Journal of Viral Hepatitis, 2015

doi:10.1111/jvh.12380

Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection T. Tu,1,2,3 W. S. Mason,4 A. D. Clouston,5 N. A. Shackel,2,3,6 G. W. McCaughan,2,3,6 M. M. Yeh,7 E. R. Schiff,8 A. R. Ruszkiewicz,9 J. W. Chen,10 H. A. J. Harley,11 U. H. Stroeher1 and A. R. Jilbert1 1Department of Molecular and Cellular Biology, School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia; 2Centenary Institute, Sydney, NSW, Australia; 3Sydney Medical School, University of Sydney, Sydney, NSW, Australia; 4

Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA; 5Centre for Liver Disease Research, School of Medicine, Faculty of

Health Sciences, University of Queensland, Brisbane, QLD, Australia; 6A. W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Sydney, NSW, Australia; 7Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA; 8Schiff Liver Institute and Center for Liver Diseases, Miller School of Medicine, University of Miami, Miami, FL, USA; 9Department of Anatomical Pathology and Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia; 10South Australian Liver Transplant Unit, Flinders Medical Centre, Adelaide, SA, Australia; and 11Department of Gastroenterology and Hepatology, Royal Adelaide Hospital, Adelaide, SA, Australia Received August 2014; accepted for publication November 2014

SUMMARY. Hepatocyte clone size was measured in liver samples of 21 patients in various stages of chronic hepatitis B virus (HBV) infection and from 21 to 76 years of age. Hepatocyte clones containing unique virus–cell DNA junctions formed by the integration of HBV DNA were detected using inverse nested PCR. The maximum hepatocyte clone size tended to increase with age, although there was considerable patient-to-patient variation in each age group. There was an upward trend in maximum clone size with increasing fibrosis, inflammatory activity and with seroconversion from HBV eantigen (HBeAg)-positive to HBeAg-negative, but these differences did not reach statistical significance. Maximum hepatocyte clone size did not differ between patients with and without a coexisting hepatocellular carcinoma. Thus, large hepatocyte clones containing integrated HBV DNA were detected during all stages of chronic HBV infection. Using laser

INTRODUCTION HBV chronically infects ~400 million people worldwide [1,2]. Patients with chronic HBV infection have a ~25% Abbreviations: 2D, 2-dimensional; 3D, 3-dimensional; AVT, antiviral therapy; BG, beta-globin; dslDNA, double-stranded linear DNA; HBeAg, hepatitis B virus e-antigen; HBsAg, hepatitis B virus surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; ID, identifier; invPCR, inverse nested PCR; LCC, large cell changes; LMD, laser microdissection; ND, not detected; NR, not repeated; rnd, randomly generated number; SA, survival advantage; SD, standard deviation; SEM, standard error of mean; Tm, melting temperature; VCJ, virus–cell junction. Correspondence: Allison Jilbert, Department of Molecular and Cellular Biology, School of Biological Sciences, University of Adelaide, SA 5005, Australia. E-mail: [email protected]

© 2015 John Wiley & Sons Ltd

microdissection, no significant difference in clone size was observed between foci of HBV surface antigen (HBsAg)-positive and HBsAg-negative hepatocytes, suggesting that expression of HBsAg is not a significant factor in clonal expansion. Laser microdissection also revealed that hepatocytes with normal-appearing histology make up a major fraction of the cells undergoing clonal expansion. Thus, preneoplasia does not appear to be a factor in the clonal expansion detected in our assays. Computer simulations suggest that the large hepatocyte clones are not produced by random hepatocyte turnover but have an as-yet-unknown selective advantage that drives increased clonal expansion in the HBV-infected liver. Keywords: clonal expansion of hepatocytes, hepatitis B virus, hepatocellular carcinoma, inverse nested PCR, laser microdissection, virus–cell DNA junction.

