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Molecular Oncology, Markers, Clinical Correlates |
1 The Royal Veterinary and Agricultural University, Department of Pharmacology and Pathobiology, Frederiksberg C, Denmark, and 2 Erasmus MC, Department of Medical Oncology, Josephine Nefkens Institute, GE Rotterdam, the Netherlands
ABSTRACT
Purpose: In the present study, we investigated the association between tumor tissue levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and prognosis in patients with primary breast cancer and analyzed whether TIMP-1 may be useful as a prognostic marker in combination with urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1).
Experimental Design: In cytosolic extracts of 2984 primary breast tumors, total levels of TIMP-1 were determined using an established, validated ELISA. Levels of uPA and PAI-1 have previously been determined in the extracts.
Results: Univariate survival analysis showed a significant relationship between higher levels of TIMP-1 (continuous log-transformed variable) and poor prognosis [recurrence-free survival (RFS), overall survival (OS); P < 0.001]. Performing isotonic regression analysis, we identified a cut point to classify tumors as TIMP-1-low or TIMP-1-high. Using this cut point, high levels of TIMP-1 were significantly associated with shorter survival in univariate analysis, both in the total patient group (RFS, OS; P < 0.001), in the node-negative subgroup (RFS, hazard ratio = 1.28, P = 0.006), and in the node-positive subgroup (RFS, hazard ratio = 1.43, P < 0.001). In multivariate analysis, including uPA and PAI-1, TIMP-1 was significantly associated with shorter RFS, both when included as a continuous log-transformed (P = 0.03) and as a dichotomized variable (P = 0.002).
Conclusions: This study validates previous findings that tumor tissue levels of TIMP-1 are associated with prognosis in patients with primary breast cancer. It confirms that TIMP-1 may be useful as a prognostic marker in combination with uPA/PAI-1 and adds substantial positive information on the use of TIMP-1 as a prognostic marker in breast cancer.
Introduction
Breast cancer is one of the most common malignant diseases among Western women. When diagnosed with breast cancer, prognosis is assessed in every patient to plan appropriate treatment. Today, nodal status, tumor size, grade of malignancy, age, and hormone receptor status are accepted as useful parameters for determining prognosis (1)
. All patients regarded at high risk of experiencing recurrence of disease are offered systemic adjuvant treatment. Using the above-mentioned parameters, systemic adjuvant treatment is offered to as many as 90% of the patients. This implies that a number of patients are overtreated, especially in the group of lymph node-negative patients of whom 
60–70% are cured by surgery alone. Systemic adjuvant treatment may be associated with adverse side effects, and an improvement of prognostic stratification of breast cancer patients, in particular of lymph node-negative patients, is needed to assure that fewer patients will be overtreated in the future.
Members of the urokinase plasminogen activation system urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have proven to be of use as prognostic markers in patients with primary breast cancer and have recently reached level of evidence I (2) for prognostic markers (3 , 4) . Using uPA and PAI-1 in the process of prognostic stratification, an improvement is achieved (5) ; however, a complete separation of low-risk and high-risk node-negative patients is not possible, and accordingly, additional prognostic markers are needed.
An association between tumor levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and poor patient outcome has previously been demonstrated in breast cancer at both mRNA (6 , 7) and protein levels (8 , 9) . TIMP-1 is one of four known endogenous inhibitors of matrix metalloproteinases (MMPs; reviewed in Ref. 10 ), and its presence has been documented in a variety of body fluids and tissues (11) . Lately, a more complex role in the process of growth and dissemination of cancer cells has been implicated for TIMP-1 as other functions have been demonstrated; these include inhibition of apoptosis (12, 13, 14) , promotion of growth (15 , 16) , and presumably, regulation of angiogenesis (17, 18, 19, 20) .
