Expression of Matrix Metalloproteinase-7 is an Unfavorable Prognostic Factor in Intrahepatic Cholangiocarcinoma
Intrahepatic cholangiocarcinoma (IHCC) is a highly malignant neoplasm, but the prognostic factors of IHCC are not yet fully understood. The matrix metalloproteinases (MMPs) are known to be related to tumor viability. The aim of this study was to evaluate the prognostic significance of clinicopathological and immunohistochemical characteristics of resected IHCC.
Patients and Methods
From 1996 to 2006, we surgically treated 35 patients with IHCC. Clinicopathological and immunohistochemical characteristics, including expression of MMPs, vascular endothelial growth factor, and epidermal growth factor receptor in the resected specimens, were investigated, and overall survival rates were evaluated with regard to the characteristics using univariate and multivariate analyses.
Univariate analysis revealed the significant prognostic factors to be preoperative serum CEA and CA19-9, intraoperative transfusion, tumor size, surgical margin, lymph node metastasis, invasion of portal and hepatic vein, intrahepatic metastasis, UICC stage, and expression of MMP-7. Subsequent multivariate analysis indicated that MMP-7 was an independent prognostic factor (hazard ratio (HR), 4.698; 95% confidence interval (CI), 0.057–0.866; P = 0.03) along with intrahepatic metastasis (HR, 5.694; 95% CI, 0.029–0.706; P = 0.017).
MMP-7 expression is associated with a poor prognosis in patients with resected IHCC.
KeywordsCholangiocarcinoma Matrix metalloprotease Matrix metalloprotease-7 Prognostic factor
Intrahepatic cholangiocarcinoma (IHCC) is a highly malignant neoplasm originating from the bile duct system with markers of cholangiocyte differentiation.1,2 The incidence of IHCC is increasing worldwide, and it is the second most common form of primary liver cancer next to that of hepatocellular carcinoma. Despite advances in diagnosis and treatment, most patients present with advanced metastatic lesions and are not amenable to surgical extirpation or liver transplantation.3, 4, 5, 6, 7 Furthermore, the current chemotherapy regimen that is used to treat IHCC offers very limited benefit in terms of patient survival. When compared with other malignancies, IHCC is generally characterized by strong proliferation, invasion, and early metastasis.
Matrix metalloproteinases (MMPs) are zinc-dependent neutral endopeptidases, which participate in degradation of extracellular matrix proteins.8,9 They play roles in many physiological processes, such as bone remodeling and organogenesis, and have additional roles in the reorganization of tissues during pathological conditions such as inflammation, invasion, and metastasis of cancer cells.8,10 Many recent studies have provided evidence that the biological activities of various cell surface molecules are proteolytically modulated by several MMPs, including MMP-2, -9, and -7.11, 12, 13 These MMPs are likely to regulate cellular functions by activating, inactivating, or releasing membrane proteins. Such regulation of cell surface proteins, as well as MMP-catalyzed degradation of the extracellular matrix (ECM), a natural barrier against tumor invasion, is important to tumor invasion and metastasis.
MMP-7, the smallest of the MMPs, has broad substrate specificity and has been demonstrated to degrade or process a variety of matrix and non-matrix molecules.14 Unlike most MMPs, which are expressed by stromal cells, MMP-7 is principally expressed by epithelial cells.15 In human cholangiocarcinomas, this enzyme appears to be one of the most important MMPs because the expression of MMP-7 correlates highly with the malignant and metastatic potentials of the cancers.16
In this study, we investigated the association of clinicopathological characteristics of resected IHCCs with prognosis. To improve the prognosis in IHCC, we believe that a fuller, more detailed understanding of the prognostic factors of IHCC is required.
