Clonal expansion of normal-appearing human hepatocytes during chronic hepatitis B virus infection

Abstract

Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A. 102:1139-1144, 2005; W. S. Mason et al., J. Virol. 83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.

FIG. 1.

Inverse PCR assay for integrated HBV DNA. An example of an integration junction between host DNA and the right end of DSL DNA, at nt 1831, is shown at the top. To detect integration junctions near this location in viral DNA, total DNA from infected livers was cleaved with NcoI, which cuts HBV DNA at nt 1374 and host DNA at locations that differ according to the integration site. The cut DNA was circularized by incubation with T4 DNA ligase and was cut with the BsiHKAI restriction endonuclease to produce linear molecules in which virus-cell junctions are flanked by viral sequences. These fragments were serially diluted into 96-well PCR trays and were amplified by nested PCR using the virus-specific primers described in Materials and Methods. PCR products were cut from the gels and sequenced to locate the integration site.

FIG. 2.

H&E-stained liver tissues from HCC patients. Formalin-fixed, paraffin-embedded liver tissues (nontumorous) from patients P1, P2, P4, and P5 were stained with H&E and Masson's trichrome stain (MT) in order to assess the degree of deposition of collagen fiber, which is stained blue. Separate blocks of tissue were frozen for inverse PCR, as shown in Fig. 5. Magnifications, ×200 (H&E) and ×40 (Masson's trichrome stain). P3 liver tissue (not shown) was similar in appearance to P2 liver tissue.

FIG. 3.

Large hepatocyte clones in liver samples from patients 1 to 5. Four frozen liver fragments from each patient were extracted and analyzed. A summary of the large clones found in these samples is shown. Integration sites corresponding to clones of 1,000 or more hepatocytes are summarized in Table 3 and, in greater detail, in Table S5 in the supplemental material.

FIG. 4.

Evidence that HBV DSL DNA is the precursor to integrated HBV DNA. The junction sites for each distinct integration event are plotted. As can be seen by comparison to a map of the viral genome, numbering from the unique EcoRI site, all integration junctions but two occurred upstream of the 5′ end of the minus strand, which defines the right end of DSL DNA. None were detected immediately downstream, although virus-cell junctions were common in this region when integration junctions near the left end of HBV DSL DNA were analyzed (21).

FIG. 5.

Inverse PCR for integrated HBV DNA in fixed liver tissue from an HBV carrier. Fixation and embedding of frozen liver tissue for sectioning was carried out after the removal of tissue fragments for the experiments described in the legend to Fig. 3 and in Tables 1 and 2; thus, these integration events were distinct from those summarized there and in Table S5 in the supplemental material. (A) Representative area of an ethanol-fixed, H&E-stained 5-μm-thick liver tissue section from patient 3. (B) Inverse PCR of DNA extracted from an immediately adjacent 5-μm-thick section containing ∼20,000 hepatocytes. Inverse PCR for integrated HBV was carried out on one-eighth of the total extracted DNA. Following the inversion reaction (Fig. 1), the DNA was distributed to a 96-well tray for nested PCR. Results of gel electrophoresis of the PCR products are shown. The bands were extracted and sequenced. The results are summarized in Table 4 and are presented in detail in Table S6 in the supplemental material.