In a previous study, which included 341 patients with primary breast cancer, we demonstrated an association between high levels of tumor tissue TIMP-1 and poor prognosis in univariate and in multivariate survival analysis. Moreover, prognostic information obtained from measurements of tumor tissue TIMP-1 levels was independent of and additive to that obtained from measurements of PAI-1 (8) . However, for prognostic markers to be safely used in the clinic, sufficient evidence on their usefulness must be obtained and our findings warrant additional investigation.
The present validation study was conducted as a European Organization for Research and Treatment of Cancer-Receptor and Biomarker Group collaborative study, including 2984 patients with primary breast cancer. Total levels of TIMP-1 were determined by ELISA in cytosol extracts of 2984 primary breast tumors, and TIMP-1 levels were related to patient outcome and clinicopathological data. Moreover, as levels of uPA and PAI-1 have been determined previously in these tumor tissue extracts (21) , we investigated whether measurements of tumor tissue TIMP-1 levels may be used in combination with measurements of uPA and PAI-1 when predicting prognosis.
Patients and Methods
Patients and Tissue Samples.
TIMP-1 levels were determined in cytosol preparations (as described below) from 2984 primary breast carcinomas collected between 1978 and 1995. The study design was approved by the medical ethical committee of the Erasmus University Rotterdam, the Netherlands (protocol no. MEC 02.953). Selection of samples [according to the criteria described elsewhere (22)
] was based on the availability of stored cytosol extracts (in liquid nitrogen), which remained after routine estrogen receptor (ER) and progesterone receptor (PgR) analyses. Inoperable T4 tumors [staging according to the International Union against Cancer TNM classification (23)
], tumors obtained after neoadjuvant treatment, and tumor specimens from a biopsy only were not included in the present study. Furthermore, patients admitted to the institute >100 days after primary surgery and patients with distant metastasis at the time of primary surgery (M1 patients) were excluded.
Median age of the patients at the time of surgery (modified mastectomy = 1658 patients, breast-conserving treatment = 1326 patients) was 57 years (range, 22–94 years). Lymph node status was known for 2958 patients; 1433 patients (48%) were lymph node positive; and 1525 (51%) were lymph node negative. Of the 1433 node-positive patients, 745 (25%) had 1–3 positive nodes and 688 (23%) had >3 nodes involved. A total of 1170 patients (39%) was premenopausal, and 1814 (61%) were postmenopausal at the time of primary surgery. Radiotherapy was applied to 2120 patients (71%): on the breast/thoracic wall in 1840 patients and/or on the axilla in 740 patients, respectively, parasternal and/or supraclavicular lymph nodes in 846 patients. T1 tumors (
2 cm) were present in 1267 patients (42%), T2 tumors (2–5 cm) in 1399 patients (47%), T3 tumors (>5 cm) in 186 patients (6%), and operable T4 tumors in 132 patients (4%). The differentiation grade was based on histological and cellular characteristics, as stated in the reports of the regional pathologists, and it is not based on a central pathological review of all tumor samples and, thus, reflects daily practice. Pathological examination was performed as previously described (24)
, and the histological differentiation grade was poor in 1603 patients (54%), moderate in 533 patients (18%), good in 46 patients (2%), and unknown for 802 patients (27%). None of the 1525 node-negative patients received systemic adjuvant therapy. Adjuvant chemotherapy (mainly cyclophosphamide/methotrexate/5-fluorouracil) was given to 478 patients (mainly premenopausal patients), whereas 340 patients received adjuvant hormonal therapy (mainly postmenopausal patients), either alone (310 patients) or in combination with chemotherapy (30 patients).
All patients were examined routinely every 3–6 months during the first 5 years of follow-up and once a year thereafter. The median follow-up period of patients alive (n = 1837) was 98 months (range, 1–231 months). Of the 2984 patients included, 1398 (47%) showed evidence of disease (including locoregional relapse) and count as failures in the analysis of recurrence-free survival (RFS). A total of 190 patients (6%) died without evidence of disease and were censored at last follow-up in the analysis of RFS. Nine-hundred fifty-seven (32%) died after a previous relapse. Thus, a total of 1147 patients (38%) was failures in the analysis of overall survival (OS).
