The Prognostic Value Of EMMPRIN And ADAM17 Overexpression In Patients With Extrahepatic Cholangiocarcinoma

Research BackgroundExtrahepatic cholangiocarcinoma (ECC) is an uncommon but not rare malignancy which derived from the extrahepatic bile duct epithelium. Anatomically, ECC can be divided into distal cholangiocarcinoma and hilar cholangiocarcinoma, based on the location of tumor. En bloc resection of the tumor (including negative histological resection margins) is the only chance for patients to obtain long time survival. However, it is difficult to get a free bile duct margin because of the rather short length of the bile duct and the tumor microscopic spread along with the bile duct wall. Previous researches had found that multiple clinicopathologic factors were associated with patients’ outcome, including depth of tumor invasion, lymph-node-metastasis, surgical margin, histological differentiation, and so on. The rate of radical resection was increased followed by the improvements of surgical techniques. Unfortunately, the patients who undergo resection were often associated with a poor prognosis. It was shown by previous researches that the 5-year survival rate was approximately 30%. Thus, an exploration of the new molecular pathogenesis of ECC may play pivotal roles in the guiding clinical treatment and improvement of prognosis.It is widely known that the tumorigenesis and progression was a complex and multistep signal conditioning processes, involving degradation and proteolytic remodeling of the extra-cellular matrix, cellular signal-regulated activities and angiogenesis factors. Degradation and proteolytic remodeling of the tumour extra-cellular matrix is a critical step in tumour metastasis and multiple proteolytic enzymes were involved in this process, including MMPs (matrix metalloproteinase) family, cathepsins and plasmin. Moreover, degradation and proteolytic remodeling of the extracellular matrix is the initiating event in the formation of the tumour microenvironment. Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) plays an important role in the synthesis of extracellular matrix metalloproteinase (MMPs). The abnormally high expression level of EMMPRIN was correlated with poor prognosis of cancers, including intrahepatic cholangiocarcinoma, renal carcinoma, colorectal cancer, osteosarcoma, and hepatocellular. Zhao et al silenced the expression level of EMMPRIN in ovarian carcinoma cells by siRNA treatment showed reduced expression of Wnt5a, p70s6k, Akt, VEGF, survivin, Bcl-xL, and MMP-9 than mock and control cells at both mRNA and protein levels, and resulted in a lower growth, apoptotic induction, Gl arrest, decreased migration and invasion, and suggested that EMMPRIN(CD147) might be involved in the pathogenesis, differentiation, and progression of ovarian carcinomas, possibly by modulating cell cycle, migration, proliferation, invasion, and apoptosis. ADAMs (a disintegrin and metalloproteinase) family is membrane-anchored protein, which has been implicated involving protein ectodomain shedding and hence the rapid modulation of key cell signalling pathways in the tumor microenvironment. It has been revealed that ADAMs is a potent sheddase of six out of the seven known EGFR(epidermal growth factor receptor) ligands (transforming growth factor (TGF) a, HB-EGF, amphiregulin, betacellulin, epiregulin and EGF), which regulated the EGFR activity in a wide variety of tumour. EGFR activity is a key component in the tumorigenesis and progression, and its expression associated with poor prognosis of patients with extrahepatic cholangiocarcinoma. As a primary member of the ADAM protein family, ADAM17 (tumor necrosis factor-alpha-converting enzyme) plays an important role in activating the EGFR. Experiments have corroborated that ADAM 17 regulates biological characteristic of cancers through signaling pathways. For instance, ADAM17 promote prostate cancer cell invasion and proliferation via EGFR-MEK-ERK and EGFR-PI3K-AKT pathway. ADAM17 regulates epidermal growth factor receptor (EGFR) expression via the activation of Notch1 in Non-Small Cell lung cancer. Meanwhile, growing evidences point to overexpression of ADAM 17 was correlated with poor prognosis of human cancers. In addition, recent reports suggested that there may be ADAM17-EGFR-EMMPRIN asis regulated tumor biological characteristics.However, there were no related literatures on expression of EMMPRIN and ADAM 17 in ECC field. This study was focused on the expression of EMMPRIN and ADAM17 in ECC and their prognosis, and provide preliminary theoretical basis for the following molecular studies of ECC.Part 1 Relationship between EMMPRIN, ADAM17 expression and clinicopathological parameters of patients with extrahepatic cholangiocarcinomaObjectiveTo observe the expression of EMMPRIN and ADAM17 in ECC, the relationship between EMMPRIN, ADAM17 expression and clinicopathological parameters of patients with ECC, and the relevance of EMMPRIN and ADAM17 expression in ECC.MethodsClinical data and specimens from 78 patients with ECC who underwent surgical resection at Navy General Hospital between March 2003 and December 2013 were identified from a database maintained by the Department of Hepatobiliary Surgery. The diagnosis of ECC was validated by the Department of Pathology in Navy General Hospital. None of them had history of malignancies and received preoperative anti-cancer