| Background and objectives:In the United States, prostate cancer (PCa) is one of the most common urological cancers and the second leading cause of cancer death in men. Although patients with localized prostate cancer can often be successfully treated with radical prostatectomy or radiation therapy, hormone refractory and metastatic disease are usually the causes of prostate cancer deaths. It is often difficult to identify patients who will progress, recur and require additional treatments in the clinical work. Prostate cancer prognosis varies significantly in patients according to clinical stage, pathological grade and Gleason score. More and more sensitive prostate cancer novel molecular markers that are associated with biological aggressiveness and providing valuable information in the diagnosis and treatment of the disease are particularly important. Until now, effective treatment of metastatic disease is one of the major therapeutic challenges in prostate cancer treatment. In recent years, many studies have focused on identifying nomograms and establishing a model to include various prognostic parameters to predict prostate cancer outcome. Prostate cancer cells with high-metastatic or low-metastatic potential vary in their biological properties, such as proliferation, adhesiveness, invasiveness and motility. These variations are the results of both germ line variation between individuals and somatic alterations of genes and gene expressions in cancer cells. For these reasons, considerable efforts have been made to discover new molecular markers that can accurately predict the onset of disease recurrence and lead to better targeted and more effective treatment. Extracellular matrix metalloproteinase inducer EMMPSRIN (also known as CD147), a member of the immunoglobulin family, is a glycoprotein enriched on the surface of many types of tumor cells. CD147has been demonstrated to be involved in different kinds of tumor invasion and metastasis via stimulating matrix metalloproteinase(MMPS) synthesis in neighboring fibroblasts, in malignant cell proliferation via the activation of ERKl/2and p38mitogen-activated protein kinases, in enhancing angiogenesis via vascular endothelial growth factor, in inducing chemoresistant tumor cells via the production of hyaluronan and in the resistance of cancer cells to anoikis through inhibition of Bim. Reduction of CD147expression increased DNA fragmentation and decreases cellular viability, as demonstrated by activation of caspase-3. Moreover, CD147expression is reported to correlate with the clinical prognosis of patients with breast carcinomal2and other types of cancers. Inhibition of CD147gene expression with RNA interference could reduce tumor cell invasion and tumorigenicity, as well as increased chemosensitivity to paclitaxel. However, the distribution of CD147in benign prostate tissues, metastatic lesions and different prostate cancer stages, especially the correlation between the CD147expression and the prostate-specific antigen (PSA) failure-free survival and the distant metastasis-free survival are yet to be evaluated. In our study, we exaimed the CD147expression in formalin-fixed and paraffin-embedded (FFPE) specimens from240prostate cancer patients who were confirmed to have Prostate cancer with a range of Gleason scores (GS) and well characterized clinicopathological properties. The correlation of PSA failure-free survival, metastasis-free survivals and the overall survival of all the prostate cancer patients were analyzed to investigate the influence of CD147expression in cancer progression. Our study is aim to demonstrate the involvement of CD147in prostate cancer progression and prove if it can become an independent predictor of biochemical recurrence, development of metastasis and reduced overall survival in prostate cancer. Materials:Patients populationTwo hundred and forty consecutive patients with prostate cancer who underwent radical prostatectomy at the Massachusetts General Hospital (MGH, Boston, MA) from September1993to March1995were included in our study. Patients who received adjuvant or neoadjuvant hormonal or radiation treatment prior to cancer recurrence were excluded. All hematoxyolin-eosin (H&E)-stained sections from each case were reviewed by pathologist, and the Gleason score was reassigned based on the current grading recommendation provided by the International Society of Urological Pathology. The tissue blocks containing the index prostate cancer (tumor focus with the highest GS) were selected for inclusion in the tissue microarray (TMA). Relevant clinicopathological data collected included age, preoperative PSA, GS, American Joint Committee on Cancer (AJCC) T stage, surgical margin status, PSA recurrence, PSA recurrence date, postoperative metastasis status, metastasis date, overall survival and operative date. The PSA failure-free survival was defined as the time interval between the initial surgery and the day of the first appearance of detectable PSA in patients with two consecutive rises of PSA. The distant metastasis-free survival was defined as the time interval between the initial surgery and the day of the