Assays of ER, PgR, Total Protein, uPA, PAI-1, and TIMP-1 in Tumor Tissue Extracts.
Tumor tissues were stored in liquid nitrogen and pulverized in the frozen state with a microdismembrator as recommended by the European Organ-ization for Research and Treatment of Cancer for processing of breast tumor tissue for cytosolic ER and PgR determinations (25)
. The resulting tissue powder was suspended in European Organization for Research and Treatment of Cancer receptor buffer [10 mM dipotassium chloride EDTA, 3 mM sodium azide, 10 mM monothioglycerol, and 10% v/v glycerol (pH 7.4)]. The suspension was centrifuged for 30 min at 100,000 x g to obtain the supernatant fraction (cytosol). ER and PgR levels were determined by ligand binding assay or with enzyme immunoassay as described previously (26)
. The cutoff point used to classify tumors as ER or PgR positive and negative was 10 fmol/mg protein. Total cytosolic protein was quantified with the Coomassie brilliant blue method (Bio-Rad Laboratories, Hercules, CA) with human serum albumin as a standard. The cytosolic levels of uPA and PAI-1 were determined with ELISAs as described before (27
, 28)
. Total levels of TIMP-1 in the cytosols were measured by use of an established kinetic total TIMP-1 ELISA (29)
. Briefly, immunoassay plates were coated with sheep polyclonal anti-TIMP-1 antibody (30)
, and captured TIMP-1 was detected using a specific anti-TIMP-1 monoclonal antibody, which detects both free TIMP-1 and complexes of TIMP-1 and various MMPs [MAC 15 (31)
] and an alkaline phosphatase-conjugated rabbit antimouse antibody (DAKO, Glostrup, Denmark). Recombinant human TIMP-1 was used as a standard for calculation of concentrations and for spiking in recovery experiments.
Before use of the total TIMP-1 ELISA, the assay was carefully validated for measurements of TIMP-1 in cytosol extracts. The validation process included investigation of reproducibility (intra-assay variation, interassay variation), of linearity between dilution and the signal obtained in the assay, and analysis of recovery of spiked recombinant TIMP-1 signal in cytosol extract. For validation purposes, a number of different cytosol samples as well as a cytosol pool were used.
Before measurements of TIMP-1 levels in the cytosol extracts, previously diluted to 1 mg protein/ml in European Organization for Research and Treatment of Cancer receptor buffer, all extracts were further diluted (22-fold) in sample dilution buffer [50 mM phosphate (pH 7.4), 100 mM sodium chloride, 10 mg/ml BSA (Fraction V; Sigma-Aldrich, Steinheim, Germany), and 0.1% (v/v) Tween 20]. The TIMP-1 concentration in individual samples was normalized against total protein content of the sample.
Statistics.
The strength of the associations of TIMP-1 with continuous variables was tested with Spearman rank correlation. The strength of the association of TIMP-1 (used as a continuous variable) with other variables (used as grouping variable) was tested with the nonparametric Wilcoxon rank-sum test or Kruskal-Wallis test, followed by a Wilcoxon-type test for trend across ordered groups where appropriate. Survival probabilities were calculated by the actuarial method of Kaplan and Meier (32)
. Both univariate and multivariate analyses were performed using the Cox proportional hazards model. The likelihood ratio test in the Cox regression models was used to test for differences and for interactions. In our search for the best categorization of TIMP-1, we used isotonic regression analysis (28
, 33)
. The proportionality assumption was investigated using a test based on the Schoenfeld residuals (34)
. The residuals are retrieved and a smooth function of time fitted and then tested whether there is a relationship. All computations were done with the STATA statistical package, release 8.0 (STATA Corp., College Station, TX). All Ps are two-sided. The level of statistical significance was set at 0.05.
Results
Assay Validation.
