The In Situ Detection Of HBVcccDNA In FFPE Liver Tissues Of Patients With Hepatocellular Carcinoma

Objective: Primary hepatolcellular carcinoma（HCC）is one of the commonhighly malignant tumors, with second highest incidence for all cancers inChina. Epidemiology has confirmed that infection of HBV is the independentrisk of HCC. Covalently closed circular DNA (cccDNA) is an importantreplicative intermediate in the life period of HBV, which serves as the originaltemplate for viral and pregenomic messenger RNA (pgRNA),has been generallyconsidered as the basic reason of persistent viral infection and liver progressivepathological changes. Studies in vitro and animal researchs have shown thatthere is a closely relationship between the HBVcccDNA and hepatocellularcarcinoma. With the PCR development, quantitative detection techniques ofintrahepatic cccDNA have been matured. But there are less reports about in situdetection of cccDNA from liver tissues of patients with HCC domestic andoverseas. Therefore, we developed a novel in situ method to detect theintrahepatic cccDNA in tumor and adjacent non-tumor tissues in HCC patientsand to investigate the significance in HCC progression and provide theory forprevention and treatment of primary liver cell carcinoma.Methods: The pairedtumor and adjacent non-tumor tissues tumor tissues were obtained from20HCC patients hospitalized in Beijing302Hospital during July2011-July2012. Thesamples were fixed by10%formaldehyde, paraffin imbedded and serialsectioned at5μm. The liver tissue sections were firstly treated by plasmid-safeATP-dependent DNase (PSAD) so as to digest relaxed circular DNA（rcDNA）prior to RCA. Four pairs of primers were designed for mediating RCA for thefirst round amplification of HBV cccDNA specifically according to completegenome sequence B and C. After RCA, HBV cccDNA was further amplified bya pair of selective primers labeled digoxigenin that targets the gap regionbetween the two direct repeat regions (DR1and DR2) of the virus via in situPCR (IS-PCR) to Further amplificate cccDNA.HBV cccDNA was stained byImmunohistochemistry and examined under a microscope.FFPE liver biopsytissues from the patient with HCV, health adult and transgenic mice. In addition,FFPE liver biopsy tissues from the patient with HCC, which were amplified insitu omitting specific primer, labled primers, phi29polymerase and Taq DNApolymerase respectively, instead of ddH2O, were also examined for evaluationof the specificity of the assay as negative control.Results: HBVcccDNA wasexpressed in17out of20subjects，located in the nuclear compartment withclustering，staining purplish red or blue-violet. The positive rate of HBVcccDNA in HCC and their adjacent tissues were10%(2/20) and85%(17/20)respectively. The difference was signifciant (P<0.01). No positive signals weredetected in negative control.Conclusions: RCA combined with IS-PCR is aneffective and practicable method that could detect the presence of intrahepaticcccDNA sensitively and specifically. The levels of HBV replication in thetumor-adjacent tissues was prior to the tumor tissues.