Primary Study On Perineural Invasion Of Human Extrahepatic Cholangiocarcinoma

Cholangiocarcinoma is an important malignancy of biliary system, because of itsdifficult diagnosis, fast deterioration and poor prognosis. A complete surgicalresection with histological negative resection margin is the only cure for the disease.Despite of advantage of diagnosis and treatment technology in recent years, the 5years survival rate is still less than 5%. The main reason is the tendency to recurrence.Invasion and metastasis are the biological characteristics of malignancy, and alsothe most important prognosis factors of tumor. Traditionally, malignancy has fourspreading ways: direct invasion, blood vessel metastasis, lymphatic metastasis andperitoneal dissemination. Beside above four ways of spreading, cholangiocarcinomahas another special way: perineural invasion. On one side, neural invasion, consideredas an important reason for recurrence, is difficult to diagnose and cure, because ofwidespread distribution in retroperitoneal nerve plexus and adherent to the largevessels. On the other side ,the only treatment of perineural invasion is extended softtissue dissection, which brings the patients severe surgical injury and, sometimes, anobstinate diarrhea caused by augment plexus dissection. This condition is extremelydisadvantageous to the elderly. So, the perineural invasion becomes an intractableproblem in surgical treatment of cholangiocarcinoma.Although the problem is concerned by clinical doctors, little was known about the perineural invasion. Recent advancement is extending resection. But, even with the "curative resection", the long-term survival is still discouraged. Thus, many researches think that how to find a breakthrough from the viewpoint of mechanism study. Present data showed that many factors participates the neural invasion, nerve growth factor may be one of them.Under the leading and direction of Professor Zou Shengquan, we have studied on the epidemiology, genetics and etiology of cholangiocarcinoma. The present study is based on the previous results, concentrating on the importance of perineural invasion in cholangiocarcinoma malignant progression, and the role of NGF in neural invasion, trying to refer an experimental basis for further study. Part 1: Correlation analysis on nerve growth factor and clinical pathologic characteristics of cholangiocarcinomaPaper 1: The clinical pathological analysis on perineural invasion of human bile duct carcinomaObjects: To investigate the clinical characteristic of perineural invasion of human bile duct carcinoma, and the correlation between perineural invasion and abdominal or back pain, pathological type, lymphatic metastasis, as well as TNM staging.Methods: Retrospectively analyzing on 92 cases of cholangiocarcinomas in our hospital and Hebei Forth Hospital from 1998 to 2003. Then Spearman's correlation test was used to analyze the relationship between perineural invasion and abdominal or back pain, lymphatic metastasis, pathological type as well as TNM stage.Results:1. Of 92 cases, perineural invasion were positive in 35 cases. Among the latter, 11 patients complained with abdominal or back pain. In 57 non-perineural invasion cases, 22 patients complained with abdominal or back pain. There is no significant correlations between perineural invasion and abdominal or back pain (r=-0.073, P=0.492). While, the pain tightly correlated with TNM staging (r=0.264, P=0.011).2. In high or medal differentiated cholangiocacinomas, perineural invasion was positive in 13/60 cases, while in poor differentiated chlangiocarcinmas, 22/32 of cases had perineural invasion. Prineural invasion was tightly correlated with the poor differentiation.3. In 35 positive perineural invasion cases, 27/35 of cases had lymphatic metastasis. In 57 non-perineural invasion cases, 28/57 of cases had lymphatic metastasis. Perineural invasion was highly correlated with the lymphatic metastasis of cholangiocarcinoma.4. Most of the cases in our group were in I B to IIB stage, which consisted 88% of the total cases. 88.9% of cases in IIB stage had perineural invasion. The perineural invasion correlates with the TNM stage of cholangiocarcinoma.Conclusions:1. The perineural invasion correlates with the poor differentiation, lymphatic metastasis and TNM stage of human cholangiocarcinoma. So perineural invasion is an important pathological characteristic of cholangiocarcinoma and may be helpful to estimate the prognosis.2. Perineural invasion is an early event in cholangiocarcinoma development, which indicates that cholangiocarcinoma is a high invasive malignancy.3. The generation of pain is complicated. Perineural invasion is only one of causes. Thus, the complaining of abdominal or back pain is not helpful for diagnosis of perineural invasion before operation. Paper 2: The relationship between nerve growth factor and perineural invasion of human bile duct carcinomaObjects: To investigate the role of nerve growth factor and its receptor TrkA in the perineural invasion, lymph node metastasis, pathological type and TNM stage of human