Patients and Methods
Number of patients
64.9 ± 10.3
Operation time (min)
463 ± 170
Blood loss (g)
1,190 ± 750
Tumor size (mm)
58.9 ± 31.2
Surgical margin (mm)
5.6 ± 7.1
Postoperative hospital stay (days)
40.2 ± 21.6
Results of univariate analysis
<65 years old
≥65 years old
Tumor size (mm)
Well to moderate
Lymph node metastasis
Invasion of portal vein
Invasion of hepatic vein
Bile duct invasion
Immunohistochemistry for MMP-2, -7, -9, vascular endothelial growth factor (VEGF), and epidermal growth factor receptor (EGFR) was performed using an Envision+system (Dako, Cytomation, Glostrup, Denmark). Sources and dilutions of primary antibodies were as follows: anti MMP-2, -7, -9 (mouse monoclonal; diluted 1:100; Daiichi Fine Chemical Co., Ltd., Toyama, Japan), anti-VEGF (mouse monoclonal; diluted 1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-EGFR (mouse monoclonal, diluted 1:500; Santa Cruz Biotechnology, Inc.).
Formalin-fixed, paraffin-embedded serial tissue sections (4 μm) were deparaffinized and rehydrated in xylene and grade-diluted ethanol (50–100%) and submerged for 20 min in 0.3% H2O2 with absolute methanol to block endogenous peroxidase activity. After protein blocking, the sections were incubated with each primary antibody at room temperature for 2 h. The sections were then treated for 20 min with biotinylated anti-mouse IgG (Nichirei Biosciences Inc., Tokyo, Japan), followed by treatment for 30 min with streptavidin–biotin–peroxidase complex (Nichirei Biosciences Inc.), at room temperature with a PBS wash between each step. The slides were developed with diaminobenzidine-H2O2 (DAB+system, Dako Japan Inc., Tokyo, Japan). The nuclei were also lightly stained with hematoxylin.
Evaluation of Immunohistochemistry
The slides were reviewed by two independent observers who had no knowledge of patient outcomes. The expression of MMPs was evaluated semiquantitatively according to the percentage of positively stained cells: negative (positive tumor cells/total tumor cells ≤10%) and positive (positive tumor cells/total tumor cells >10%). The expression of MMPs in the stromal and inflammatory cells was not evaluated in this study. The expression of VEGF was defined as follows: negative (positive tumor cells/total tumor cells ≤30%) and positive (positive tumor cells/total tumor cells >30%). The expression of EGFR was defined as follows: negative (positive tumor cells/total tumor cells ≤10%) and positive (positive tumor cells/total tumor cells >10%).
Results are expressed as means ± SD. Postoperative survival probabilities were calculated by the Kaplan–Meier method and analyzed by the log-rank test. Difference was regarded as significant when the P value was less than 0.05. Multivariate analyses were performed using the factors identified as significant by univariate analyses. All statistical analyses were performed using Dr SPSS II software (version 11.01 J; SPSS Japan Inc., Tokyo, Japan).
Overall Survival and Results of Univariate Analysis
Results of Multivariate Analysis
Results of multivariate analyses regarding overall survival
Tumor size (<50, ≥50 mm)
Lymph node metastasis
Invasion of portal vein
Invasion of hepatic vein
Many clinicopathological factors, such as lymph node metastasis, UICC stage, surgical margin, R0 resection, cirrhosis, use of postoperative adjuvant chemotherapy, and other factors, are potential prognostic factors after resection of IHCC.18, 19, 20, 21 However, there has been no definitive way to predict prognosis of IHCC using molecules. This study was performed to determine whether expression of MMP-2, -7, -9, VEGF, and EGFR in resected specimens of IHCC can predict disease outcome. As a result, MMP-7 expression in the tumor cells was found to be a prognostic factor, in addition to that of intrahepatic metastasis.