lifetime risk of developing liver cirrhosis and/or hepatocellular carcinoma (HCC), translating into ~600 000 deaths annually. Infection acquired in the first year of life has been suggested to progress through at least three phases [3,4]. First, the immune tolerant phase characterized by high HBV DNA titres and few histological abnormalities. Antiviral therapy (AVT) has not been considered necessary during this phase [5], although this remains controversial [6,7]. Infection progresses, sometimes after several decades, to an immunologically active (immune reactive) phase, in which cumulative liver damage can occur. Seroconversion from HBV e-antigen (HBeAg)-positive to HBeAg-negative status can also occur and lead to an immunologically inactive (inactive carrier) phase, marked by sustained, low HBV DNA titres. HCC is typically confined to the two latter phases [8–10].

2

T. Tu et al.

In healthy liver, low-level hepatocyte turnover occurs through the death of ~0.075% hepatocytes/day (derived from studies of rat models and human liver [11]), and compensatory division of mature hepatocytes [12] and in rare cases possibly by division of hepatic stem cells [12] although the existence of hepatic stem cells remains controversial [13]. Hepatocyte death and division leads to clonal expansion of some hepatocyte lineages, while others are lost. The increased hepatocyte turnover associated with chronic HBV infection, especially during the immune reactive phase [14–18], should lead to larger hepatocyte clones [12,19]. Whether some hepatocyte clones have, or acquire, a selective survival or growth advantage to evade the antiviral immune response, leading to their greater clonal expansion, is unknown. The evidence that 95% in short-term infections [22,23], suggests the emergence of hepatocyte clones that are resistant to HBV and thereby are able to evade immune attack. Selective clonal expansion of cellular subpopulations is a known risk factor for many cancers, including HCC [24–28], although it is unclear whether this applies to HBV-associated hepatitis. Integrated HBV DNA has been used to measure the clonal expansion of hepatocytes [14–16,18,29]. The double-stranded linear DNA (dslDNA) form of HBV DNA can integrate at random sites in the host cell DNA by nonhomologous recombination [30,31], creating unique virus–cell DNA junctions (VCJs) that can be used to track hepatocyte lineages. Detection and mapping of integrated HBV DNA has been achieved by Southern blot hybridization for identification of large hepatocyte clones (>105 cells). VCJs have also been detected by inverse nested PCR (invPCR), which can detect hepatocyte clones (e.g. of two or more cells) that are smaller than those detected by Southern blot hybridization. Using these and other approaches, including whole genome sequencing, large clones of nontumourous hepatocytes have been demonstrated in late-stage HBV infection in patients with [16–18,32–34] and without cirrhosis [15,32–34]. HCCs have also been reported to be clonal [14,35]. In the current study, invPCR was used to measure clonal expansion of hepatocytes, to examine possible correlations of hepatocyte clone size with patient age and histological and clinical features of infection. Furthermore, individual hepatocyte foci were isolated by laser microdissection (LMD) and analysed by invPCR and by immunostaining for levels of HBV surface antigen (HBsAg) expression to determine if hepatocyte clone size is affected by expression of HBsAg with mutations in the Pre-S region [36–39]. Hepatocyte foci showing large cell change (LCC) were also isolated by LMD and tested by invPCR for the presence of hepatocyte clones.

MATERIALS AND METHODS Ethics statement Ethics approval was obtained from the Royal Adelaide Hospital Human Research Ethics Committee, the Southern Adelaide Clinical Human Research Ethics Committee, and the Institutional Review Boards of the University of Miami, the University of Washington, and the Fox Chase Cancer Center. Informed and written consent was obtained from all patients who were given a unique identifier (ID) to maintain patient confidentiality. All clinical investigation was conducted according to the principles expressed in the World Medical Association Declaration of Helsinki.

