AIM: To explore the etiology and pathogenesis of human primary intrahepatic cholangiocarcinoma, the expression of HBV genes and HBV-antigens was detected in the cancerous tissue and its surrounding hepatic tissues.

METHODS: HBV-antigens were detected by immunohistochemical technique and HBV genes were examined with in situ hybridization.

RESULTS: In 20 cases of cholangiocarcinoma, the positive detection rate of HBxAg, pre-S1, pre-S2, HBsAg and HBcAg was 75%, 40%, 40%, 10% and 0%, respectively, and in the surrounding hepatic tissues of 19 cases the positive rates were 84.2%, 47.9%, 47.9%, 31.6% and 31.6%. Among 40 cases of cholangiocarcinoma, the positive rate of HBVDNA, x-gene, pre-s gene, s gene and s gene fell on 77.5%, 70.0%, 47.5%, 40% and 42.5%, respectively, and of the surrounding hepatic tissues in 33 cases, 87.9%, 84.8%, 63.6%, 69.7% and 66.7%.

CONCLUSION: The development of human primary intrahepatic cholangiocarcinoma bears a close relationship with chronic persistent HBV infection. Particularly, the x gene of HBV and its protein (HBxAg) might play an important role in pathogenesis of hepatic carcinoma.

A large number of studies indicate a close relationship between human primary hepatocellular carcinoma and hepatitis B virus (HBV) infection, which is considered generally as an important factor in the development of hepatic carcinoma[1,2]. In human primary hepatic carcinoma, hepatocellular carcinoma is more frequently encountered, while intrahepatic cholangiocarcinoma (ChC), including hepatocholangiocarcinoma (HChC), is relatively less, being 8%-10%[3]. For a long time, the etiology and pathogenesis of intrahepatic cholangiocarcinoma have been unclear. A few reports considered it to be related to infestation with clonorchiasis sinensis[4,5], but never involved with HBV infection. We used immunohistochemical technique and in situ hybridization methods to detect HBV genes and their-related antigens in the tissues of intrahepatic cholangiocarcinoma and its surrounding hepatic tissues for the purpose of exploring the etiology and pathogenesis of intrahepatic cholangiocarcinoma.

All the 40 cases of surgically resected specimens of intrahepatic cholangiocarcinoma and hepato cholangiocarcinoma were from the Pathologic Laboratory of the Affiliated Hospital of Fourth Military Medical University. The specimens were fixed in 10% formalin, paraffin embedded and serially sectioned 5 μm in thickness. The HBV antigens (HBxAg, HBsAg, HBcAg, pre-S1 and pre S2) were detected by immunohistochemical techniques (ABC and PAP). In situ hybridization method was adopted for detection of HBV genes (HBV-DNA, x gene, s gene, pre s gene and c gene). Of the main reagents of immunohistochemistry, the antibodies of anti-HBx, S1 and S2 were gained from Fox Chase Cancer Center, Philadelphia, USA, and ABC kit and PAP kit (including anti HBs and anti-HBc) were purchased from BioGenes Lab, USA. The ABC and PAP methods were in accordance with the instructions of the kits. For in situ hybridization, Bio-11-dUTP was purchased from the Institute of Pharmacology of Beijing Medical University. The gene primers of s, pre s and c of HBV were acquired from Fox Chase Cancer Center, Philadelphia, USA. The colony of E. Coli- C600-HBV (containing PCP 10) was supplied by the Research Institute of Liver Diseases of Beijing Medical University. The colony of PMM15-HBV was furnished by the Basic Medical Institute of Military Medical Academy, Beijing. E. Coli, DNA polymerase I, restriction endonuclease EcoRI, Bam HI and Bgl II were purchased from Hua Mei Biotechnology, Co. As for the preparation of probes, the plasmids of PMM15-HBV and PCP-HBV were extracted through lysis by alkali. PCP 10-HBV was cleaved by EcoRI and various sized gene fragments of 3.2 kb were harvested by freeze-squeezing method, and then, cleaved with Bam HI and Bgl II. 584 X gene fragments of nucleotide were obtained by PMM15-HBV. The probes of HBV-DNA and X gene were labeled through nicking translation, and the fragments of C gene, S gene and pre S gene of HBV were labeled with PCR. Control groups were set up including positivity, negativity, blank and replacement.

In human primary hepatic carcinoma, intrahepatic cholangiocarcinoma is relatively less encountered, and few reports were involved in the study of etiology and pathogenesis. This disease was considered to be related to colloidal thoriun dioxide (ChO2)[6]. Hou reported that the development of intrahepatic cholangiocarcinoma bore relationship with Clonorchis sinensis infection by mechanic stimulation of worm-moving and chemical irritation of the bile and tyrosinase from the worm body[6]. In our study, these patients had[5] neither taken colloidal thorium dioxide, nor infected with Clonorchis sinensis. Moreover, the cancerous and surrounding hepatic tissues showed no pathological pattern of parasitic infection. In the cancerous and surrounding hepatic tissues in this series, the detection rate of HBV antigens was obviously high, being 85%, especially the HBxAg, being above 80%. The detection rate of HBV-DNA was 82.5%. Among the genes of X, pre S, C and S, X gene had the highest detection rate of 77.5%. By pathological examination of the surrounding hepatic tissues, CAH was 66.7%, CPH 21.2% and CIR 12.1%. 75.0% of the patients with hepatic carcinoma had a history of chronic hepatitis. This result indicates that the development of intrahepatic cholangiocarcinoma is closely related to HBV infection. The persistent HBV infection plays an important role not only in hepatocellular carcinoma, but also in intrahepatic cholangiocarcinoma.

Up to now, the mechanism of development of intrahepatic cholangiocarcinoma by HBV is still unclear. The HBV and reverse transvirus are homologous in the origin of evolution[7], and it is generally considered that the X gene of HBV and its protein product may play an important role in malignant transformation. It has been discovered that the sera of patients with hepatic carcinoma contained higher anti-HBx with a detection rate of 85.7%[8]. The X gene region might be the important region or HBV integrated into the chromosomes of primary hepatic carcinoma, and the integration of DNA of the host cells with HBV-DNA was completed through the peculiar recombination in the X gene region[9]. The translation product of X gene has a transactive expression of genes[10]. The experiment of transgenic mice manifested that the HBx might directly induce primary hepatic carcinoma[11]. These facts state clearly that HBV may possess probably the direct carcinogenic effect, and HBx gene is the importain factor. Thus, intrahepatic cholangiocarcinoma may be closely related to HBV infection.

About 80% hepatocellular carcinoma cases are accompanied by liver cirrhosis. It can be assumed that HBV infection may induce chronic active hepatitis, further progress to live cirrhosis and finally result in malignant transformation. Fewer intrahepatic cholangio-carcinoma cases are complicated with liver cirrhosis[12]. In our study, only 12% of intrahepatic cholangiocarcinoma were associated with liver cirrhosis, while 87.8% with chronic active hepatitis and chronic persistent hepatitis. This indicates that intrahepatic cholangiocarcinoma is not chiefly developed through liver cirrhosis but mainly through persistent hepatitis. The precise relationship of HBV and development of intrahepatic cholangiocarcinoma remains to be further studied.