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Molecular Oncology, Markers, Clinical Correlates |
Department of Surgery, Chirurgische Klinik und Poliklinik Klinikum Innenstadt, Ludwig-Maximilians University, 80336 Munich [B. P., W. S., R. S-H., O. T., W. M.]; Departments of Thoracic Surgery [B. P., W. S., O. T.] and Pathology [W. W.], Asklepios Fachkliniken München-Gauting, 82131 Gauting; and Division of Molecular Oncology, University Hospital Eppendorf, 20246 Hamburg [K. P.], Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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80% of all
of the lung cancer patients (1
, 3)
. The standard treatment
for early-stage NSCLC is surgery, which results in a 5-year survival of
only 50–60% in stages I and II (4
, 5)
. Approximately
40% of stage I patients who are postoperatively staged as tumor free
relapse within 24 months after surgery (6)
. Better
understanding of the molecular mechanisms of lung cancer progression
might help to identify this group of patients, with potential
consequences for adjuvant therapy. Several steps of tissue destruction are considered essential for spread of tumor cells. Degradation of extracellular matrix and penetration of basement membranes is known to play an important role in tumor invasion and metastasis (7) . One group of proteolytic enzymes that has been associated with this ability is the MMP family (7 , 8) . Several recent reports confirmed involvement of MMPs in NSCLC (9, 10, 11) . Specific MMP inhibitors will soon be available for advanced clinical trials evaluating adjuvant therapy for NSCLC (12) . MMP-2 (also known as gelatinase A) has been implicated in lymphatic and vascular invasion of NSCLC (9) , but prognostic value of MMP-2 expression in NSCLC has thus far not been documented. Thus, this study was performed to assess the impact of MMP-2 expression on prognosis and outcome of operable NSCLC. The expression of MMP-2 in 193 specimens of primary surgery for NSCLC was examined by immunohistochemistry to analyze possible correlation with relapse and survival after long-term follow-up. This is the first report demonstrating that MMP-2 overexpression is an independent risk factor for poor prognosis in NSCLC.
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PATIENTS AND METHODS |
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The median follow-up duration was 71.5 months (range, 12–120 months). Follow-up studies included physical examination, chest X-ray, and blood tests at 3-month intervals and an additional thoracic CT scan, abdominal ultrasound, and bronchoscopy in 6-month intervals. Close follow-up was documented by contacting family practitioners with questionnaires concerning local relapse, distant metastasis, and death. If possible, a relapse was confirmed at our institution, and the patient was admitted for subsequent therapy.
Immunohistochemical Analysis.
The presence of MMP-2 was analyzed by immunohistochemical staining
using the LSAB method. Briefly, paraffin sections were dewaxed,
rehydrated, and subsequently incubated with 0.08% trypsin (Sigma,
Taufkirchen, Germany) for 30 min at 25°C. Endogenous peroxidase
activity was blocked by treating the specimens with 30% hydrogen
peroxide for 10 min. Nonspecific antibody binding was prevented by
commercial blocking agent (LSAB-kit, Dako, Hamburg, Germany). Excess
blocking agent was drained, and the sections were incubated overnight
at 4°C with anti-MMP-2 primary antibody AB809 (Chemicon Inc.,
Temecula, CA).
The polyclonal rabbit antibody recognizes the hinge region of both the latent 72-kDa proenzyme and the active 62-kDa form of MMP-2 as well as MMP-2 in complex with tissue inhibitor of metalloproteinase 2. No cross-reactivity against other MMP family members was observed. AB809 (1 mg/ml) was used in 1:200 dilution with background-reducing antibody diluent (Dako). For the negative controls the primary antibody was replaced with rabbit nonimmune IgG (Southern Biotechnology Associates Inc., Birmingham, AL). Immunohistological staining was continued by incubating the slides with biotinylated antimouse/antirabbit secondary antibody solution (LSAB kit, Dako) for 30 min at 25°C. Peroxidase was introduced using a streptavidin conjugate (LSAB kit, Dako). Between all of the stages of the procedure the specimens were thoroughly rinsed with 0.1 M Tris-HCl buffer (pH 8.2). Peroxidase reactivity was revealed using aminoethyl carbazole (Sigma) dissolved in dimethylformamide and 0.1 M acetate buffer (pH 5.2), creating a red-brown staining with 30% hydrogen peroxide. Finally, the sections were counterstained with hematoxylin and mounted in Kaiser’s glycerol gelatin.
