| BackgroundEndometrial carcinoma, also referred to as uterine cancer, represents the most frequently diagnosed malignancy of the female genital tract and ninety-seven percent of all cancers of the uterus arise from the glands of the endometrium. The incidence varies among regions and in contrast to that in less developed countries, it is ten times higher in North America and Europe. Its incidence has raised year after year in China. A dualistic model of endometrial carcinogenesis has been proposed since1980s based on light microscopic appearance, clinical behavior, and epidemiology,which includes type Ⅰ and type Ⅱ cancers. Most endometrioid carcinomas are well to moderately differentiated and arise on a background of endometrial hyperplasia. These tumours, also known as type1(low-grade) endometrial carcinomas, have a favourable prognosis. They are associated with long-duration unopposed oestrogenic stimulation. About10%of endometrial cancers are type2(high-grade) lesions. Women with such tumours are at high risk of relapse and metastatic disease. These tumours are not oestrogen driven, and most are associated with endometrial atrophy. The histological type is either poorly differentiated endometrioid or non-endometrioid. Obesity, hypertension, diabetes, nulliparity, menopause postponement etc are the high risk factors associated with endometrial cancer, but the pathogenenesis is not fully known so far. A multi-step endometrial carcinogenesis involving coordinated intervention of hormonal regulation, gene mutation, adhesion molecules, apoptosis, imbalance between metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is currently accepted. Most cases of EC are diagnosed in transitional years of menopausal and postmenopausal middle-aged women above the age of50.The disease often causes abnormal bleeding as a first symptom and is therefore usually detected in its early stages (FIGO stage Ⅰ). When diagnosed at early stages of the disease, this type of cancer is a highly curable malignancy with a5-year relative survival rate of more than80%. However, there are a group of patients with a high risk of cancer recurrence or metastatic spread.Lymph metastasis and direct invasion is the main route of spread for endothelial carcinoma. Especially, partial and pelvic lymph node metastasis should happen earlier when the deeper endometrial invasion, more malignant degree, or worse histological grade existed, which will directly affect the result of therapy. Lymph metastasis firstly occurs in adjacent stroma of solid tumors in respect that tumor cells produce some tissue factors that can promote micro-lymphatic hyperplasia and hydrolyze stromal component, which will lead to the invasion and instant metastasis spread of solid tumors by lymph cycle.Matrix metalloproteinases(matrix metalloproteinase, MMPs) are a group of structurally related proteins that degrade extracellular matrix and basement membrane components. Increased MMP expression has been detected in a wide range of cancers, and it has been correlated with tumor invasion and metastasis. MMP-9is one of MMPs what has been studied most extensively. It is one of the main members of the family of MMPs. The ability of MMP-9(gelatinase B) to degrade type IV collagen and fibronectin, present in both vascular and subepithelial basement membranes, has been firmly established. MMP-9plays a critical role in breakdown of extracellular matrix in normal physiological processes, such as endometrial physiologic characteristics, embryonic development, reproduction, and tissue remodeling. MMP-9in the form of enzyme secretion, must be activated to degrade matrix collagen and other proteins. Generally considered fibrinolytic enzyme in fibrinolytic enzyme activator activation is under the action of fibrinolytic enzyme, the latter to cascade amplification waterfall effect MMP-9to activate substrate biodegradation. Once activated, MMP9is thought to promotes the metastasis of tumor cells.ObjectivesSo far, there have been few studies concerning matrix metalloproteinases in endometrial cancer. In some of the previous studies, MMP-1and MMP-2or MMP-7, MMP-8, and MMP-9or MT1-MMP (membrane-type matrix metalloproteinase-1) proteins or mRNAs have been detected in endometrial cancer tissue. Increased amount or activity of MMP-9has been found by different methods (sandwich immunoassay, zymography, ELISA, and in situ hybridization for mRNA) in cancer-affected endometrium compared to a benign one. Overexpressed MMP-9in EC may accelerate tumor growth by inducing angiogenesis and enhance local cell invasion and metastasis by degrading the extracellular matrix. However, some issuses were still controversial about MMP-9expression correlated to clinicopathologic features in endometrial cancer, including the prognostic value of MMP-9. Di Nezza et al. used immunohistochemistry assay in EC tissues to find MMP-9staining scores in tumor cells were significantly associated with the presence of myometrial invasion and vascular/lymphatic invasion. Furthermore, Aglund et al. showed that increased expression of MMP-9protein was associated with histologic grade and clinical stage. Karahan et al. found that MMP-9overexpression was correlated with vascular and lymphatic invasion (P=0.001and P=0.012, respectively). Lopata’s group has confirmed elevated levels of latent and active forms of MMP-2and MMP-9in uterine lavage samples from endometrial cancer patients. However,in the study of Honkavuori et al, they did not find any correlations between MMP-9immunostaining and conventional prognostic indicators of endometrial cancer. Therefore, the aim of the present study was to analyze the expression of matrix metalloproteinase-9(MMP-9) in endometrial cancer and its correlation with clinicopathologic features, including the survival of patients with endometrial cancer. Our research will provide the clue and way to deeply research to regulation mechanism of endometrial cancer.Methods1. Sample collectionFrom2002to2007, formalin-fixed and paraffin em-bedded samples of30normal