Liver tissues, tissue processing and histopathology Non-HCC liver tissue from 21 patients with chronic HBV infection (Table 1) was studied. Following collection, liver tissues were immediately snap-frozen in liquid nitrogen and stored at
80 °C. For immunostaining, LMD and DNA extraction from sections, liver tissue fragments were fixed in 70% ethanol, dehydrated, embedded in paraffin wax and sectioned at 5-lm intervals. Ethanol was used as a fixative to preserve cellular DNA. Liver tissues were also fixed in 4% formalin, then wax-embedded, sectioned and stained with haematoxylin and eosin, periodic acid Schiff-diastase, Gordon and Sweet’s reticulin stain or haematoxylin van Geison’s stain by the Centre of Neuropathology, SA Pathology.

Extraction of total DNA Two 5-lm sections of 70% ethanol-fixed liver tissue were placed into a 1.5-mL tube, then dewaxed with 1 mL of xylene for 2 9 5 min, washed with 1 mL of ethanol for 2 9 5 min and then vacuum-dried. Dewaxed liver tissue sections or, in other instances, ~5-mg snap-frozen liver tissue fragments, were digested in 400 lL of digestion solution (100 mM NaCl, 0.5% SDS, 50 mM Tris pH 7.5, 10 mM EDTA, 2 mg/mL proteinase K) in a shaking thermomixer at 55 °C for 2 h. Total DNA was extracted with 400 lL of 25:24:1 UltraPure phenol:chloroform:isoamyl alcohol (Life Technologies, Carlsbad, CA, USA), then ethanol precipitated with 0.3 M sodium acetate (pH 4.6) and 800 lL of ethanol at
20 °C overnight. DNA pellets were washed with 70% ethanol and dissolved in 50 lL of elution buffer (Qiagen, Venlo, Limburg, Netherlands).

Immunostaining of HBsAg and LMD Five-lm sections of 70% ethanol-fixed and wax-embedded liver tissue were mounted onto glass slides and used for immunostaining of HBsAg. Sections were dewaxed in xylene, washed in ethanol, rehydrated in PBS and then treated © 2015 John Wiley & Sons Ltd

Age

21

22

29

40 44 46

48 48 48 52 54 54

Patient ID*

(A) P1

P2

© 2015 John Wiley & Sons Ltd

P3

P4 P5 P6

P7 P8 P9 P10 P11 P12

M M M M M F

M M M

M

M

F

Sex

4 4 4 4 2 4

4 4 4

0

0

1

Fibrosis (/4)

3 1 3 1 1 3

0 3 1

0

1

1

Activity (/3)

METAVIR score†










+







?








+











AVT‡




HCC‡


+ ? + + ?

+



+




+

Serum HBeAg‡

A D A D C C

D A C

C

D

A

HBV genotype§

190 1.2 2 groups. To determine whether HBV DNA integration occurred preferentially in a particular chromosome, the frequency of VCJs in each chromosome was normalized to chromosome length and then analysed by Grubb’s test for outlier detection (http:// graphpad.com/quickcalcs/Grubbs1.cfm). The size of hepatocyte clones detected in the same patient by invPCR were compared using two-tailed Wilcoxon’s matched-pairs signed-rank test.

RESULTS Analysis of liver tissues for hepatocytes clones InvPCR was performed on DNA extracted from ~5-mg fragments of frozen liver tissue from 16 patients (Table 1A, Data set S1). Hepatocyte clones, which had been previously detected by invPCR in liver tissue from five additional patients, P17 to P21 [15], were also analysed (Table 1B, Data set S1). Repeated VCJs (Figure S2) were detected in liver tissue from 10 of the 16 patients analysed in this study, with estimated hepatocyte clone sizes ranging from 48 to 38 800 cells (Table 1A). Including the previously analysed samples from the five patients, P17 to P21, clonal expansion of hepatocytes was detected in 15 of 21 patients. Five of the six patients without detectable hepatocyte clones had cirrhosis. Hepatocyte clones may be difficult to

8

T. Tu et al.

(a)

patient variation and the trend to increasing clone size with age did not reach statistical significance.