Recently, many targeted therapies against EGFR, VEGF, and human EGFR type 2 (HER2) such as cetuximab, lapatinib, erlotinib, and bevacizumab have been used for treatment of gastrointestinal cancers. Development of targeted agents in biliary tract cancer (BTC) including IHCC has lagged behind other types of tumors, and there are a few phase II studies examining early experience of efficacy and safety of targeted therapies for BTC patients.22, 23, 24, 25, 26 Chemotherapy has been the main therapeutic modality in locally advanced or metastatic BTC, and a randomized, controlled, phase III trial of 410 patients with BTCs has established the combination of gemcitabine and cisplatin as a new global standard for the treatment of locally advanced or metastatic BTC.27 Although the results are encouraging, the study strongly suggests that BTC remains very difficult to treat with current methods, and molecular targeted therapy is urgently needed for this deadly disease. According to phase II studies of the targeted therapies, cetuximab may be useful for BTC when combined with gemcitabine and oxaliplatin. Gruenberger et al.22 reported that cetuximab plus gemcitabine/oxaliplatin (GEMOX) was well tolerated and had encouraging antitumor activity, leading to resection in one third of patients. These findings warrant further study of cetuximab plus GEMOX in a large randomized trial. In the present study, the rate of positivity for EGFR was 74.3%, and the 5-year survival rates of EGFR(+) and EGFR(−) patients were 45.9% and 33.3%, respectively. Univariate analysis revealed that EGFR(+) patients had a tendency for poor prognosis; however, the difference between EGFR(+) and EGFR(−) patients was not statistically significant (P = 0.145).
MMPs can collectively degrade all components of the ECM and play important roles in embryo development, morphogenesis, tissue remodeling, as well as tumor invasion and metastasis.28,29 With regard to tumor invasion, the extracellular proteases, especially MMPs, appear to be crucial for this process. In fact, the invasion of cancer cells into nearby stroma, across blood vessel walls, and through normal epithelial cell layers has been shown to be promoted by various MMPs.28, 29, 30 Among the MMPs, MMP-7 is well-known for its involvement with many malignant tumors. MMP-7 is associated with malignancies in the colon, pancreas, and biliary tract.31,32 By degrading ECM components such as type-IV collagen, fibronectin, and laminin, MMP-7 plays important roles in invasion and metastasis of carcinoma cells.33 Interestingly, cleavage of cell surface proteins other than ECM components by this MMP may also contribute to tumorigenic effects. For example, MMP-7 activates EGFR by releasing a ligand of epidermal growth factor and tumor growth factor-α.34 Moreover, by processing E-cadherin and thereby inducing loose and then tight aggregation of tumor cells, this proteinase induces homotypic adhesion of human colorectal cancer cells and enhances the metastatic potential in vivo.35 By disrupting tight junction structure and a consequent induction of cell dissociation, MMP-7 might enhance metastasis as well as invasion of IHCC cells.36
Some in vitro and in vivo studies have been performed to determine how MMPs contribute to progression of BTC including IHCC. It has been reported that the serum level of MMP-7 is elevated in BTC patients, and this appears to have a potential to differentiate BTC from benign biliary tract diseases.37,38 Moreover, histological expression of MMP-7 has been reported to be an unfavorable postoperative prognostic factor of BTC.39,40 Also, higher expression of MMP-9 is suggested to be associated with the potential of invasion41; however, MMP-9 has not been reported to be a prognostic factor. With regard to MMP-2, it is controversial whether this MMP is associated with poor prognosis.41,42 The multivariate analysis in the present study showed that immunohistochemical expression of MMP-7 in IHCC tissues was associated with poor prognosis, and expression of MMP-2 and -9 was not associated with it. This is the first report, to our knowledge, in which multivariate analysis shows the clinical significance of MMP-7 expression in patients with IHCC. In this study, the Kaplan–Meier life table indicated that 5-year survival was 4 times greater in patients without MMP-7-expressing tumors compared with that in patients with MMP-7-expressing tumors (72.7% vs. 18.3%). Therefore, we believe that patients with MMP-7 expressing tumors of IHCC will have poorer prognoses and should be followed up more closely after the operation. In addition, MMP-7 could be a potential therapeutic target. If specific inhibitor of MMP-7 expression by small RNA and so on is developed in the future,43 it may be useful to improve the prognosis in combination with standard chemotherapy.
In conclusion, the present study suggested that MMP-7 is associated with a poor prognosis in IHCC patients. However, this study included a small number of patients, and further studies enrolling a larger number of patients will be required to confirm the results of this study.
Conflict of interest
All authors declare no conflicts of interest.
No financial support was received for this study.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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