Evaluation of the Specimens.
The slides were examined under a Leitz laborlux light-microscope
(Leitz, Wetzlar, Germany) using objectives with x10 and x40
magnifications (Fig. 1)
. Macrophages in
the sections served as positive controls. Staining intensity is
unreliable because it varies between batches, and, therefore, it was
not useful for analysis of MMP-2 expression. Immunostaining was
analyzed semiquantitatively by verifying the distribution of stained
cancer cells in the center of the tumor. The tumors were divided into
three groups: +, homogeneous staining of cancer cells; +/-,
heterogeneous staining of cancer cells; -, no staining of cancer
cells. In further analyses homogeneous staining (+) was considered
positive and heterogeneous (+/-) or no immunostaining (-) was
considered negative concerning overexpression of MMP-2. Staining
specificity was assessed by evaluation of the negative controls in
which the primary antibody had been replaced with normal rabbit
nonimmune IgG. This assessment of negative controls revealed unspecific
staining in four cases that were excluded from further analyses. All
slides were examined by two independent observers who were unaware of
the clinical data. The slides with discrepant evaluations were
reevaluated, and a consensus was reached.
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2
test.
For analysis of follow-up data, life table curves were calculated with
the Kaplan-Meier method, and survival distributions were compared by
log-rank statistics. Relapse and death hazard rates were estimated by
dividing the number of events by persons at risk and years of
follow-up. The Cox proportional hazards model was applied for
multivariate analysis using the SPSS software program (SPSS Inc.,
Chicago, IL). For this analysis all of the variables were dichotomized.
Age as the only continuous variable was dichotomized at the median (60
years). The threshold for statistical significance was chosen as
P = 0.05. |
RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evaluation of Immunohistochemical Staining.
The majority of the 189 specimens exhibited either completely negative
or highly homogenous staining of cancer cells. No staining of MMP-2 was
observed in 81 specimens (42.9%), and staining of MMP-2 was homogenous
in 64 tumors (33.9%). The remaining 44 specimens (23.2%) exhibited an
intermediate pattern of MMP-2 staining. For further analyses homogenous
staining of MMP-2 was considered positive, and intermediate or no
staining of MMP-2 was considered negative concerning MMP-2
overexpression. Assessment of immunohistochemical MMP-2 detection in
fibroblasts revealed a negative or weak, heterogeneous staining of
fibroblasts in all of the specimens.
Analysis of Survival and Relapse.
In total, overexpression of MMP-2 was observed in 64 patients (33.9%).
There was no significant correlation with clinicopathological
parameters such as tumor extension, lymph node involvement, staging,
grading, tumor histology, age, or sex. Counter to one’s expectations,
MMP-2 overexpression was not associated with the presence of lymph node
metastasis or tumor extension (Table 1)
.
However, univariate analysis revealed a relationship between
overexpression of MMP-2 and unfavorable outcome in operable NSCLC (Fig. 2)
. Among the 175 eligible patients there
was a tendency toward an increased cancer-related death rate (0.070
versus 0.058; P = 0.06) and relapse rate
(0.072 versus 0.058; P = 0.09) associated
with MMP-2 overexpression. Subsequent stratification according to
postoperative stage and lymph node status showed that in patients with
no lymph node involvement MMP-2 overexpression predicted for both
shortened disease-free and shortened cancer-related survival (Fig. 3)
. The log-rank test Ps for
cancer-related and disease-free survival in N0
disease were 0.04 and 0.03, respectively.
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. The Cox regression
analysis was performed with a total of 90 eligible patients because of
prior exclusion of 6 patients with cancer-unrelated death or incomplete
follow-up. Overexpression of MMP-2, tumor size, age, and tumor
histology were tested for independence of a possible prognostic value.