Before measuring TIMP-1 levels in the 2984 tumor tissue extracts, the assay was thoroughly validated using cytosol extracts. The signal obtained in the ELISA showed good linearity upon dilution of the cytosols ranging from 1:10 to 1:340 (Fig. 1)
. The recovery of spiked TIMP-1 signal assessed in 1:51 diluted cytosols ranged from 69 to 122% (mean 89%, n = 8). Furthermore, the spiked TIMP-1 signal showed good linearity upon dilution. Using a cytosol pool, reproducibility of the assay was assured through the demonstration of low intra-assay and interassay variations, i.e., intra-assay variation of duplicates (n = 91) was 4.2%, and the interassay variation of a pooled cytosol sample was 5.2% (91 duplicates run on individual ELISA plates).
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, ranging from 0 to 336 ng/mg protein (median, 14.0 ng/mg protein). Table 1
shows the median and the 25th and 75th percentile levels of TIMP-1 in subgroups of tumors and their relationship with patient and tumor characteristics. The tumor level of TIMP-1 was higher in postmenopausal patients (P < 0.001) and was positively related with age (rs = 0.20, P < 0.001), ER (rs = 0.18, P < 0.001), PgR (rs = 0.13, P < 0.001), uPA (rs = 0.29, P < 0.001), and PAI-1 (rs = 0.37, P < 0.001). Furthermore, higher TIMP-1 levels were found in tumors of node-positive patients (P = 0.01) and in larger tumors (P < 0.001). The level of TIMP-1 was not significantly related with tumor grade (P = 0.23).
|
|
. All of the clinicopathological parameters showed a significant association with OS and, except for menopausal status and ER status, also with RFS.
|
] and OS [HR (95% CI) = 1.32 (1.17–1.49), P < 0.001, Fig. 3B
]. Taking into account all failures during the total follow-up period, the proportional hazards assumption was not violated.
|
. The association of TIMP-1 with a poor RFS [HR (95% CI) = 1.43 (1.24–1.65), P < 0.001] and OS [HR (95% CI) = 1.33 (1.15–1.55), P < 0.001] was stronger in node-positive patients (Fig. 4, C and D)
compared with node-negative patients [HR (95% CI) = 1.28 (1.07–1.52), P = 0.006 for RFS and 1.22 (0.99–1.50), P = 0.06] for OS, respectively (Fig. 4, A and B)
.
|
. In the analyses, larger tumor size, the number of involved lymph nodes, poor tumor grade, and young premenopausal age were associated with poor prognosis. PgR positivity was associated with a favorable OS, whereas ER did not significantly add to the model after the inclusion of PgR. After inclusion of TIMP-1 as a dichotomized variable (cut point 11.71 ng TIMP-1/mg protein) to the multivariate models, the increase in 
2 (
2) was 31.6 in the analyses of RFS (df = 1, P < 0.001) and 8.7 in the analyses of OS (df = 1, P = 0.003). Analysis of TIMP-1 as a log-transformed continuous variable showed a significant contribution as well, i.e., in the model for RFS with a 2 of 28.6 (df = 1, P < 0.001) and in the model for OS with a 2 of 8.9 (df = 1, P = 0.003). When TIMP-1 was added to the base models, including the established strong prognostic factors uPA and PAI-1 [categorized using two cut points for each, as described before (21)
], TIMP-1 still significantly contributed to the multivariable models for RFS both when included as a dichotomized variable (2 = 9.8, df = 1, P = 0.002; Table 4
) and as a log-transformed continuous variable (2 = 4.7, df = 1, P = 0.03). When uPA and PAI-1 were included in the model for OS, TIMP-1 did not further contribute to the model, neither when analyzed as a dichotomized variable (P = 0.57, Table 3
), nor as a continuous variable (P = 0.31).
