Japanese Journal of Clinical Oncology Advance Access published online on July 29, 2008
Japanese Journal of Clinical Oncology, doi:10.1093/jjco/hyn065
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© The Author (2008). Published by Oxford University Press. All rights reserved
Clinicopathologic Evaluation of Immunohistochemical CD147 and MMP-2 Expression in Differentiated Thyroid Carcinoma
Department of General Surgery, Xiangya Hospital, Central South University, Changsha, China
For reprints and all correspondence: Huihuan Tang, Department of General Surgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China. E-mail: tanhui{at}medmail.com.cn
Received March 24, 2008; accepted June 24, 2008
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Abstract |
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Objective: CD147 is one of the molecules involved in regulating the expression of matrix metalloproteinases (MMPs). The goal of this study was to analyze the expression levels of CD147 and MMP-2 in differentiated thyroid carcinomas (DTCs) tissues, as well as their associations with the clinicopathologic features of DTC patients.
Methods: CD147 and MMP-2 expression in 156 patients who underwent operation for DTC (100 with papillary thyroid carcinomas and 56 with follicular thyroid carcinomas) were examined by immunostaining on paraffin-embedded tumor specimens. The Spearman correlation was calculated between the expression levels of CD147 and MMP-2 in DTC tissues. Then, the association of their expression with clinicopathologic characteristics was analyzed.
Results: CD147 and MMP-2 were expressed mainly in cancerous lesions, but also expressed in some normal tissues. A total of 55 and 58 in 156 (35.26 and 37.18%, respectively) cases showed low CD147- and MMP-2-positive expression; 52 and 50 in 156 (33.33 and 32.05%, respectively) cases showed intermediate CD147- and MMP-2-positive expression and 49 and 48 in 156 (31.41 and 30.77%, respectively) cases showed high CD147- and MMP-2-positive expression. The Spearman analysis indicated that the expression level of CD147 was positively correlated with that of MMP-2 significantly (rs = 0.86, P = 0.02). Positive CD147 and MMP-2 immunostaining associated significantly with extrathyroidal invasion (P = 0.02, 0.03), lymph node metastasis (P = 0.01, 0.01) and depth of tumor invasion (P < 0.01, =0.01).
Conclusions: The results have been demonstrated that the expression of CD147 and MMP-2 may be an important feature of DTC. The detection of these two markers may increase the ability of clinicians to investigate the progression of DTC patients.
Key Words: differentiated thyroid carcinoma • CD147 • MMP-2 • immunohistochemistry • clinicopathology
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INTRODUCTION |
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Thyroid carcinomas are rare malignancies in China and roughly divided into differentiated thyroid carcinomas (DTC) and undifferentiated thyroid carcinoma according to histologic criteria (1). Surgical resection is the main modality of treatment for DTC, including papillary thyroid carcinomas (PTC) or follicular thyroid carcinomas (FTC), with generally favorable results: the disease-specific overall survival rate at 10 years is
80–90% (2). However, a small number of these patients develop metastases and recurrent disease, and this disease course is often correlated with a worse clinical outcome. Several clinicopathologic factors have been reported to be significant in the prognosis of DTC, such as age at diagnosis, local tumor size, extracapsular invasion and distant metastasis (3–6). However, it is the lacking of markers that could predict patients with a low risk of suffering a poor outcome from those with a higher risk of suffering a poor outcome. Therefore, it is important to find biological factors that affect disease recurrence and the survival of patients with DTC. Extracellular matrix metalloproteinase inducer (CD147/EMMPRIN) is a member of the immunoglobulin superfamily of adhesion molecules and is able to activate several matrix metalloproteinases (MMPs), which are the members of zinc-dependent proteolytic enzymes and play a central role in the processes of local invasion and distant metastasis of tumors due to their ability to breakdown basement membranes and most extracellular matrix (ECM) components (7–9). Many metastasis-associated and regulatory elements have been known to be influenced by MMPs. So, CD147 expression is very important for the progression of tumors. There have been a larger number of observations on CD147 and MMPs expression between tumor entities, such as squamous-cell carcinomas, pancreatic carcinoma, renal carcinoma, hepatoma, medullary breast adenocarcinomas and glioblastoma, which are all presented with a particular high incidence of CD147 and MMPs expression (10–12). However, there seems to be a paucity of research concerned with CD147 expression in thyroid carcinomas and its correlation with MMPs. For this reason, the goal of the present study was to investigate the immunohistochemical expression of CD147 and MMP-2 in DTC tissues.
