Expression Of Matrix Metalloproteinase Regulator, RECK, And Its Clinical Significance In Osteosarcoma

Expression of matrix metalloproteinase regulator, RECK, and its clinical significance in osteosarcomaIntroductionOsteosarcoma is a disease which predominantly targets the adolescent age group and the most common primary malignant bone tumor in general. In spite of advances in imaging techniques, surgery, and especially combination chemotherapy, the overall survival rate over 5 years is still approximately 65%, and 25%-50% of patients with initial diffusion succumb to lung metastasis. The rate of mortality for osteosarcoma is basically the same as that some decades ago. Prognostication in individual cases remains a problem. It would be helpful if objective instruments were available for predicting the chance of survival or chemotherapy response, especially early in treatment, preferably even before surgery.The RECK (reversion-inducing cysteine-rich protein with Kazal motifs) gene was originally isolated as a novel transformation suppressor gene by cDNA expression cloning. The RECK gene is extensively expressed in normal tissues and various tumor-derived cell lines, but down-regulated in tumor tissues. It suggest a potential importance of RECK in the diagnosis and therapy of malignant tumors. The RECK gene encodes a glycosylphosphatidylinositol (GPI)-anchored glycoprotein (molecular weight,110,000) as a negative regulator of matrix metalloproteinases (MMPs), including at least MMP-2, MMP-9, and MT1-MMP. Restoration of RECK in tumor-derived cell lines strongly suppresses their abilities to invade, metastasize, and angiogenesis. Positive correlation between residual RECK expression in tumor tissues and survival of the patients have been documented in a variety of cancers.In this study, we aimed to determine whether the expression of RECK is a predictor of the clinical behavior of osteosarcoma and predict a high-risk group of patients who would benefit from new treatment strategies.Objective:To explore the expression of RECK in osteosarcoma To reveal the possible role as prognostic indicatorMATERIALS AND METHODS1 Patients and Tumor Samples.We retrospectively identified 61 patients with primary osteosarcoma occurring between 2001 and 2005 who met the following criteria:(1) no metastasis at presentation;(2) no history of previous treatment;(3) adequate preoperative and postoperative chemotherapy;(4) tumor excision with a wide or radical margin;(5) presence of incisional biopsy specimens available for immunohistochemical staining (IHS). Among the 61 selected patients, there were 42 males and 19 females with an average age of 21.4 years (range,4-53 years). Following initial biopsy, all patients underwent multiagent, neoadjuvant chemotherapy and definitive surgery. The locations of the tumor were the femur (28 cases), tibia (30cases), proximal humerus (2 cases), pelvic bone (1 case). The histologic subtypes were osteoblastic in 49 patients, and other in 12 patients.The tumors were classified as intracompartmental (StageⅡA) in 16 patients, extracompartmental (StageⅡB) in 43 patients and Stage I in 2 patients. Chemotherapy was given before and after surgery.The chemotherapy protocol was the modified T12 protocol. The surgical margins of the tumor specimens were histologically defined according to the system of Enneking[11]. The margins were radical in 8 patients and wide in 41 patients. During option of surgical procedures, the location and extent of the tumor and the life expectancy of the patient took into account. Limb-salvage procedures were performed in 42 patients (85.7%), whereas 7 patients (14.3%) underwent amputation. All tumors specimens were immediately fixed in 10% formalin and then embedded in paraffin.The specimens we analyzed were from incisions of biopsy before chemotherapy. The paraffin blocks were evaluated again to select a representative area. The extent of tumor necrosis was evaluated according to a previously described semiquantitative method. The response of chemotherapy was considered good if the extent of tumor necrosis was>90% and poor if it was<90%. Follow up duration was defined as the date of initial presentation to the date of death or last visit. The minimum followup for survivors was 8 months2. Reagents2.1 RECKpolyclonal antibodies against RECK (H-300, rabbit