Detection of hepatitis B virus covalently closed circular DNA in paraffin-embedded and cryo-preserved liver biopsies of chronic hepatitis B patients

Abstract

Background: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may become an important predictor for treatment outcome or long-term follow-up.

Aim: To detect cccDNA in formalin-fixed, paraffin embedded (FFPE) and to compare with cryo-preserved liver tissue.

Methods: Biopsies of 56 chronic hepatitis B patients were collected. Cryo-preserved and FFPE liver biopsies were available from 37 out of 56 patients. Paraffin was extracted with 1 ml xylene, followed by 100% alcohol and acetone. For the detection of cccDNA, selective primers were used. For quantification of hepatocytes a commercial Taqman beta-actin control kit was used.

Results: The cccDNA was detected in 80% of FFPE and in 100% of cryo-preserved liver specimens. Recovery of hepatocytes and cccDNA was approximately a 100-fold lower in FFPE liver tissue, but intrahepatic cccDNA levels were comparable. In FFPE and cryo-preserved liver tissue, intrahepatic cccDNA levels correlated strongly with HBV DNA, hepatitis B e antigen (HbeAg), and plasma cccDNA levels. HbeAg positive chronic hepatitis B patients had significantly higher intrahepatic cccDNA levels compared with HBeAg negative patients (P<0.05). In HBeAg positive patients, no difference in intrahepatic cccDNA levels were seen between patients with active (histological activity index score>3; HBV DNA>20 000 IU/ml) and inactive hepatitis (histological activity index score</=3). In HBeAg negative chronic hepatitis B patients, intrahepatic cccDNA levels were significantly higher in patients with active hepatitis (P=0.004 and 0.001).

Conclusion: Recovery of hepatocytes and cccDNA in FFPE tissue was lower, but intrahepatic cccDNA in FFPE biopsies were comparable with cryo-preserved liver tissue. Therefore, FFPE liver tissue is an attractive alternative for cccDNA analysis when cryo-preserved tissue is not available.