detection of metastatic lesions. The overall survival was deternined from the date of surgery to the time of the last follow-up or death. Our study received approval by the human study ethics committees at MGH, Boston, MA and of the Mistry of Public Health of PR China.Tissue microarray constructionThe tissue microarray was constructed by using a manual tissue-array instrument (Beecher Instruments, Silver Spring, MD). Briefly, the H&E-stained slides were reviewed for accuracy of Gleason score. The index tumor defined as the largest and/or highest GS was marked on the slide. Tissue cylinders with a diameter of0.6mm were punched from selected area of each donor block and brought into a recipient paraffin block. Each block contained normal prostate tissue derived from normal peripheral zone significantly away from the tumor, and total of20normal prostate tissues were included as the control samples for the immunohistochemical analysis. After construction of the TMA blocks, H&E-stained sections were made for histologic evaluation.Methods:Immunohistochemical analysisThe TMA paraffin-embedded tissues were cut at5um and then deparaffinized with xylene and rehydrated for further H&E or peroxidase3,3’-diaobenzidine immunohistochemical staining using DAKO EnVision System (Dako Diagnostics, Zug, Switzerland).Briefly, after a brief proteolytic digestion and a peroxidase blocking of tissue slides, the slides were incubated overnight with the primary antibody against respective target protein at a dilution of1:1,000at4C. After washing, peroxidase labeled polymer and substrate-chromogen were then used to visualize the staining of the interested proteins. Normal prostate tissues were used as control for immunohistochemical staining. After a hematoxylin counterstaining, each stained slide was scored by two experienced pathologists independently in a blinded fashion without any information regarding the clinicopathological data and clinical outcomes of the patients. The scores of the two pathologists were compared and any discrepant scores were resolved through re-exaiming the slide by both pathologists simultaneously to achieve a consensus score. The numbers of positive-staining cells showing immunoreactivity on the cell membranes and cytoplasm in10representative microscopic fields (or the tumor cells in the entire core if tumor cells are limited) were counted, and the percentage of positive cells was calculated. Tumor specimens were scored as positive if greater than5%of the tumor cells exhibited immunoreactivity. Statistic Analysis:The software of SAS9.2version (SAS Institute, Cary, NC) was used for statistical analysis. Continuous variables were expressed as X±s Statistical analysis was performed independently by two biostatisticians with Fisher’s exact test for any2×2tables and Pearson v2test for non-2x2tables. The Cox proportional hazards model was used to evaluate the association of CD147expression with PSA failure-free survival, distant metastasis-free survival and overall survival in univariate and multivariate analyses. The underlying time axis for the Cox model is years of survival. Survival curves were estimated based on the Kaplan-Meier method and assessed with log-rank test. The distribution of each clinical feature was compared between CD147-positive and CD147-negative group, with the use of two sample t test foncontinuous variables and χ2test for categorical variables. Differences were considered statistically significant when p value was less than0.05(two-sided).In the univariate analyses with Cox proportional hazard model of CD147expression, confidence interval, CD147status (CD147positive vs. CD147negative), Gleason score (6,7,8,9and10), preoperative PSA levels ng/ml (continuous value PSA levels<4ng/ml is considered normal in clinical practice), AJCC pathological tumor stage (pT2and pT3), surgical margin (positive vs. negative), PSA failure (biochemical recurrence) were included. The CD147status, GS, preoperative PSA, AJCC pathological tumor stage, surgical margin and the status of PSA failure were included in the multivariate analysis. Values of p<0.05are considered significant.Results:CD147expression and localization in patients with Prostate cancerTo determine whether CD147expression could be linked to prostate cancer progression, we stained prostate tissue sections using the antibody that specifically recognizes CD147(provided from the Fourth Military Medical University, Shanxi, China). In general, we found that this antibody stained the membrane and cytoplasm of prostate cancer cells and gave evenly distributed staining patten with various intensities. The positive expression rate of CD147in tissue samples from prostate cancer patients [113/240(47.08%)] was significantly higher than that in normal prostate tissues [1/20(5%)](p=0.006). We noticed that in some sections of normal prostate tissues very faint CD147staining was observed when compared to tumor tissues obtained from Prostate cancer patients and was considered in the negative range. We analyzed240radical prostatectomy specimens represented in TMA from Prostate cancer patients. The clinical characteristics of these patients and