Computer simulation of random death and division within the hepatocyte population

(b)

Fig. 1 Maximum hepatocyte clone size detected by invPCR. (a) Distribution of maximum hepatocyte clone size by individual patient age (data from Table 1). (b) Geometric mean (+/
standard error of mean, SEM) of the maximum hepatocyte clone size by age, grouped by decade of life. detect in cirrhotic nodules, potentially due to a low incidence of integrated HBV DNA [50]. To confirm the size of the hepatocyte clones detected by invPCR, we independently measured hepatocyte clone size in patients P12 and P6 using qPCR. Hepatocyte clone P12 i, detected by invPCR to be a clone of 38 880 cells, was estimated by qPCR to be ~33 800 cells. Similarly, hepatocyte clone P6 i, detected by invPCR to be a clone of 26 460 cells, was estimated by qPCR to be ~31 700 cells. These results provide an independent confirmation of the size of hepatocyte clones detected by invPCR.

The maximum detected hepatocyte clone size tended to increase with age The maximum detected hepatocyte clone sizes for all patient samples are shown in Fig. 1. The largest clones were found in the oldest patients (Fig. 1a). This trend is also clear when the geometric mean of the maximum clone sizes is determined for patients grouped by decade of life (Fig. 1b). However, there was considerable patient-to-

Next, we carried out computer simulations to determine whether it was likely that the observed hepatocyte clones were produced by random hepatocyte turnover. Our simulation of random hepatocyte turnover showed a linear increase in maximum clone size with patient age (Fig. 2a). Further, if only random hepatocyte turnover was considered it showed that the maximum hepatocyte clone size expected in an 80-year-old patient, experiencing 6.5 liver turnovers/year from birth, was ~4100 cells. Liver growth was also modelled using similar computer simulations. We considered a 10-fold increase in liver size from birth to 14 years of age [51,52] and a death rate of 0.075% hepatocytes per day, consistent with liver turnover in healthy individuals [11] and the normal liver enzyme levels observed in HBV-infected immune tolerant teenage patients [7]. In these simulations, liver growth both with and without hepatocyte death did not significantly increase clone size above that simulated by assuming the maximum likely average turnover for a HBV-infected patient of 6.5 liver turnovers/year used above (data not shown). Although to a greater degree in late-stage disease, hepatocyte clones composed of >4100 cells were detected in our studies using invPCR in patients in all age groups (Fig. 1b). Thus, neither random liver turnover nor liver growth fully explained the observed hepatocyte clones in our patient cohort, suggesting that other factors regulate clonal expansion in many, if not all, patients. When we performed simulations where hepatocytes were given a SA, even a 2% SA stimulated the outgrowth of large hepatocyte clones (Fig. 2b), suggesting the expansion of hepatocytes with a SA in our patient cohort.

Maximum hepatocyte clone size did not correlate with disease progression We next attempted to find associations between maximum hepatocyte clone size and markers of disease progression, such as HBeAg sero-conversion, the incidence of HCC and levels of fibrosis and inflammatory activity (Fig. 3). While there was a trend of increased maximum clone size in HBeAg-negative compared to HBeAg-positive patients (Fig. 3a), this was not statistically significant, possibly due to low numbers of HBeAg-positive patients with detectable VCJ. Furthermore, maximum hepatocyte clone size differences between patients with HCC and non-HCC, various stages of fibrosis (METAVIR F0-F4) and inflammatory activity (METAVIR A0-A2) were not statistically significant (Fig. 3b–d).

© 2015 John Wiley & Sons Ltd

Clonal expansion of hepatocytes in chronic HBV

9

(a)

(b)

