Abstract
OBJECTIVE To investigate expression of the tissue factor (TF) and matrix metalloproteinase-9 (MMP-9) in breast cancers, and to assess their expression in relation to possible prognostic significance.
METHODS The expression of TF and MMP-9 in 71 breast cancer specimens were determined by EnVision immunohistochemistry, and the positive expressions related to the patient clinical outcome.
RESULTS Positive rates of TF and MMP-9 staining were respectively 43.7% and 42.3%. K-M monofactorial analysis showed that the 5-year survival rate of the patients with a positive expression of TF and MMP-9 was lower than those with negative expression (P < 0.05). However, the COX multifactorial analysis indicated that TNM staging and lymph node metastasis were the prognostic factors for breast cancer patients, and that TF and MMP-9 could not be used as the independent prognostic factors (P > 0.05).
CONCLUSION The positive rates of TF and MMP-9 were considerably high in breast cancers, which could provide useful information for patient prognosis.
keywords
Introduction
There is a high expression of the tissue factor (TF) and matrix metal-loproteinase-9 (MMP-9) in the histocytes of many tumors. It has been shown that expression in these patients significantly correlates with the biological behavior. However, whether the level of their expression can be regarded as a prognostic factors for breast cancer patients remains unclear. In our study, TF and MMP-9 expression in the breast cancers of patients in our hospital and the patient’s prognosis were analyzed.
Materials and Methods
Materials
Our study involved 71 cases of breast cancer that were filed in the Pathology Department of the First Hospital of Beijing University in 2001. Paraffin sections of all the cancers and a complete set of clinical data were available. All patients were female, with ages ranging from 29 to 72 years, and an average of 51. Clinical stages of these cases included 23 Stage-I, 38 Stage-II and 10 Stage-III cases. Based on the pathological types, there were 3 cases with ductal carcinoma in situ (DCIS), 2 with infiltrating lobular carcinoma (ILC), and 66 with infitrating ductal carcinoma (IDC). The mode of operation included simple excision in 3 cases, breast-conserving surgery in 3, modified radical operation in 63 and radical operation in 2. Post-operative radio-chemotherapy was conducted in 10 of the 71 cases, simple chemotherapy in 31 and simple radiotherapy in 1. In addition, endocrine therapy was performed in 39 of the total cases in our study.
Main reagents
First antibody: the mouse-antihuman TF monoclonal antibody (TFMab) was purchased from the US ADI Co., and the mouse-antihuman MMP-9 monoclonal antibody (Mab) was from the Santa Cruz Co. Second antibody: horseradish enzyme labeling sheep anti-mouse IgG multimer was bought from the Santa Cruz Co., and the PV-9000 immunohistochemical EnVision kit was from the Beijing Zhngshan Golden Bridge Bio-Tech Co., Ltd.
Immunohistochemical staining
After a 10% neutral formalin fixation of the samples, routine dehydration and paraffin imbedding were conducted. The 4-μm paraffin sections were heated for 7 h or more at 60°C followed by immunohistochemical staining.
Based on instruction in the kit, the EnVision method was used to detect the expression of TF. Dimethylbenzene deparaffinage, gradient alcohol hydration and microwave coctoantigen repair were conducted. Using the TF mAb first antibody (1:1000), MMP-9 first anti-body (1:500) and sheep anti-mouse IgG second antibody (1:4000), incubations were respectively conducted. DAB staining was performed after PBS washing, and the sections were mounted after a Mayer’s hematoxylin after-stain. LoVo cell staining of large-intestine cancer with a high level of TF expression was employed as the positive control, and PBS was used to replace the first antibody for negative control staining.
Assessment of results
The methods of Contrino et al.[1] and Bartsch et al.[2] were referred to for positive criteria: 10 high fields were observed for each case; the TF localized at the cellular membrane, with the brownish-yellow color as the criterion of positiveness. The MMP-9 localized in the cytoplasm of the cancer cells, with brown fine granular form as the criterion of positiveness. If the positive cells were less than 10% of the total cells in any field, the results were designated as negative. If the positive cells were more than or equivalent to 10%, the results were considered positive (Figs.1 and 2.)
Positive expression of TF staining in breast cancer ( EnVision, ×400).
Positive staining of MMP-9 in breast cancer (EnVision, ×400).
Statistical analysis
SPSS13.0 software was used to conduct a Pearson correlation analysis to assess the relationship of the TF and MMP-9 expressions, age, tumor size, lymph node metastasis, TNM Staging (UICC, revised in 1988), pathological type, ER, PR and HER-2 to our patient’s life expectancy. We also conducted K-M monofactorial analysis and multifactorial COX model survival analysis. Statistical significance was accepted if the P value was less than 0.05.
Results
TF and MMP-9 expressions in the breast cancers
In the breast cancers, the positive rates of TF and MMP-9 staining were respectively 43.7% (31/71) and 42.3% (30/71) (Figs.1 and 2). Those with either a positive TF or MMP-9 staining reached up to 53.5% (38/71), and those with a positive expression of both TF and MMP-9 amounted to 32.4% (23/71). There was a significant correlation in the expressions of the two proteins (coefficient correlation r = 0.563, P < 0.01, Table 1).
Relationship between TF expression and MMP-9 expression in the breast cancers.