treatment, such as chemotherapy or radiotherapy. Additionally,20 cases of pericarcinoma tissues were used as a normal control. The expression of EMMPRIN and ADAM17 was detected using the immunohistochemical staining method and compared with clinicopathological parameters via chi-square test, and evaluated the relevance between the expression of EMMPRIN and ADAM17 through Spearman’s method in patients with ECC.ResultsThe EMMPRIN and ADAM 17 proteins mainly located on the membrane and in the cytoplasm of the tumor tissues and their adjacent tissues. The highly expression of EMMPRIN and ADAM17 protein was showed in 46(59.0%) and 41 (52.6%) of 78 patients with ECC, respectively. However, the EMMPRIN and ADAM17 protein was lowly or negatively expression in adjacent tissues of tumor. The EMMPRIN and ADAM17 expression was statistical significance difference between the tumour tissues and their adjacent tissues(P<0.001). The EMMPRIN expression was related with a number of clinicopathological parameters including age(<65, P=0.023), histological differentiation(poor, P=0.010), T classification(T3+T4, P=0.001), lymph-node-metastasis(Positive, P=0.044), and vascular invasion(Positive, P=0.010), but not related to gender, tumour size, tumor location, Pre-CA19-9 and perineural invasion. The highly expression of ADAM17 protein were significantly correlation with tumour size(Size≥3cm, P=0.047), T classification(T3+T4, P=0.007), lymph-node-metastasis(Positive, P<0.001), and vascular invasion(Positive, P=0.018), but not associated with age, gender, tumor location, differentiation, Pre-CA19-9, and perineural invasion. It was statistically significant that the relevance between the expression of EMMPRIN and ADAM17 in patients with ECC (r=0.356, P=0.001).ConclusionsThe EMMPRIN and ADAM 17 proteins mainly located on the membrane and in the cytoplasm of cell, and its overexpression which might be involved in invasion and progression of ECC. There was a correlation between the expression of EMMPRIN and ADAM17 in ECC.Part 2 Influences of EMMPRIN and ADAM17 overexpression on prognosis in patients with extrahepatic cholangiocarcinoma after resectionObjectiveTo investigate the relationship between EMMPRIN, ADAM17 overexpression and the prognosis of patients with ECC after resection.MethodsThe deadline for follow-up was April 2014. The date of tumor recurrence and death for 78 patients were recorded. The cumulative disease-free survival and overall survival curves among parameters were analyzed via the Kaplan-Meier method and compared by log-rank test. Parameters of statistically significant (P<0.05) by univariate analysis were selected for multivariate analysis, which was performed using the Cox proportional hazards model.ResultsOverall survival rates were 32.5% and 23.2% at 3 years and 5 years for 78 patients with ECC, respectively. Univariate analysis revealed that the highly expression of EMMPRIN and ADAM17 protein were markedly related to poor prognosis (EMMPRIN:3-year OS 18.4% VS 50.2%,5-year OS 18.4% VS 31.3%, P=0.017; ADAM17:3-year OS:18.1% VS 44.6%,5-year OS:18.1% VS 30.0%. P=0.006). Furthermore, the tumor location (Hilar, P=0.016), tumour size (Size≥3cm, P=0.002), differentiation(poor, P=0.012), T classification(T3+T4, P=0.028), lymph-node-metastasis(Positive, P=0.004), surgical margin(Rl+R2, P<0.001), perineural invasion(Positive, P=0.048) and vascular invasion(Positive, P=0.001) were associated with poor outcome. Multivariate COX analysis indicated that a number of parameters including EMMPRIN and ADAM17 overexpression were independent prognostic factors of overall postoperative survival for patients with ECC (EMMPRIN:HR 2.729,95% CI 1.234-6.035, P=0.013; ADAM17:HR 2.050,95% CI 1.037-4.051, P=0.039).In 49 patients with ECC after curative resection, the 3 years and 5 years disease-free survival rates were 37.5% and 30.0%, overall survival rates were 45.2% and 32.3%. Univariate analysis revealed that the highly expression of EMMPRIN was markedly related to overall survival for patients with ECC after curative resection, but not to disease-free survival (3-year OS:26.2% VS 71.4%, 5-year OS:26.2% VS 44.5%, P=0.036; 3-year DFS:29.3% VS 50.0%,5-year DFS: 29.3% VS 33.3%, P=0.108). The overexpression of ADAM17 protein was inversely associated with disease-free survival and overall survival for patients with ECC after curative resection (3-year DFS:25.4% VS 50.0%,5-year DFS:0% VS 50.0%; P=0.006.3-year OS:24.5% VS 63.6%,5-year OS:24.5% VS 42.8%; P=0.003). Besides, the tumour size (≥3cm, P=0.009) and lymph-node-metastasis (Positive, P=0.002) were related to disease-free survival, and the overall survival were associated with tumour size (≥3cm, P=0.013), differentiation (Poor, P=0.030) and lymph-node-metastasis (Positive, P=0.003). In multivariate analysis by using the COX proportional hazards model, the ADAM17 overexpression (DFS:HR 2.635, 95% CI 1.186-5.856, P=0.017; OS:HR 2.629,95% CI 1.071-6.452, P=0.035) and lymph node metastasis were independent prognostic factors of disease-free survival and overall survival for patients with ECC after curative resection.ConclusionsThe EMMPRIN and ADAM17 overexpression were significantly correlation with poor prognosis of patients with ECC after resection. The ADAM17 overexpression was independent prognostic factor of overall survival for patients with ECC after curative resection, and could be used as a molecular marker of tumour recurrence.