Fig. 2 Maximum hepatocyte clone size (mean +/- standard deviation, SD) predicted using a computer simulation. (a) The effect of random liver turnover on hepatocyte clonal expansion was simulated as described in Materials and Methods. The largest clone size in an 8 000 000 (2003) cell LiverArray (n = 20) was measured at each liver turnover for the equivalent of 80 patient-years, with an estimated 6.5 liver turnovers/year (Materials and Methods) for a total of ~520 liver turnovers. (b) Effect of a selective SA on clonal hepatocyte expansion. The overall hepatocyte death rate was as in Panel A, except that the effects of a SA are now included. Maximum clone size was measured over the equivalent of 80 patient-years (n = 20) containing 80 randomly chosen cells with 0% (crosses), 2% (hollow triangles), 5% (asterisks), 10% (plus signs) or 25% (hollow squares) SA. In simulations containing cells with a SA of 2%, 5%, 10% and 25%, an average maximum clone size of 10 000 cells is reached within 350, 150, 100 and 40 liver turnovers, respectively. When converted using our estimated rate of 6.5 liver turnovers/year (Materials and Methods), these liver turnovers correspond to a minimum patient age of ~53, ~23, ~15 and ~6 years, respectively.

The majority of clones appear to be derived from histologically normal hepatocytes DNA extracted from a pair of 5-lm liver tissue sections was analysed by invPCR for the presence of clones. Although the DNA extracted from the tissue sections was ~10-fold less than from 5-mg liver fragments, VCJs were detected in tissue sections from 14 of the 17 patients tested. As expected, clones were not detected in the patients with cirrhosis P5, P14 and P16 (Table 2A). The estimated 3D size of the largest hepatocyte clones detected in the liver tissue sections of each patient (Table 2A, Text © 2015 John Wiley & Sons Ltd

S1) did not significantly differ from the maximum clone size detected by invPCR in the 5-mg tissue fragments from the same patients (P = 0.55, two-tailed Wilcoxon’s matched-pairs signed-rank test). Liver tissue sections from patients P6, P17, P19, P20 and P21 contained multiple large clones, allowing for more detailed study of the cell type containing the VCJ. Nonhepatocyte areas, including infiltrating inflammatory cells, fibrotic tissue, bile ductules, central veins and portal tracts, were isolated by LMD from adjacent liver tissue sections of each of these patients and were tested by invPCR. The nonhepatocyte areas contained no detectable VCJs,

10

T. Tu et al.

(a)

(b)

(c)

Fig. 3 Clonal expansion of hepatocytes was not associated with disease progression. Maximum hepatocyte clone sizes found in DNA extracts from ~5-mg pieces of liver tissue were grouped by serum HBeAg status (a), HCC status (b), METAVIR fibrosis score (c) and METAVIR inflammatory activity score (d). The geometric means of the hepatocyte clone sizes for each group are shown in dashed lines. Differences in mean clone size shown in (a) and (b) were analysed by two-tailed Mann–Whitney U-test but did not reach statistical significance (P = 0.159 and P = 0.931, respectively). Differences in mean clone size shown in (c) and (d) were analysed by one-tailed Kruskal–Wallis test and again did not reach statistical significance (overall P = 0.269 and P = 0.102, respectively). All VCJs are shown in Table 1A and 1B, and are fully listed in Data set S1.

patients P18, P19 and P21. A total of 15 hepatocyte foci with LCC (2, 6 and 7 foci/patient, respectively) were isolated from adjacent serial sections by LMD and analysed by invPCR. Only a single VCJ, P21 ii, was detected. In contrast, eight distinct VCJs were detected from hepatocyte foci with normal histology in these patients (Table 2B). Further, no foci of hepatocytes with small cell change, a dysplastic histophenotype strongly associated with HCC progression [53], were found in our tissue sections. Thus, hepatocytes with normal histology appear to make up the majority of cells undergoing clonal expansion as detected by invPCR.