the statistical analysis of CD147-positive expression and various clinicopathological features are analyzed. We found that CD147expression was significantly correlated with Gleason score (p=0.0002), positive surgical margin status of Prostate cancer patients (p<0.0001), reduced PSA failure-free survival (p<0.0001), reduced metastasis-free survival (p<0.0001) and reduce overall survival (p<0.0002). When Kaplan-Meier analysis was conducted, we found that over expression of CD147is significantly associated with reduced PSA failure-free survival, distant metastasis-free survival and overall survival.CD147expression and surgical outcomeUnivariate analysis revealed that CD147expression (p<0.001, p=0.0023and p=0.0006, respectively) and Gleason score (p<0.001, p=0.0013and0.0006, respectively) were significant prognostic factors for PSA failure-free survival distant metastasis-free survival and overall survival in patients with prostate cancer. On the other hand, our analysis showed that preoperative PSA concentration (p<0.001), clinical stage (p=0.0023) and surgical margin (p=0.0036) were significant prognostic factors for PSA failure-free survival but did not show any correlative relationship with the distant metastasis-free survival and overall survival in patients with prostate. We then used the Cox proportional hazards multivariate model to analyze the association of clinicopathological factors and CD147staining with PSA failure-free survival, distant metastasis-free survival and overall survival. Multivariate analysis indicated that CD147expression and Gleason score are both independent predictors of PSA failure-free survival, distant metastasis-free survival and overall survival in patients with prostate cancer. We also found that preoperative PSA concentration was shown to be independent predictors of PSA failure-free survival. However, it could not be considered as independent predictor for distant metastasis-free survival or overall survival in patients with prostate cancer, whereas pathological tumor stage and surgical margin preoperative were not independent predictors for PSA failure-free survival, the distant metastasis-free survival or the overall survival. These results suggested that CD147expression directly contributes to the malignant potential of prostate cancer.CD147expression and Gleason score, Pathological gradeGleason score is considered as one of the most reliable factors for prediction of prostate cancer disease progression. The need for additional tests to complement and improve on the GS would be extremely helpful to identify patients who need to be treated aggressively and who can safely be monitored for disease progression. In our study, we explore the potential correlation of CD147over-expression levels with the survival outcome in Prostate cancer patients with different Gleason score. Both univariate and multivariate analyses revealed that CD147expression and Gleason score were both significant prognostic factors for PSA failure-free survival, metastatic-free survival and overall survival in patients with prostate cancer. Kaplan-Meier survival curves indicated that CD147expression is associated with reduced PSA failure-free survival in the GS6and7groups of patients,but not in the GS8group of patients. Interestingly, even though CD147was not significantly associated with the overall survival in patients with higher GS (GS=7, p=0.1757and GS≥8, p=0.3151), the Kaplan-Meier survival curve indicated a correlative relationship between CD147expression and the reduced overall survival in patients at lower GS (GS≤6, p=0.0160). Moreover, the Kaplan-Meier survival curves also showed that the CD147over-expression was not only a significant predictor of both PSA failure- free survival and overall survival in higher pathological stage Prostate cancer (p<0.0001and p=0.0028, respectively), but also in lower pathological stage prostate cancer (p<0.0001and p=0.0253, respectively).Conclusion:1. Compared with the normal prostate cells, CD147is over-expressed in prostate cancer cells.2. CD147expression is correlated with Gleason score, surgical margin status, PSA failure, metastasis failure and overall survival.3. CD147positive expression may reduce the PSA-free survival rate, metastasis-free survival rate and overall survival rate.4. CD147expression may serve as an independent prognostic factor for prostate cancer besides Gleason score.5. CD147expression can not only predict the PSA-free survival rate and overall survival rate in pT2stage prostate cancer, but also play the same role in pT3stage prostate cancer.6. CD147expression can predict the PSA-free survival rate and overall survival rate in GS≤6group, but not in the high GS group.7. Our data demonstrated that CD147may be involved in Prostate cancer progression. It may also play a role in the future assessment of newly diagnosed prostate cancer, as it may add to the current prognostic model including preoperative PSA, GS, AJCC tumor stage and surgical margin status.8. The hypomethylation of CD147is a potential mechanism which induces high expression of CD147in prostate cancer. |
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