discontinuous6135.0
±
2.4.2―Total14226.5
±
22.3ap<001bp
±
<0.05.
Laminin in Lung Adenocarcinoma.
The distribution of laminin
in a tumor-associated basement membrane showed an immunohisto
chemically continuous or discontinuous pattern, and the pattern in
normal tissue-associated basement membrane underlying the respira
tory epithelia and surrounding the vascular structure was always
continuous (Fig. 1B). Of the 115 patients there were 54 (47%) with a
continuous pattern and 61 (53%) with a discontinuous basement
membrane pattern. There was no statistically significant difference
among various clinicopathological factors.
Correlation of Cathepsin B Expression with Laminin Degrada
lion. In the serial section, laminin disappeared in the areas in contact
with cathepsin B-positive tumor cells (Figs. 1, A and B). To search for
possible degradation of the basement membrane by the tumor cells,
the proportion of cathepsin B-positive tumor cells was determined
according to continuity of the laminin distribution patterns. The mean
labeling percentages of cathepsin B were 35.0 ±24.2 in tumors
with a discontinuous pattern of laminin and 21.9 ± 16.9 in those
with a continuous pattern, with a statistically significant difference
(P < 0.01)
Impact
. @>,_
.
±
±
(Table
1).
on Survival.
The overall
5-year
survival
rates of patients
with high and low cathepsin B percentages were 26% and 77%,
respectively (P < 0.01), 5-year survival rates for various clinicopath
ological factors are given in Table 2. In the univariate analysis,
patients with high cathepsin B were associated with a poor prognosis
5 years after surgery for sex, stage I, stage IIIB, well differentiated,
moderately differentiated, complete resection and laminin (P < 0.05).
The result of multivariate analysis using the Cox life table regres
sion model is shown in Table 3. T-factor, degree of differentiation,
expression of cathepsin B, and tumor size proved to be independent
prognostic factors (P < 0.05).
@;T@@@T:
DISCUSSION
The basement membrane is a major barrier to tumor invasion and
metastasis. Liotta (18) described a three-step theory of invasion into
the extracellular matrix. In these processes, malignant neoplastic cells
Fig. 1. A, immunostaining for cathepsin B in lung adenocarcinoma. Note the immu
attach to the basement membrane, after which the attached tumor
noreactive products of cathepsin B in tumor cells (arrows) in the infiltrating area of tumor.
B, immunostaining of laminin in the same case of A. Note the discontinuous basement
cells secrete hydrolytic enzymes that locally degrade the matrix,
membrane of laminin in three areas (arrows) in contact with cathepsin B-positive tumor
and finally, the tumor cells move into the proteolytically modified
cells. The continuous basement membrane of laminin was found in contact with cathepsin
matrix.
B-negative tumor cells. X130.
6134
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CATHEPSIN
B AND LAMININ
Because cathepsin B plays an important role in these processes
(19), it was reported to be closely associated with tumor invasion and
metastasis. Cathepsin B directly degrades: (a) collagen (20); (b)
collagen by activation of collagenase (21); and (c) laminin (22). When
a cytosolic assay for mRNA of cathepsin B was done using human
colon carcinoma, the level in carcinomatous tissue was elevated over
that in normal tissue (23). In case of gastric carcinoma, the enzyme
activity of cathepsin B is closely related to the progression of disease
(10). We noted the same tendency in our immunohistochemical study
with lung adenocarcinoma; the more progressive the stage of the
disease, the greater the increase in cathepsin B.
The urokinase-type plasminogen activator belongs to serine pro
teinase, and it transforms plasminogen into plasmin, which is active
on a number of substances and is an important factor linked to the
prognosis of patients with lung adenocarcinoma (24). No other pro
teinase has been shown to be a prognostic factor thus, our findings are
the first of a correlation between cathepsin B and laminin. For the
patients for whom cathepsin B is overexpressed there is a progressive
destruction of laminin, one component of the basement membrane. It
was reported that the loss of laminin significantly led to an increase in
the incidence of metastasis and that survival time was reduced in the
cases of lung (6) and rectal cancer
(25).
We found that the survival rate of patients with a high expression
of cathepsin B was significantly shorter than it was in those with a low
expression of cathepsin B. Particularly in stage I disease, the expres
sion of cathepsin B provides useful and precise information on the
prognosis. In general, even in stage I disease the recurrence rate in
patients treated by surgery is about 40% (26). It may be one of the
reasons why the patient with stage I disease might have been hema
togenous micrometastasis at the time of operation. In the case of a
Table
2 Sur@'iva1rates ofpatients with lung adenocarcinoma
degreeVariables
of cathepsin B expressione
(%)PSexMale
Cathepsin B
High
33<0.01Low
73Female
No. of patients
High
30<0.01Low
94II
High
0NW'Low
High
15<0.05Low
60IV
High
19NSLow
10
5
High
33<0.01Low
34
41
0.0214
Size0.6127
1.8454
0.3617
1.1846
0.012
0.013
1.02170.000
0.029
higher expression of cathepsin B, cancer invasion and metastasis are
more advanced than we had expected; therefore it may be a case of
stage IV disease, from the view of biological behavior.
Both cathepsin B and laminin stain easily so paraffin embedded
tissue sections can be used. Immunohistochemical techniques have an
advantage over cytosolic assays for analyzing cathepsin B. We ex
amined the overexpression of cathepsin B in cancer cells using a light
transmission microscope, but the enzyme activity cannot distinguish
between cathepsin B in alveolar macrophages from that in cancer
cells. In contrast, one might observe cathepsin B expressed in cancer
cells without enzyme activity because the activity of cathepsin B can
be inactivated by low molecular weight, endogenous cysteine protein
ase inhibitors (27). So we must take such inhibitors into consideration
on the studying for cathepsin B.
In summary, the expression of cathepsin B is related to the aggres
siveness of the tumor and the prognosis is poor. These findings can
serve as a pertinent prognosis factor to determine the postoperative
therapeutics when the tumors have been completely resected.
ACKNOWLEDGMENTS
We thank Mariko Ohara for comments on the manuscript.
5. Terranova, V. P., Liotta, L. A., Russo, R. 0., and Martin, 0. R. Role of laminin in the
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6
53DifferentiationWell
—1.0170
0.1694
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20
48
3
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Odds
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High
45<0.01Low
Table 3lamininVariableEstimated
Multivariateanalysis of clinicopathologicalfactors.
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Cathepsin B Expression and Laminin Degradation as Factors
Influencing Prognosis of Surgically Treated Patients with Lung
Adenocarcinoma
Takashi Inoue, Teruyoshi Ishida, Kenji Sugio, et al.
Cancer Res 1994;54:6133-6136.
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