HBsAg expression is not associated with larger hepatocyte clones

(d)

confirming that hepatocytes are the main cell type contributing to the clones detected in whole liver tissue sections. Foci of hepatocytes with LCC, a histological, non-preneoplastic change in hepatocytes associated with various liver diseases [53], were observed in liver tissue sections of

To determine whether increased expression of HBsAg was associated with increased rates of clonal expansion as reported previously [36–39], foci of ‘HBsAg-positive’ hepatocytes (containing multicellular clusters of HBsAg-positive hepatocytes) and foci of ‘HBsAg-negative’ hepatocytes (containing HBsAg-negative hepatocytes and 0.05, two-tailed Mann–Whitney U-test) in hepatocyte clone size was observed in HBsAg-positive vs HBsAg-negative hepatocyte foci (Table 2B). This suggests that HBsAg expression does not confer a survival or growth advantage. Interestingly, in each of these patients P6, P17, P20 and P21, 6 VCJs (P6 ii, P6 v, P17 iii, P17 xii, P20 i and P21 i) were detected in geographically adjacent or nearby foci isolated by LMD (Table 2B; Figure S3). Identical VCJs were not detected in geographically separate foci (Table 2B; Figure S3), suggesting that the foci that contain an identical VCJ may have evolved by clonal expansion of hepatocytes following the initial integration event. In these cases, using the LMD technique, hepatocyte foci containing identical VCJ © 2015 John Wiley & Sons Ltd

Clonal expansion of hepatocytes in chronic HBV

11

Table 2 Hepatocyte clones detected by invPCR of DNA extracted from (A) a pair of 5-lm liver tissue sections and (B) foci isolated from liver tissue sections by LMD

Patient ID

Age

Tissue blocks analysed*

(A) P1 P2

21 22

1 2

P3 P4

29 40

1 2

P5 P6

44 46

4 1

P7 P11 P12 P13

48 54 54 56

1 2 4 4

P14 P16 P17

58 65 37

2 1 1

P18 P19

46 66

1 1

P20

70

1

P21

76

1

Clone ID†

Estimated 2D clone size‡

Minimum estimated 3D clone size§

– P2 iv P2 v P2 vi P2 vii P2 viii P2 ix P3 xi P4 i P4 ii P4 iii – P6 i P6 vii P7 iv–vi P11 i P12 ii–v P13 i P13 ii P13 iii P13 iv P13 v P13 vi P13 viii P13 ix – – P17 i P17 ii P17 iii – P19 i P19 ii P20 i P20 ii P21 i P21 ii P21 iii

ND 12 6 4 4 4 4 NR 648 216 216 ND 312 48 NR 100 NR 1728 648 432 216 216 144 48 48 ND ND 2430 810 90 NR 810 270 540 90 270 90 90

ND 41 14 8 8 8 8 NR 26 920 5180 5180 ND 8990 543 NR 1631 NR 117 200 26 920 14 651 5180 5180 2820 543 543 ND ND 195 460 37 620 1390 NR 37 620 7240 20 480 1390 7240 1390 1390

Total number of analysed foci¶ Patient ID

HBsAg-positive

HBsAg-negative

Focus ID¶

Clone ID†

Estimated 2D clone size‡

Minimum estimated 3D clone size§,**

(B) P6

13

17

3N 4P 4N

P6 i P6 ii P6 v

624 320 208

15 502 5693A 2983B (continued)

© 2015 John Wiley & Sons Ltd

12

T. Tu et al.

Table 2 (continued) Total number of analysed foci¶ Patient ID

P17

HBsAg-positive

11

HBsAg-negative

12

Focus ID¶

Clone ID†

7N

P6 vi P6 v P6 ii P17 viii P17 iii P17 v P17 xii P17 xv P17 vii P17 xiii P17 xvi P17 iv P17 ix P17 xvii P17 xix P17 xx P17 x P17 xxv P17 xxvi P17 vi P17 iii P17 xii P17 xviii P17 xxi P19 v P19 iii P19 iv P20 iii P20 i P20 v P20 i P20 iv P21 vi P21 ii P21 i P21 iv P21 iii P21 i P21 i

12N 1N 2N

4N

5N

7N

8N

P19

12

12

P20

7

15

P21

9

11

8P 8N 9P 2N 5P 5N 7P 9N 3N 4N 5P 5N 6N

Estimated 2D clone size‡ 96 NR NR 16 NR 24 NR NR 16 NR NR 72 16 NR NR NR 16 NR NR 16 NR NR NR NR NR 80 32 16 NR NR 16 16 NR 48 NR 48 NR NR 32

