Studies On The Expression And Significance Of CXCL12-CXCR4 In Extrahepatic Cholangiocarcinoma (EHCC)

Cholangiocarcinoma is a malignant tumor composed of cells resembling those of the biliary tract epithelium, first reported by Durand Fardel in 1840. Clinically, cholangiocarcinoma is quite few, accounts for only 3% of all gastrointestinal cancers. The overall incidence of cholangiocarcinoma is 1.2/100,000 individuals, with two-thirds of all cases occurring in patients more than 65 years old. And a nearly 10-fold increase in the incidence of cholangiocarcinoma could be seen in subjects more than 80 years of age. Extrahepatic cholangiocarcinoma (EHCC) arises from the extrahepatic bile ducts(excluding gallbladder), comprises 90%-94% of all cholangiocarcinoma. The incidence of cholangiocarcinma is increasing by 5% every year in China. Up to now, surgery is the most effective therapeutic method for EHCC. Three-year survival rate is only 35% to 50% even after radical resection. Lack of effective comprehensive therapeutic methods is still the challenge for biomedical workers.A number of molecules involved in the metastasis processes of cholangiocarcinoma cells have been studied. Among them, chemokines has caused the concern of oncological field. Chemokines is a superfamily of small proteins and divided into four subfamilies: CC, CXC, C, and CX3C based on the position of their NH2-terminal cysteine residues. They can bind to G protein-coupled receptors on target cells, and may control tumor dissemination and promote tumor growth by directly promoting cell proliferation or neovascularization. CXC chemokine ligand-12 (CXCL12, or stomal cell-derived factor-1, SDF-1) and its specific receptor CXCR4 have gained considerable interest because of their roles in carcinogenesis, invasion, metastasis and recurrence as well as proliferation of malignant cells. However, there have been no reports on the putative roles of CXCR4-CXCL12 interraction on carcinogenesis and invasion of cholangiocarcinoma.To further explore the putative role and mechanisms of CXCR4-CXCL12 on the development and progression of EHCC, the following works were carried out in this study: 1. Surgical procedure and prognostic factors of extrahepatic cholangiocarcinoma were retrosepectively reviewed. 2. The expression of CXCR4-CXCL12 system in EHCC and their clinicopathological significance were studied with IHC, RT-PCR and Western bloting. 3. The impacts of CXCR4-CXCL12 on invasion, lymphanigogenesis and metastasis of EHCC was studied by means of IHC and RT-PCR. 4. Effects of accumulation of FOXP3+ Treg on immunotoleance and prognosis in EHCC was studied.The study includes the following four parts:1 Surgical procedure and prognostic analysis of extrahepatic cholangiocarcinoma: a report of 161 casesObjective: To explore the impact of clinical features and surgical procedure on survival of extrahepatic cholangiocarcinoma (EHCC) and the prognostic factors in patients with EHCC after curative resection. Methods: From 1995 to 2006, 161 EHCC patients underwent operations in the Second Hospital, Hebei Medical University were included in this study. The clinical features, diagnosis, surgical procedure and follow-up data were retrospectively analyzed. Factors influencing postoperative survival were analyzed using Cox multiple stepwise regression model.Results: Of the 161 patients, 110 underwent radical resection and 32 underwent palliative resection, 19 underwent drainage or laparotomy. The overall 1,2-,3-and 5-year survival rates were 74.9%, 45.3%, 36.5% and 11.1% respectively. Cox analysis results showed hepatic infiltration,portal vein and/or hepatic artery invasion and lymph node metastasis were independent prognostic factors after radical resection (P<0.05). The Radical resection may help to improve the survival of EHCC. Partial hepatectomy or/and pancreatoduodenectomy in combination with Skeletonization may be of help in increasing radical resection rate and improvement of postoperative survival. 