polyclonal antibody; 1:50, Santa Cruz Biotechnology Inc, sc-28918)2.2 ChemMateTMEnVisio+/HRP/DAB Rb&McChemMateTMEnVisio+/HRP/DAB Rb&Mc DAKO Biotechnology Inc.3. ImmunohistochemistryRECK expression was determined by using Streptavidin-Perosidase immunohistochemistrical method. Serial 4-μm sections were cut from formalin-fixed, paraffin-embedded blocks and placed on silan-coated slides. After deparaffinization, sections were incubated in 3% H2O2 for 20 min to inactivate endogenous peroxidase. Deparrafinized and rehydrated specimens were heated in 10 mM citrate buffer (pH 6.0) for 10 min in an autoclave at 105℃. After being cooled to room temperature for 30 min, specimens were incubated with normal horse serum for 20 min at room temperature followed by incubation with polyclonal antibodies against RECK (H-300, rabbit polyclonal antibody; 1:50, Santa Cruz Biotechnology Inc, sc-28918) for 16 h at 4℃. The sections were washed three times for 5 min in PBS and incubated for 1 h with biotinylated antirabbit IgG secondary antibodies. After rinsing, immune complexes were visualized by the standard avidinbiotin-peroxidase complex (ABC) method. After counterstaining with Meyer's hematoxylin, the slides were dehydrated and mounted.The staining was evaluated independently by two pathologists, and the data from the two investigators were averaged. We interpreted immunoreactivity in a semiquantitative manner by an intensity proportion scoring system similar to that previously described for RECK expression scoring in non-small-cell lung cancer and Ezrin expression in osteossarcomas[13 14]. The score was calculated by summing intensity score and proportion score providing a score between 0 and 6.The proportion score was as follows: 0=no positivity; +1=less than 25% tumor cell positivity; +2=25 to 50% tumor cell positivity; +3=more than 50% tumor cell positivity. The intensity score was as follows: 0=no staining; +1=weak staining; +2=intermediate staining; +3=strong staining.We further divided RECK-positive specimens into low and high expression according to the score; specimens with scores 1 to 4 were regarded as low expression, whereas specimens with scores 5 or 6 were regarded as high expression.4. Statistical AnalysisData were compared by theχ2 test Kaplan-Meier method was used to analyze the postoperative survival rate, and the differences in survival rates were assessed by the log-rank test. Any factor influencing prognosis in univariate analysis was analyzed in a multivariate analysis using Cox's proportional hazard regression model with a forward conditional stepwise procedure to determine whether the factor was acting independently. All calculations were performed with SPSS version 13.0 software, and p<0.05 was considered significant.5. Results5.1 The expression of RECK in osteosarcomaThe expression of RECK canbe found in most patients. RECK immunoreactivity was found in the cytoplasm of tumor cells.5.2 RECK Expression and Clinical CharacteristicsOn the basis of summing intensity score and proportion score of RECK in the primary tumors,27 patients were classified as RECK-weak patients and 34 patients as RECK-strong patients. No correlation was revealed between the RECK status and age, sex, tumor location, or subtype (Table 1). RECK expression was correlated with metastasis (p=0.003) and recurrence(p=0.001). 5.3 Prognostic Factors and Postoperative SurvivalUnivariate analyses of prognostic factors demonstrated that the RECK expression, metastasis, histological response, Ennecking stage and recurrence were significant prognostic factors(Table 2). No correlations were found between prognosis and age, sex, tumor location, or subtype. The 5-year survival rate of RECK-strong patients was 82.4%, which was significantly higher than that of RECK-weak patients.5.4 Multivariate Analysis of Prognostic FactorsTo assess whether the prognostic value of RECK was attributable to prognosis, COX regression analysis was performed with all variables. Reduced RECK expression was an independent and significant factor to predict a poor prognosis(P =.006; hazard ratio,0.253; 95% confidence interval,0.095-0.676; Table 3).6. ConclutionRECK status is a useful prognostic factor in osteosarcoma, and an independent prognostic factor contributing to the determination of more adequate therapy strategies for each patient.