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Separate multivariate analyses for RFS in subgroups of node-negative [HR (95% CI) = 1.33 (1.11–1.58), 2 = 9.9, df = 1, P = 0.002] and node-positive patients [HR (95% CI) = 1.40 (1.22–1.61), 2 = 21.8, df = 1, P < 0.001] showed that TIMP-1 as a dichotomized variable significantly added to the model, including age and menopausal status, tumor size, and grade. However, after including uPA and PAI-1 as well to the multivariate model, TIMP-1 did not contribute in the analysis of node-negative patients [HR (95% CI) = 1.13 (0.93–1.36), 2 = 1.5, df = 1, P = 0.22], whereas it still added to the model of node-positive patients [HR (95% CI) = 1.25 (1.07–1.45), 2 = 8.6, df = 1, P = 0.003].
Discussion
The present study confirms the main finding of our previous study—namely that higher levels of tumor tissue TIMP-1 are significantly associated with poor prognosis in patients with primary breast cancer.
The association between high levels of a protease inhibitor and poor prognosis may be somewhat surprising as proteolytic systems such as the MMP system have important roles in enabling and facilitating cancer growth and dissemination (reviewed in Refs. 10 , 35 ). Consequently, one would expect high levels of an endogenous protease inhibitor to be protective against growth and spread of malignant cells. This is, however, not always the case; in breast cancer patients also, high tumor tissue levels of the protease inhibitor PAI-1 are associated with a poor prognosis (3 , 4) .
The association of TIMP-1 with poor prognosis has been reported in several cancer forms (reviewed in Ref. 36 ), and because biological functions other than MMP inhibition have been reported for TIMP-1, it could be speculated that these functions are responsible for the unexpected association with poor prognosis. It has been reported that TIMP-1 stimulates growth in several cell types (16 , 37) and furthermore that TIMP-1 is capable of inhibiting apoptosis—both through stabilization of extracellular matrix (12) and independently of MMP inhibition (13 , 14) . Both increased proliferation, through promotion of growth, and inhibition of apoptosis are characteristic of the uncontrolled growth and spread of tumor cells. Moreover, a regulatory role in angiogenesis has been suggested for TIMP-1; however, whether TIMP-1 inhibits (19 , 20) or promotes angiogenesis, through an influence on generation of angiogenic and antiangiogenic factors (17 , 38) , has not been determined. Recent work questions the role of TIMP-1 in angiogenesis altogether as it has been demonstrated that endothelial tubulogenesis is largely dependent on MMPs, which are not inhibited by TIMP-1 (18) .
In the present study, we made an attempt at identifying a cut point for dividing tumors into TIMP-1-low and TIMP-1-high ones, respectively. Using this cut point, in univariate and multivariate survival analysis, we demonstrated significant associations between high TIMP-1 tumor levels and shorter survival. The use of a different cut point has been reported in the literature (9) ; it is questionable, however, whether this can be compared with the one identified in the present study. TIMP-1 levels in the study by McCarthy et al. (9) are different from those reported here—perhaps because of the use of different ELISAs. Furthermore, different types of tumor tissue extracts have been used in the different studies. Thus, the cut point identified in the present study still needs confirmation in an independent patient group.
Additional univariate survival analyses were performed dividing the patients into quartiles (data not shown). A similar analysis in a previous study revealed a marked tendency for very high levels of TIMP-1 in a tumor to be associated with a prognosis similar to that associated with low intermediate levels (8) . In the present study, the quartiles analysis of the total patient group did not show this tendency. However, when looking separately at the clinically important subgroups of lymph node-negative and lymph node-positive patients, we noticed in the lymph node-negative group that high intermediate tumor tissue TIMP-1 levels were associated with prognosis similar to or better than the one associated with low intermediate tumor tissue TIMP-1 levels. In the subgroup of lymph node-positive patients, although, high intermediate and very high TIMP-1 levels were associated with essentially the same poor prognosis. Thus, associations between tumor tissue TIMP-1 and prognosis seemed to vary with lymph node status. It has been suggested that the balance between anti-MMP, tumor-suppressing activities of TIMP-1, and the tumor-promoting ones (promotion of growth, inhibition of apoptosis) is regulated by the amount of TIMP-1 present in the tumor environment (36) . Also, the timing of TIMP-1 expression as well as the presence of a putative TIMP-1 receptor on the cell surface has been suggested as important determinants of the net effect of TIMP-1 (36) . This, however, does not explain the difference in findings in the lymph node-negative and lymph node-positive subgroups. It could be speculated that this is merely an indicator of the large number of biological differences between individual breast tumors.