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PATIENTS AND METHODS |
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Patients and Tissue Samples
Surgical specimens were obtained from 156 patients with DTC (100 with PTC and 56 with FTC), who had undergone a resection of primary thyroid carcinoma at the Department of Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, PR China, from January 1992 to January 1997. They included 28 males and 128 females with aging from 16 to 88 years (46.16 ± 18.92 years). The resected thyroid tissues had been macroscopically examined to determine the location and size of the tumors. Samples obtained from the thyroid lesions and dissected lymph nodes were fixed in 10% formalin and routinely processed for paraffin embedding. Histologic sections, cut at 4 µm, were stained with hematoxylin and eosin, and immunoperoxidase procedures (avidin–biotin complex method). Histologic sections were independently reclassified by two experienced pathologists according to histologic typing of the WHO as PTC or FTC (13) to examine the extent and mode of invasion in the thyroid, lymph node metastasis and histologic subtype. Only differentiated carcinomas were selected for this study, whereas all poorly differentiated and anaplastic carcinomas were excluded. In addition, normal thyroid tissues were offered by the Pathology Department of Xiangya Hospital, Central South University, Changsha, Hunan, PR China. The study was approved by the Research Ethics Committee of Xiangya Hospital, Central South University, Changsha, Hunan, PR China. Informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards.
Immunohistochemical Staining and Assessment
Immunohistochemical staining was carried out on tissue microarray sections using the avidin–biotin method. A commercially available kit (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA) and monoclonal antibodies to CD147 and MMP-2 (Santa Cruz Biotechnology, CA, USA) were used. One paraffin-embedded block of thyroid tissue was selected from each case and cut into 4 µm sections. Deparaffinized sections were treated with methanol containing 3% hydrogen peroxide for 10 min before conducting antigen retrieval using a microwave oven at 95°C for 5 min and cooling at 25°C for 2 h. After washing with PBS, blocking serum was applied for 10 min. The sections were incubated with an anti-CD147 and -MMP-2-monoclonal antibody overnight at 4°C. After washing with PBS, a biotin-marked secondary antibody was applied for 10 min followed by a peroxidase-marked streptavidin for an additional 10 min. The reaction was visualized by using 3,3’-diaminobenzidine tetrahydrochloride. The nuclei were counterstained with hematoxylin. Positive and negative immunohistochemistry controls were routinely used. Reproducibility of staining was confirmed by reimmunostaining via the same method used in multiple, randomly selected specimens.
To evaluate the expression of CD147 and MMP-2, three independent observers without knowledge of any clinicopathologic data examined immunostaining. The number of positive cells that showed immunoreactivity on the cell membranes and cytoplasm in 10 representative microscopic fields was counted and the percentage of positive cells was calculated. The criteria used for assessment were as previously reported (14), where 0 (negative, <5%), 1+ (low, 6–25%), 2+ (intermediate, 26–50%) and 3+ (high, >51%) of the tumor cells stained.
Statistical Analysis
SPSS12.0 software for Windows (SPSS Inc, USA) was used for statistical analysis. Continuous variables were expressed as 
± s. Statistical analyses were performed with Fisher’s exact test for any 2 x 2 tables and Pearson 
2-test for non-2 x 2 tables, and P values were given. The P values of <0.05 were considered to be statistically significant.