2 The clinicopathological and prognostic significance of CXCL12 and CXCR4 expression in EHCCObjective: CXCR4-CXCL12 expression in EHCC has seldomly been reported. This study was to investigate the correlation of CXCR4-CXCL12 expression to clinicopathological factors and prognosis in EHCC.Methods: The CXCR4 and CXCL12 expression in EHCC, benign neoplasm and non-tumor bile duct epithelium was studied with Immunohistochemical staining , RT-PCR and Western blot.Results:The positive expression rate of CXCL12 in 82 cases of EHCC,7 cases of benign bile duct neoplasms, 12 cases of non-tumor bile duct epithelium was 92.7%,57.1% and 16.7 respectively. The expression of CXCL12 in EHCC and benign bile duct neoplasms was significantly higher than that in non-tumor bile duct epithelium (P<0.01). The positive rate of CXCR4 in EHCC ( 75.6%) was significantly higher than that in benign bile duct neoplasms and non-tumor bile duct epithelium( 28.6% and 0, P<0.01). The positive expression rate of CXCL12 was slightly higher in the stageⅡ~Ⅳcases than that in stageⅠcases (42.7% vs 18.3%. 26.01%, P=0.068). The positive rate of CXCR4 was slightly higher in the stageⅡ~Ⅳcases than that in stageⅠcases (22.4% vs 7.3%) but no statistical significance was found. RT-PCR results showed that positive expression percentage of CXCL12 mRNA in stageⅡandⅢcases were higher than that in stageⅠcases(0.50±0.15 vs 0.24±0.02, P<0.05) and the same results were found between the cases with lymph node metastasis and without groups (0.32±0.4 vs 0.09±0.02, P<0.01). Positive expression percentage of CXCR4 mRNA in stageⅡandⅢcases were higher than that in stageⅠcases(0.43±0.07 vs 0.25±0.08, P<0.05). The expression of CXCR4 mRNA in the cases with lymph node metastasis was significantly higher than that without groups (0.44±0.07 vs 0.23±0.03, P<0.05). Western blot results showed there was no significant difference in CXCR4 expression among the cases with and without lymph node metastasis, with different stage and differentiation.CXCR4 expression was positively correlated with CXCL12 expression in stageⅡ~Ⅳ cases, but the correlation did not exist in stageⅠcases.3 The effect of CXCL12-CXCR4 expression on metastasis and lymphangiogenesis in EHCCObjective: To investigate the expression of VEGF-C and matrix metalloproteinase (MMP-9), and correlation of their expression to clinicopathological viariables and pognosis in EHCC and to analyze the relationship between its expression and that of CXCL12.Methods: Immunohistochemistry, RT-PCR and Western blot were used to study the expression of VEGF-C and MMP-9 in EHCC, benign neoplasm and non-tumor bile duct epithelium.Results: The positive expression rates of VEGF-C and MMP-9 in EHCC and benign bile duct neoplasms were significantly higher than that in non-tumor bile duct epithelium ( 87% and 57.1% vs 16.7%, P<0.05).The positive expression rate of MMP-9 in EHCC, benign bile duct neoplasms and non-tumor bile duct epithelium was 87%;71.4% and 16.7 respectively, the former two was significantly that that in non-tumor bile duct epithelium (P<0.01). There was significant correlation between VEGF-C expression and stage, lymph node metastasis, differentiation in EHCC. The positive expression rate of VEGF-C was higher in the stageⅡ~Ⅳcases than that in stageⅠcases (36.4% vs 11.7% , P<0.05), in cases with lymph node metastasis than that without (27.6% vs 21.1%, P<0.05) and in the poorly differentiated than that in the well-moderately differentiated cases(P<0.01). The positive rate of MMP-9 was significantly higher in the stageⅡ~Ⅳcases than that inⅠcases (42.9% vs 20.8%, P<0.05). RT-PCR results showed that positive expression percentage of VEGF-C mRNA in cases with lymph node metastasis were significantly higher than that without(0.24±0.03 vs 0.17±0.05, P<0.05). Positive expression percentage of MMP-9 mRNA in stageⅡ,Ⅲcases were higher than that in stageⅠcases(0.43±0.02,0.23±0.02, P<0.05), and the same results were found between that with