Follow-up results
In our study, the loss of a visit occurred in only 1 of the 71 patients because she failed to inform the hospital staff of a change of address. The follow-up rate reached 98.6%. The longest follow-up time was 60 months, with an average survival time of 58.4 months. The 5-year survival rate was 88.7%, with 3 cases of survival with a tumor and a 5-year disease-free survival rate of 84.5% (Fig.3). There were 8 deaths among the 71 patients, all of whom died of breast cancer 12 to 60 months after surgery.
Overall survival curve and disease-free survival curve of the 71 patients.
Statistical results
Using the follow-up results from all the patients, SPSS 13.0 software was employed to conduct the survival analysis with the life-table method. For the survival curves of the total survival and disease-free survival rates, see Fig.3.
The TF and MMP-9 expression rates of the 71 patients were used as the reference factors for a K-M monofactorial survival analysis. The results confirmed that the survival rates of the patients with positive expressions of TF and MMP-9 were significantly lower compared to that of the patients with negative expression. There was a statistical difference between the two rates, with P values of 0.015 and 0.006, respectively (Table 2, Figs.4 and 5). K-M monofactorial analysis was conducted using the patient’s age, tumor size, lymph node metastasis, TNM staging, pathological type, ER, PR and HER-2. See Table 3 for the results, which show there was a statistical significance in relating lymph node metastasis and TNM staging with the survival rates (P values of 0.000 and 0.011, respectively).
K-M monofactorial survival analysis of TF and MMP-9 expressions.
K-M survival curve of the patients with negative and positive expression of TF (P = 0.015).
K-M survival curve of the patients with negative and positive expression of MMP-9 (P = 0.006).
K-M monofactorial survival analysis of other factors.
Concerning the interaction among the variables, the above-mentioned factors were introduced to the COX models for a multifactorial regression analysis, so as to emphasize independent prognostic value of the variables (Table 4). These results showed that there was no statistical significance in the relationship of TF and MMP-9 expressions with the survival rates (P > 0.05), but there was a statistical significance in relating the TNM staging and lymph node metastasis on the survival rates. The P values were respectively 0.008 and 0.028.
COX Multi-factorial survival analysis.
Discussion
Current basic research has shown that TF and MMP-9 play key roles in infiltration and metastasis by the tumor cells. TF, also known as thromboplastin or CD142, is a single-strand transmembrane glycoprotein with a molecular weight of 47 kDa. The gene assignment of TF is at 1p22-23, and TF has a physiological function of participating in blood clotting. However, it can promote invasion and metastasis of the tumor cells, and angiogenesis of the tumor by blood clotting and signal-transduction pathways[3,4]. The gene assignment of the MMP-9, also called the gelatinase B, is at 20 q11.2-q13.1. It is secreted in a proenzyme form, with a molecular weight of 92 kDa. Upon activation of MMP-9, a type-IV collagenase is formed, the type IV collagen and gelatin close to the extra-cellular matrix on the tumor surface are degraded, resulting in tumor infiltration and metastasis[5,6].
It has been reported[7] that the positive rate of MMP-9 expression exceeded 70% in breast cancers, and some experts found that there was a correlation between the expression of TF and MMP-9 in the large intestine carcinomas[8]. Our study indicated that the breast cancer patients with a positive expression of TF and MMP-9 went beyond 40% of the totals, and those with a positive expression of both TF and MMP-9 exceeded 30% of the total patients, with a significant correlation between the two proteins. Therefore, TF and MMP-9 may become important breast cancer immunohistochemical indices.
Ueno et al.[9] found that the expression of TF in breast cancers was an independent prognostic factor for predicting life expectancy, and could be of effective and prospective value for assessing remote metastasis and recurrence in the breast cancer patients. It was reported by Ranuncolo et al.[10] that there was a continuously low MMP-9 level in the breast cancer patients without a postoperative relapse, however, the MMP-9 level of the patients with a tumor recurrence increased at a period from 1 to 8 months before the recurrence. A study by Liu et al.[11] revealed that there was a positive correlation between MMP-9 mRNA expression and certain factors, such as tumor size, clinical stages, lymph node metastasis and prognostic index, having a definite impact on the patient’s prognosis. All these assays showed that TF and MMP-9 expression might be of value as prognostic factors for tumor patients.
In our laboratory assays, monofactorial analysis indicated there was a significant correlation between TF and MMP-9 expression and the patient’s survival rate. Nevertheless the multifactorial analysis failed to confirm that TF and MMP-9 could be considered as independent prognostic factors for breast cancer, most probably because the information on the patient’s age, TNM staging, pathological type and immunohistochemistry etc. in the test group could not be included in the monofactorial analysis. In the multifactorial analysis, at the same time, the small number of total cases resulted in a considerable discrepancy. Further extended studies with a larger sample size are needed to reach definitive conclusions from our study.
In short, the positive expressions of TF and MMP-9 were very high in the breast cancers studied, and monofactorial analysis has shown that there was a correlation between the high expression and the patient’s prognosis, suggesting the TF and MMP-9 might play a key role in carcinogenesis and progress of breast cancer. Further investigations are needed to determine if TF and MMP-9 are suitable as independent prognostic factors for breast cancer patients.
- Received October 9, 2007.
- Accepted January 3, 2008.
- Copyright © 2008 by Chinese Anti–Cancer Association
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