The method of tissue extraction should also be considered, especially when comparing with other studies. In the present study, an extraction buffer without detergent was used; using this type of buffer, cytosol extracts are obtained. The prognostic value of a marker may be influenced by the extraction procedure as shown for uPA by Jänicke et al. (39) ; it was speculated that the use of detergent freed uPA bound to the uPA receptor, thereby increasing the amount of extracted uPA. Thus, an investigation of optimal extraction conditions should be performed for TIMP-1, more so because cell surface binding has been suggested for TIMP-1 (40) , and we are currently inves-tigating this matter. It should be emphasized, however, that the association between high tumor tissue levels of TIMP-1 and poor prognosis is present in this study using cytosol extracts as well as in our previous one where detergent extracts were used (8) .
Furthermore, it should be investigated whether plasma samples could be used for prognostic evaluation instead of tumor extracts; this has proven possible in patients with colorectal cancer as high preoperative plasma TIMP-1 levels are significantly associated with poor prognosis in these patients (41) . As tumor tissue is heterogeneous in composition and as only a very limited amount may be available, plasma would be preferable if possible.
This study also investigated whether TIMP-1 may be used as a prognostic marker in combination with uPA and PAI-1. In the multivariate model, including uPA and PAI-1, TIMP-1 added significantly to the model for RFS, and also, no statistically significant interactions between TIMP-1 and members of the uPA system were identified. TIMP-1 is therefore to be considered a candidate for improving prognostic stratification, also when used in combination with the strong prognostic factors uPA and PAI-1.
The results of this study contribute significantly to the total amount of information hitherto obtained on the use of TIMP-1 as a prognostic marker in breast cancer. One major advantage of this study is the inclusion of 3000 patients. Furthermore, none of the 1525 lymph node-negative patients received any adjuvant systemic therapy, and thus, the observed prognostic value of TIMP-1 could not have been obscured by possible treatment effects.
Three studies, including the present one, have demonstrated the association between high tumor tissue levels of TIMP-1 protein and poor prognosis in patients with primary breast cancer (8 , 9) . Moreover, this study as well as our previous one (8) indicate that TIMP-1 may be useful as a prognostic marker in combination with uPA and PAI-1. To safely recommend the use of TIMP-1 as a prognostic marker for clinical decision-making, prospective studies are warranted. To begin with, a prospective study could be performed in relationship to a prospective therapeutic trial as suggested by Hayes et al. (2) in the Tumor Marker Utility Grading System for evaluation of the clinical use of tumor markers. Such a study would then have to be followed by a study having the test of TIMP-1 as a prognostic marker as the primary goal or by a meta-analysis of previous studies. Finally, besides collecting sufficient evidence for the use of TIMP-1 for clinical decision-making, procedures for quality assurance should be established.
FOOTNOTES
Grant support: Danish Cancer Society, the Dutch Cancer Society (Nederlandse Kanker Bestrijding, project DDHK 2000-2256), the "Fonden til fremme af klinisk eksperimentel cancerforskning specielt vedrørende cancer mammae," and the Else and Mogens Wedell-Wedellsborg Foundation.
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.
Note: This work was a European Organization for Research and Treatment of Cancer Receptor and Biomarker Group Validation Study including 2984 patients.
Requests for reprints: Anne-Sofie Schrohl, The Royal Veterinary and Agricultural University, Department of Pharmacology and Pathobiology, Ridebanevej 9, DK-1870 Frederiksberg C, Denmark. Phone: 45-35-28-37-49; Fax: 45-35-35-35-14; E-mail: sofie{at}kvl.dk
Received 10/ 7/03; revised 12/16/03; accepted 1/ 2/04.
REFERENCES
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