lymph node metastasis and that without (0.91±0.1 vs 0.11±0.04, P<0.01). The expression of MMP-9 in the poorly differentiated group was higher than that in the well-moderately differentiated cases(0.20±0.03 vs 0.08±0.02, P<0.01). CXCL12 expression was positively correlated to expression of VEGF-C, MMP-9, both VEGF-C and MMP-9 was independent prognostic factors of EHCC.4 Relationship of CXCL12-CXCR4 expression and accumulation of FOXP3+ Treg within EHCCObjective: FOXP3+ Treg play a central role in self-tolerance and suppress effective antitumor immune responses.The aim of this study was to investigate the relationship between expression of Treg, COX-2 and the clinical significance, prognosis of EHCC, and explore the correlation between expression of COX-2, CXCL12, CXCR4 and Treg.Methods: The expression of FOXP3+ Treg and COX-2 in EHCC, benign neoplasm and non-tumor bile duct epithelium was studied with immunohistochemical staining.Results: The numbers of Treg in 94 EHCC patients was 3.37±0.39 per HPF (high-power fields) and 0.43±0.53 in benign neoplasm, while no Treg in non-tumor bile duct epithelium was found. The numbers of Treg was significantly increased in EHCC as compared with that in benign neoplasm and non-tumor bile duct epithelium (P < 0.01). The increased Treg was not significantly correlated to clinicopathologic factors. A better prognosis was observed in patients with a lower numbers of Treg, and this was independent of other survival factors (P < 0.05). Positive expression rate and strong positive expression rate of COX-2 in EHCC was 81.8% and 22.1%. The positive expression rate of COX-2 in benign neoplasm and non-tumor bile duct epithelium was 57.1% and 16.7% repectively, no strong positive cases were found. The expression of COX-2 was significantly increased in EHCC as compared with that in non-tumor bile duct epithelium (P < 0.01). The increased expression of COX-2 was not significantly correlated with clinicopathologic factors. There was no correlation between CXCL12, CXCR4 and Treg. There was significant correlation between COX-2 and Treg.Conclusions:1. Hepatic infiltration,portal vein and/or hepatic artery invasion and lymph node metastasis were independent prognostic factors for EHCC after radical resection.2. The Radical resection may help to improve the survival of EHCC. Partial hepatectomy or/and pancreatoduodenectomy in combination with skeletonization may be of help in the increase of radical resection rate and improvement of postoperative survival.3. There is an increasing tendency in CXCL12 and CXCR4 expression from non-tumor bile duct epithelium, benign bile duct neoplasms to EHCC, suggesting CXCL12 and CXCR4 were involved in the carcinogenesis of EHCC. The expression of CXCL12 and CXCR4 was closely related with clinical stage and lymph node metastasis in EHCC. Thus both of them play roles in the progression and metastasis of EHCC.4. CXCL12 expression was positively correlated to expression of VEGF-C and MMP-9 in EHCC. VEGF-C and MMP-9 expression increased from non-tumor bile duct epithelium, benign bile duct neoplasms to EHCC. The expression of VEGF-C and MMP-9 was positively correlated with clinical stage, limph node metastasis and differentiation of EHCC. VEGF-C and MMP-9 may be important factors in the development and progression of EHCC. Both VEGF-C and MMP-9 was independent prognostic factor of EHCC.5. There was no correlation between CXCL12, CXCR4 and Treg in EHCC, yet the COX-2 expression was correlated with and Treg.6. The numbers of Treg was significantly increased in EHCC as compared with that in benign neoplasm and non-tumor bile duct epithelium. The increased Treg was not significantly correlated with certain clinicopathologic factors. A better prognosis was observed in patients with a lower numbers of Treg, and this was independent of other survival factors in EHCC.7. The increased expression of COX-2 was not significantly correlated with clinicopathologic factors and prognosis in EHCC.