cholangiocarcinoma.Methods: Expression of NGF and TrkA were detected by SP immunohistochemical technique in 92 cases of cholangiocarcinoma. Then Spearman's correlation test was used to analyze the relationship between NGF positive expression and prineural invasion, lymph node metastasis, pathological type as well as TNM stage. Results:1. Of 92 cases, NGF expressed in 65 cases. Both NGF and TrkA expressed in 50 cases, while 22 cases both negative. The expression of two factors correlated tightly (r=0.542, P<0.01). Not only expressed in bile duct cancer cells, TrkA also expressed in perineurium.2. In 35 positive perineural invasion cases, 91.4% of cases expressed NGF. In 57 negative perineural invasion cases, 42.1% of cases expressed NGF. Positive expression of NGF correlates with the perineural invasion of cholangiocarcinoma.3. In NGF positive cases, 45/65 of cases had lymph node metastasis, while in NGF negative cases, only 10/27 of cases had lymph node metastasis. Positive expression of NGF correlates with the lymph node metastasis of cholangiocarcinoma.4. In high or medal differentiated cholangiocacinomas, 37/60 of cases expressed NGF, while in poor differentiated chlangiocarcinmas, 28/32 of cases expressed NGF. As the differentiation level descending, the expression of NGF elevated.5. Most of the cases in this group were in I B to IIB stage, which consisted 88% of the total cases. As the TNM stage rising, the expression rate of NGF going up. 100% of cases in IIB stage expressed NGF. The positive expression of NGF correlates with the TNM stage of cholangiocarcinoma. Conclusions:1. The positive expression of NGF correlates with the perineural invasion and TNM stage of human cholangiocarcinoma. So, detection of NGF in cholangiocarcinma may be helpful to estimate the prognosis of the disease.2. It suggested that NGF may enhance the proliferation of cholangiocarcinoma cells by autocnne loop, and stimulate the perineural invasion by paracrine loop. Part 2: Culture and identification of cholangiocarcinoma cells highly expressing β-NGFPaper 3: Construction and identification of full-length human β-NGF expression vectorObject: To construct and identify full-length human β-NGF expression vector and make an experimental basis for transfection.Method: Total RNA was obtained from normal human brain. PCR primers were designed according to the coding sequence of β-NGF gene and structure of pcDNA3.0, and the Hind III and Xba I digestion site were also included. RT-PCR was used to amplify the β-NGF cDNA. After purification of PCR fragment, reconstruction of target gene and plasmid pcDNA3.0 was performed. Then the recombinant DNA was identified by PCR confirmation and Hind III and Xba I double enzyme digestion.Result:1. β-NGF cDNA was obtained by RT-PCR, the length of target fragment was 749bp.2. The PCR identification of recombinant pcDNA3.0-NGF showed that the recombinant DNA contained β-NGF gene.3. The identification by restrictive double enzyme digestion showed that the target gene has been inserted into pcDNA3.0 carrier.Conclusion: The full-length human β-NGF expression vector pcDNA3.0-NGF was constructed successfully. The recombinant DNA could be used in followed experiments. Which refer an experimental basis for the study on the role of NGF in chilangiocarcinoma. Paper 4: The pcDNA3.0-NGF Transfection of β-NGF gene with human cholangiocarcinoma cell QBC939 and its identificationObject: To identify the function of pcDNA3.0-NGF plasmid, and culture human cholangiocarcinoma cell highly expressing β-NGF stably.Method: Using DOSPER Liposomal Transfection Reagent (Roche Comp) to transfect human cholangiocarcinoma cells QBC₉₃₉ with 15μg/ml of pcDNA3.0-NGF plasmid. After selection by 800ug/ml G418, positive cell clone was obtained. The result was identified by RT-PCR, immunochemistry and Western blot assays, respectively.Result:1. After transfection of pcDNA3.0-NGF plasmid, the expression of β-NGF mRNA in chlangiocarcinoma cell QBC939 was enhanced, which was much higher than control group and mock-transfection group. The OD value detection indicated that the difference between transfection group and control group was significant (t=16.525, P<0.001)..2. Immunochemistry detection indicated that the positive expression of β-NGF represented filemot particles in cholangiocarcinoma cells. Before transfection, the expression of β-NGF was week, while after transfection, the expression of β-NGF markedly increased.3. Western blot detection revealed that the expression of β-NGF was enhanced and the expression of β-Actin did not change, which indicated that pcDNA3.0-NGF could up-regulate the expression of β-NGF in cholangiocarcinoma cells.Conclusion:1. The transfetion of pcDNA3.0-NGF really could enhance the expression of β-NGF, which indicated the success of construction of the plasmid.2. Post-transfected cholangiocarcinoma cells could stably express β-NGF, which refer an experimental basis for further study. Part 3: The effect of nerve growth factor on the in vitro proliferation and invasion of human cholangiocarcinoma cellsPaper 5: The effect of nerve growth factor on the