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RESULTS |
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Immunohistochemical Detection of CD147 and MMP-2 in DTC Tissues
CD147 and MMP-2 were expressed mainly in cancerous lesions (Fig. 1a, b, d and e), and also expressed in some normal tissues (Fig. 1c and f). CD147 immunostaining was observed on the cell membranes and cytoplasm, as well as MMP-2 on the cytoplasm, in all DTC specimens with varying levels of percentage tumor staining and intensities. A total of 55 and 58 in 156 (35.26 and 37.18%, respectively) cases showed low CD147- and MMP-2-positive expression; 52 and 50 in 156 (33.33 and 32.05%, respectively) cases showed intermediate CD147- and MMP-2-positive expression and 49 and 48 in 156 (31.41 and 30.77%, respectively) cases showed high CD147- and MMP-2-positive expression.
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Association Between CD147, MMP-2 Expression and Clinicopathologic Characteristics of DTC
The association of CD147 and MMP-2 expression with the clinicopathologic features of DTC patients is shown in Tables 1 and 2. The positive expression of CD147 and MMP-2 was not related with the gender, tumor size, location of tumor and histologic type of DTC patients. The positive expression of these two markers tended to be associated positively with extrathyroidal invasion (P = 0.02, 0.03), lymph node metastasis (P = 0.01, 0.01) as well as with depth of tumor invasion (P < 0.01, P = 0.01), Especially, the incidences of CD147- and MMP-2-positive expression were significantly higher in tumor tissues with distant metastasis than that without it (P < 0.01).
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The Spearman analysis indicated that the expression level of CD147 was positively correlated with that of MMP-2 significantly (rs = 0.86, P = 0.02).
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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To our knowledge, the metastasis and the invasion of tumors are the complicate processes with many steps that include basement membrane disruption, stromal infiltration, intravasation and extravasation, and invasion of a target organ by tumor cells. All these require the degradation or remodeling of ECM macromolecules by proteolytic enzymes, among them, MMPs are particularly implicated in the metastastic cascade (14,15). MMP-2 is a member of the MMP family and proved to play a crucial role in tumor invasion because of its ability to degrade basement membrane collagens. It has been demonstrated that the increased MMP-2 expression is correlated with the invasive properties of tumor cells in vitro and with the malignant phenotype in vivo. Moreover, in vivo observations have indicated that in most carcinomas, stromal cells, particularly fibroblasts, are the principal source of production of MMP-2 (16–20).
Even though quiescent fibroblasts usually produce relatively low amounts of MMPs, it is likely that tumor-associated fibroblasts are stimulated to produce the elevated levels of MMPs usually present in malignant tumors. Tumor cells may interact with stromal cells via soluble or cell-bound factors, stimulating MMP production (21,22). Among these factors, CD147 has been indicated to stimulate in vitro the fibroblast production of various MMPs such as interstitial collagenase (MMP-1), gelatinase A (MMP-2) and stromelysin-1 (MMP-3) (23–25). CD147 is present in normal tissues, such as epidermis, retinal pigment epithelium, breast lobules and ductules, suggesting that CD147 may have a physiologic role in tissue remodeling by causing induction of stromal MMPs (26–28). However, CD147 expression is more prominently found in tumor proliferations.
Thyroid carcinoma constitutes a heterogeneous group of tumors with widely varying prognoses. Several studies have reported that PTC overexpresses MMP-2 and have suggested an important role for this enzyme in the progression of PTC (29–32). Our results on MMP-2 are in accordance with those of Cho Mar et al. (33), who reported that MMP-2 expression was associated with tumor invasion and metastasis in PTC and FTC. Our study also associated the CD147 expression in 156 patients with DTC and their synchronous and relapsing metastases to clinical outcome. Whereas most studies thus far investigated CD147 expression among other carcinomas, including all histopathologic grades, our study was focused on differentiated carcinomas of the thyroid only to investigate the association of CD147 expression levels with clinicopathologic characteristics of DTC. The immunohistochemical staining demonstrates positive expression of CD147 in malignant proliferations of DTC. These data are in agreement with previous reports using a different antibody (34,35).
In conclusion, the expression of CD147 and MMP-2 may play an important role in the progression of DTC. Detection of high CD147- and MMP-2-positive expression levels may benefit the prediction of progression of DTC patients at an early stage.
Conflict of interest statement
None declared.
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References |
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