endometria,30samples of endometrial atypical hyperplasia and128samples of endometrioid carcinoma were obtained in the Third Affiliated Hospital of Guangzhou Medical School, Guangzhou City, China. None of the128patients with endometrioid carcinoma received chemotherapy or radiotherapy before surgery. In the128EC cases, the median age of the patients was51.6years (range from30to85). The clinical follow-up time of patients ranged from5to102months. All specimens had confirmed pathological diagnosis and were staged according to the FIGO2009.2. ImmunohistochemistryParaffin sections (3μm) from samples of128EC and30EAH and30NE specimens were deparaffinized in100%xylene and rehydrated in descending ethanol series (100%,90%,80%,70%ethanol) and water according to standard protocols. Heat-induced antigen retrieval was performed in0.01mol/L citrate buffer for2min at100℃. Endogenous peroxidase activity and nonspecific antigen were blocked with peroxidase blocking reagent containing3%hydrogen peroxide and serum, followed by incubation with goat anti-human MMP-9antibody (1:100)(Sc6840, Santa, MA, USA) for overnight at4℃. After washing, the sections were incubated with biotin-labeled rabbit anti-goat antibody for10min at room temperature, and subsequently were incubated with streptavidin-conjugated horseradish peroxidase (HRP)(Maixin Inc, China). The peroxidase reaction was developed using3,3diaminobenzidine chromogen solution in DAB buffer substrate. Sections were visualized with DAB and counterstained with hematoxylin,1%hydrochloric acid ethanol2differentiation20s, dehydration, xylene, after a transparent mounted in neutral gum, and analyzed using a bright field microscope.3. Evaluation of stainingThe immunohistochemically stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters. The staining intensity was scored as previously described. The extent of the staining, defined as the percentage of positive staining areas of tumor cells or normal nasopharyngeal epithelial cells in relation to the whole tissue area, was scored on a scale of0to4as the following:0,<10%;1,10-25%;2,26-50%;3,50-75%;and4,>76%. The sum of the staining intensity and staining-extent scores was used as the final staining score for MMP9(0-7). For statistical analysis, a final staining scores of0-5and6-7were respectively considered to be low and high expression.4. Statistical analysesAll statistical analyses were performed using SPSS13.0software. The χ2test was used to analyze the relationship between the levels of MMP-9expression and clinicopathologic characteristics. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. The significances of various variables in survival were analyzed using multivariate cox proportional hazards model. P value of less than0.05was considered statistically significant.Results1.We measured the expression levels and subcellular localization of MMP-9protein in128archived paraffinembedded EC samples,30EAH samples and30NE samples using immunohistochemical staining. Specific MMP-9protein staining was found in the cytoplasm of non-cancerous and malignant epithelial cells. Furthermore, we observed that in42.2%(54/128) of EC samples, MMP-9protein was highly expressed. In comparison, highly expressed MMP-9protein of EAH and NE samples was26.7%and16.7%, respectively, which was significantly lower than that in the EC samples (P=0.006)2. We did not find a significant association of MMP-9expres-sion levels with patient’s age, menopausal status, FIGO stage, and depth of myometrial invasion in128EC cases. However, we observed that the expression level of MMP-9was positively correlated with the status of lymph node metastasis (Negative vs. Positive)(χ2=4.076, P=0.044), and histopathological grade (G1vs. G2vs. G3)(χ2=6.276, P=0.043) in EC patients.3. We assessed the association between the levels of MMP-9expression and patients’ survival using Kaplan-Meier analysis with the log-rank test. In128EC cases with prognosis information, we observed that the level of MMP-9protein expression was not correlated with the overall survival of EC patients. In addition, we performed multivariate analysis of the levels of MMP-9protein expression adjusted for age, family history of tumor, education, Health Insurance, career, menopausal status, FIGO stage, histological grading, depth of myometrial invasion, lymph node status, complications (including high blood pressure or diabetes mellitus), histologic subtype of EC patients. The results showed that histological grading and status of lymph node metastasis and depth of myometrial invasion, but not MMP-9, were significantly correlated with patients’ survival (P=0.021, P=0.001, P=0.004, respectively).ConclusionsIn summary, our study demonstrated that the expression level of MMP-9in endometrial carcinoma was higher than that in non-cancerous tissue. MMP-9was up regulated in endometrial carcinoma, in which indicated that it may play all important role in the genesis and development of endometrial cancer, high levels of MMP-9protein were positively correlated with the status of lymph node metastasis and the histopathological grade of EC patients. It shows that MMP-9decomposes and destroys extracellular matrix and cell membrane around EC cell surface, then promotes invade and diversion of EC. Patients with higher MMP-9expression did not correlate with EC patients’ clinical outcome. Multivariate analysis suggested that histological grading and status of lymph node metastasis and depgh of myometrial invasion, but not MMP-9expression level, were significantly correlated with patients’ survival. Yet, due to the limited sample size of patients in our investigation, further studies would be needed to verify these findings, such as increasing sample size of patients to verify our results and importing MMP-9gene into endometrial cancer cell lines and observing the effects of MMP-9on cell proliferation, migration, invasion, and metastasis in vitro as well as the rate of tumorigenesis and survival time of nude mice with transplanted tumor in vivo. |
PDF Full Text Request