It was unnecessary to consider therapy as an extra variable because it
was already taken into account by tumor size, which considers
pT3 patients who received adjuvant radiotherapy.
The multivariate regression analysis demonstrated that MMP-2
overexpression was a significant (P = 0.005)
independent prognostic predictor for shortened cancer-related survival
in node-negative patients. The relative risk for cancer-related death
in pN0 patients was 2.6-fold greater in case of
MMP-2 overexpression (95% confidence interval, 1.3–5.1). Compared
with other factors, the risk for cancer-related death in MMP-2-positive
cases was approximately as high as that in patients who were >60 years
old.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although immunohistochemical studies of MMP-2 expression in NSCLC have already been conducted (10 , 14 , 18) , no reports have thus far been published investigating the impact of MMP-2 expression on long-term survival in NSCLC (7) . In breast cancer (20) , gastric cancer (21) , and other carcinomas, overexpression of MMP-2 detected by immunohistochemistry has particularly been associated with an unfavorable outcome, suggesting for this study also to focus on overexpression of MMP-2. Tumors showing homogenous, immunohistochemical staining of cancer cells were considered positive, and specimens with intermediate staining or without staining were considered negative concerning overexpression of MMP-2. Therefore, the method for choosing the optimal cut point for differentiating between patients at high and low risk is based on previous results and knowledge of the measurement technique and not on the minimum P method, which inherits statistical danger in investigating possible prognostic factors (22) .
In this study cancer-related survival has been used as the end point to assess the prognostic value of MMP-2 expression. In contrast, other authors (20 , 23) analyzed the overall survival, which inherits the danger of misinterpretation resulting from influence of death caused by other diseases than cancer. Some other reports used the association of MMP expression with already established risk factors as indirect predictors for poor prognosis. By comparing live table curves of patients with high and low MMP-2 expression, this study revealed independent prognostic significance of MMP-2 in a direct manner. Considering all of the NSCLCs analyzed, MMP-2 overexpression was a parameter with borderline statistical significance. However, the analysis of early stages demonstrated that MMP-2 overexpression is a strong and independent predictor of poor clinical outcome in pN0 NSCLC. This observation is similar to breast cancer, in which subgroup analyses of MMP-2 expression and its prognostic value (20) showed that MMP-2 predicts poor prognosis particularly during the early cancer stages.
Even in stage I NSCLC, 40% of patients who are postoperatively staged as tumor free relapse within 24 months. Patients with early cancer stages are a potential subgroup for successful adjuvant therapeutic regimens because they have no overt tumor spread and low tumor burden. Several biological therapeutic approaches are currently being established for possible adjuvant regimens. The knowledge about correlation between expression of potential therapeutic targets and relapse or cancer-related death is a prerequisite for analysis of possible effects of adjuvant therapy in early-stage cancer. This study shows that MMP-2 overexpression correlates with early cancer-related death and indicates that MMP-2 should be considered as a potential target of MMP inhibitors in NSCLC. Because other MMP subclasses are also associated with aggressiveness of lung cancer (10) , even unspecific MMP inhibitors such as marimastat or batimastat (12 , 24) could be efficient in adjuvant therapy of NSCLC or in controlling metastatic NSCLC. Further, this type of study may provide a basis for the preselection of patients to be included in clinical trials to investigate the benefit of chemotherapy or MMP-inhibitor therapy.
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FOOTNOTES |
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1 Supported by grants from the
Sonderforschungsbereich (SFB) 469 of the Ludwig-Maximilians
University, Munich, Germany. 
2 To whom requests for reprints should be
addressed at: Department of Surgery, University of Munich, Klinikum
Innenstadt, Nussbaumstraße 20, 80336 Munich, Germany. Phone:
49-89-5160-2511; Fax: 49-89-5160-3630; E-mail: Passlick{at}lrz.uni-muenchen.de
3 The abbreviations used are: NSCLC, non-small
cell lung cancer; MMP, matrix metalloproteinase; LSAB, labeled
streptavidin-biotin.
Received 2/15/00; revised 7/17/00; accepted 7/17/00.
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