in vitro proliferation and invasion of human cholangiocarcinoma cellsObject: To investigate whether cancer cell-derived nerve growth factor can regulate the proliferation and invasion in vitro of human cholangiocarcinoma cells.Method: Using DOSPER Liposomal Transfection Reagent (Roche Comp) to transfect pcDNA3.0-NGF plasmid with human cholangiocarcinoma cells QBC939. The plasmid density of transfection was 16μg/ml, 32μg/ml, 64μg/ml and 128μg/ml, respectively. The expression of β-NGF was detected by RT-PCR and Western blot assays at mRNA and protein level, respectively. Subsequently, in vitro proliferation of transfected cells was analyzed by MTT assays. The transwell polycarbonate membrane was coated with Metrigel (BD Corp). Then, in vitro invasion of transfected cells was detected by Transwell permeable assays. Non-transfected cells and mock-transfected group acted as control groups in all the detections. Result:1. In transfected cholangiocarcinoma cells, the expression of β-NGF were markedly enhanced both at mRNA and protein levels. Comparing with non-transfected cells and mock-transfected group, the differences were significant. The expression of mRNA and protein of β-NGF augmented as the plasmid concentration increasing.2. MTT and Transwell assays showed transfection of β-NGF resulted in accelerated growth and increased invasion potential of QBC939 cells in vitro. Comparing with control group, the differences were also significant. (P<0.01)Conclusion: Cancer cell-derived β-NGF may enhance the in vitro proliferation andinvasion of cholangiocarcinoma cells. Part 4: The effect of nerve growth factor on the tumorigenicity and metastasis of human cholangiocarcinoma cellsPaper 6: The effect of cancer cell derived nerve growth factor on the tumorigenicity of human cholangiocarcinoma cellsObject: To investigate the role of cancer cell derived nerve growth factor on tumorigenicity of human cholangiocarcinoma cells.Method: Subcutaneously incubating the tansfected cholangiocarcinoma cells of different plasmid concentration into buttocks of nude mice, to build up human choalngiocarcinoma nude mice model. Recording the tumor size and survival of nude mice weekly and analyze the role of NGF on tumorigenisity of cholangiocarcinoma cells and nude mice survival. Non-transfected cells and mock-transfected group acted as control groups in all the detections.Result:1. No significant difference between frank-control group and mock-transfected group.2. Comparing with the frank-control group, the tumorigenisity of transfected cells was markedly enhanced, which showed dose-dependent effect with the plasmid concentration and β-NGF expression. 3 weeks after incubation, the tumor size differences between 64μg/ml (P<0.05) group, 128μg/ml (P<0.01) group and frank-control group were significant. 4 weeks after incubation, the significant difference also showed between 32μg/ml and frank-control group. (P<0.01)3. 8 weeks after incubation, liver metastasis in 2 nude mice of 128μg/ml group. Conclusion:1. NGF can enhance the tumorigenicity of cholangiocarcinoma cells.2. Higher expression of NGF results in faster metastasis and poor prognosis of cholangiocarcinoma. In SummaryBy clinical specimen detection, pathological data analysis and in vitro and in vivo experiments, the conclusion can be drawn as follows:1. Perineural invasion can be seen in 38% of extrahepatic cholangiocarcinomas, and correlates with the lymphatic metastasis, poor differentiation and TNM staging.2. NGF is highly expressed in cholangiocarcinoma tissues, correlating with the perineural invasion and lymphatic metastasis, as well as poor differentiation, which indicates that NGF participate in the chlangiocarcinoma malignant progression. Because NGF's high affinity receptor TrkA is expressed in cholangiocarcinoma cells, it suggests that cholangiocarcinoma cells proliferation is enhanced by autocrine loop.3. After transfected with full-length β-NGF gene, the in vitro proliferation and invasion of QBC₉₃₉ were markedly enhanced. The enhancement of proliferation and invasion was paralleled with the increased expression of β-NGF.4. In vivo experiments demonstrate that cancer cell derived β-NGF can markedly promote the tumorigenicity and metastasis of QBC₉₃₉ in nude mice.5. At present, there is no effective clinical test for evaluating the prognosis of cholangiocarcinoma. The detection of NGF may be helpful to estimation.The present study has following NEW IDEAS:1. By analyzing on clinical information and pathological data of 92 cases, we definitely explained the relationship between NGF and pathological characteristics of human extrahepatic cholangiocarcinoma: expression of NGF tightly correlates with the perineural invasion, poor differentiation, lymphatic metastasis, and TNM stage.2. Perineural invasion occurs in the early stage of cholangiocarcinoma development, which is an important biological characteristic of the cancer.3. NGF can enhance the proliferation, invasion and tumorigenicity of cholangiocarcinoma, finally results in promoted metastasis.4. Referring a potential target for further treatment research.