| Summary: | 博士 === 高雄醫學大學 === 醫學研究所 === 90 === Intrahepatic stones (IHS) and intrahepatic cholangiocellular carcinoma (CCC) are rare but endemic diseases in Taiwan and South Asia. IHS has been recognized as one risk factor to the occurrence of CCC. Many studies have reported there are some linkages between intrahepatic stones and cholangiocellular carcinoma. Data from the epidemic studies, about 5-7% of patients with intrahepatic stones developed cholangiocellular carcinomas. The incidence of cholangiocellular carcinoma in normal population is about 1 per hundred thousand. The microscopic examination in specimens of cholangiocellular carcinoma revealed many dysplastic lesions near the cancer area. The incidence of cholangiocellular carcinoma has been positively related to the duration and severity of the repeated cholangitis. The hypothesis is based on the above observation. It is postulated that intrahepatic stones could induce chronic proliferative cholangitis, which may lead to hyperplasia and dysplasia. On the rare occasion, the bile duct mucosal epithelium may further transformed into cholangiocellular carcinoma after prolonged exposure to the chronic inflammation. However, the exact mechanism of carcinogenesis at molecular biology level is still unclear.
In 1990, Fearon has proposed a genetic model for colorectal tumorigenesis.
Comparison of the frequency of genetic molecular alternations in adenoma and carcinoma has provided evidence some genetic alternations (APC and K-ras) are more likely to occur in the adenoma stage and the others (p53 and nm23) are more commonly observed in the cancer stage. Change to p53 gene may be much more closely related to malignant behavior than k-ras activation. Mutation in p53 gene has been reported in various types of cancer including colon cancer, skin cancer and breast cancer.
Since p53 gene was once selected as the molecular of year by TIME magazine, we would like to understand whether p53 mutation play an important role in the carcinogenesis of cholangiocellular carcinoma. By immunohistochemistry method and PCR-SSCP/autosequence method, we try to detect the frequency of p53 mutation in the hyperplasia, dysplasia and carcinoma stage.
Twenty two cases of benign intrahepatic stones were collected as normal control. Thirteen cases of intrahepatic cholangiocellular carcinoma and 7 cases of extrahepatic bile duct cancer from surgical resection specimen were collected as experimental groups. Immunohistochemistry staining with monoclonal antibody (Ab2; Santa Cruz, FL-393:sc-6243) against mutant p53 was performed. Twenty-one of the twenty-two cases in the control group could not demonstrate p53 staining (negative). One case with mucin producing intrahepatic stones demonstrated positive staining for p53. On the other hand, there were five cases in the cancer group demonstrated positive p53 staining. Comparison of the frequency of p53 overexpression in benign and malignant biliary diseases indicated that there was a tendency of preference of p53 overexpression in the cancer stage. When the cases were analyzed with the UICC stages, there was also a higher rate of p53 overexpression in the advanced cancer stages (III-IV) than in the early stages (I-II). However, it did not reach statistic significance (40% vs 10%, p>0.05). Later on, we collected another 33 cases of cholangiocellular carcinoma and repeated the study again, 15/33 (46%) showed p53 overexpression.
Point mutation at codon 249 of p53 was considered to be specific to aflatoxin B1-associated human hepatocellular carcinoma. Kiba has reported the incidence and pattern of mutations of the p53 gene among the same cancer type (cholangiocellular carcinoma) with different etiological backgrounds. Cholangiocellular carcinoma in Japan is related to intrahepatic stones, but in Thailand, it is related to the liver fluke Opisthorchis viverrii. In all cases but one, the p53 gene mutations were identified within highly conserved regions. We would like to know what is the type of p53 gene mutation in cholangiocellular carcinoma with intrahepatic stones in Taiwan.
Bile duct mucosal epithelium cells were obtained from five normal patients, 25 cases with benign intrahepatic stones, 10 cases with dysplastic area and 10 intrahepatic cholangiocellular carcinoma. Dysplastic area was obtained under micro-dissection technique. PCR-SSCP method was applied in searching any mutations located within exon 4 to exon 8 of p53 gene. The result of this study showed there were no detectable mutation in normal, intrahepatic or dysplastic groups. Only two of 10 cases with cholangiocellular carcinoma showed abnormal shifts of the bands in exon 4. After direct sequencing, codon 67~69 and codon 72 had mutations. Proline has been changed to become alanine after codon 72 mutation. This is a proline rich area in normal p53 gene, which may provide the binding site for –SH3 domain of tyrosine kinase. It is speculated that the mutation in codon 72 may lead to p53 protein malfunction. Because the number of cases was small, we repeated the study. Another 16 cases of CCC were collected for PCR-SSCP and DGGE (denaturing gradient gel electrophoresis) study. The result showed only 1 of 16 had aberrant band shift in exon 7. From these studies, we concluded that p53 genetic mutation was not common in CCC.
The role of centrosome abnormality in cholangiocellular carcinoma has been explored. To observe and compare the intracellular γ-tubulin staining in different tissues, confocal laser microscope has been used.
Centrosome abnormality has been identified if the numbers of centrosome is greater than 3 or if the size of it is bigger than 2 μm. 16 intrahepatic stones, 19 cholangiocellular carcinomas, 13 gallbladder cancers, 6 extrahepatic bile duct cancers were collected.
The result of this study is 1/16 (6.3%) in intrahepatic stones, 11/19 in CCC, 9/13 in BDC, 4/8 in GC demomstrated abnormal centrosome hyperamplification.
When compared to their benign counteraparts, there is a statistical significance elevation in the cancer group. When combined these three types of cancer and compared it with the clinical stages, then there is a significant higher frequency of centrosome abnormality in the late stages (III-IV, UICC)than in the early stages (I~II, UICC).
We have established a model of proliferative cholangitis in hamsters, which can provide us as research tools in studying human’s intrahepatic stones and chronic cholangitis. Taiwan is an epidemic country in IHS, but there is no such research model available so far. That is why it is important and urgent to establish such research model.
We have tried, by using transduodenal puncture, to insert a 5-0 nylon thread into the CBD. Push this thread all the way to the hilar area. 6 months after the operation, we harvest their livers, CBDs and GBs. The common bile ducts became dilated and the wall of the CBD became thick. There were some new-formed sands in the bile duct. There were some papillary foldings in the cross section of the bile duct.
There were some benefits in this model: it is easy, fast, and the mortality rate is low. The proliferative cholangitis induced in this model is similar to the pathologic pictures seen in the human’s intrahepatic stones and chronic cholangitis.
Recently the Japanese scholars have applied similar model to cancer researches. By putting on low dose chemical carcinogen on top of this model, they could induce cholangiocellular carcinoma. The most common used chemical carcinogen is N-Nitrosobis (2-oxopropyl)amine (BOP). From the literature, combined cholangitis model and BOP, cancers could be observed 16-52 weeks after the operation.
The incidence of choangiocelluar carcinoma in normal population is approximately 1 per 100,000. In a recent paper, 55 out of 1003 patients (6%), who received choledochoenteric anastomosis, developed bile duct cancers after a long period of follow-up. Choledochoenteric anastomosis could induce chronic inflammation and proliferative cholangitis. This type of disease has been occasionally reported in the literature, and we happened to come across this kind of patient several years ago. We reported this case, and this may be first time reported from Taiwan. We might improve the survival of this cancer by early detection, if we closely monitor these patients in advance.
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