Minimum estimated 3D clone size§,** 935 NRB NRA 64 NRC 117 NRD NR 64 NR NR 608 64 NR NR NR 64 NR NR 64 NRC NRD NR NR NR 712 180 64 NRE NR 64E 64 NR 331 NRF 331 NR NRF 180F

*Multiple tissue blocks were analysed for patients where tissue availability permitted. †Clone IDs were given to each unique VCJ detected, in order of clone size. Details are presented in Data set S1. ‡All samples were analysed using NcoI or DpnII invPCR designs as described in Materials and Methods. If the same VCJ was detected by both the NcoI and DpnII invPCR designs in the same DNA extract, the larger estimated 2D hepatocyte clone size was used. NR = Not repeated, VCJs that were not repeated in the DNA extract of the tissue section or hepatocyte focus. ND = Not detected, DNA extracts in which no VCJs were detected. §Minimum 3D hepatocyte clone size was calculated as outlined in Text S1. ¶HBsAg-positive and HBsAg-negative foci were isolated by LMD of liver sections from patients P6, P17, P19, P20 and P21. The total number of foci analysed from each liver tissue section is listed, but only the foci containing detectable VCJ are listed in full. Each focus was given a unique ID [a number followed by a suffix of either P (for HBsAg-positive) or N (for HBsAg-negative)] and then analysed by invPCR (Figure S3). **In some instances, identical VCJs were detected in separate adjacent hepatocyte foci. Identical VCJs are denoted by the same superscript letter (A–F).

© 2015 John Wiley & Sons Ltd

Clonal expansion of hepatocytes in chronic HBV

13

(a)

(b)

Fig. 4 VCJs are distributed throughout the host genome and the majority occur upstream of the expected right-hand end of HBV dslDNA. Repeated and total VCJs detected by invPCR of DNA extracted from ~5-mg liver tissue fragments and 5lm liver tissue sections were aligned with the human genome using BLAST (a). The frequency of detectable VCJs in each chromosome was normalized to its length. Assuming a normal distribution, the frequency of HBV DNA integration in all chromosomes did not significantly differ from each other (P > 0.05, Grubb’s test for outlier detection). Repeated and total VCJs were also aligned with the HBV genome (GenBank Accession AB241115). The cumulative incidence of VCJ was plotted against the nt position of the VCJ with respect to the EcoRI site on the HBV genome (b). In the majority of cases, the VCJ clustered before the expected right-hand end of the HBV dslDNA at nt 1832. may have been isolated as separate foci but in fact were part of a larger focus of HBsAg-positive or HBsAg-negative hepatocytes (e.g. Figure S3E, VCJ P20 i was detected in foci 4N, 5N and 6N). Our results therefore suggest that these six repeated VCJs represent true clonal expansion of hepatocytes and not multiple independent integration events. We predict © 2015 John Wiley & Sons Ltd

that integration events occurring in a completely random fashion would occur with a probability of one in 3.3 9 109 (the length of the human genome) making it highly unlikely that the identical VCJs have occurred through multiple independent integrations. For five of the six identical VCJs, the foci were exclusively either HBsAg-positive or HBsAg-

14

T. Tu et al.

negative, suggesting that the HBsAg status of hepatocytes is heritable. Accumulation of HBsAg containing Pre-S mutations [36–39] was not the cause of clonal expansion in this cohort, as HBV strains with mutations in the Pre-S region were only detected in hepatocyte foci from patient P19, and these foci did not contain any detectable VCJ (data not shown).

Analysis of virus–cell junctions BLAST analysis of repeated VCJ indicated that the majority (73%) of integrations occurred outside of transcribed regions, 44 of 162 (27%) integrations occurred within transcribed regions and only one integration (P3 ii) occurred in a coding region (Data set S1). No correlation was found between hepatocyte clone size and cellular integration site (Data not shown). The integration events were dispersed in a normal distribution (P > 0.05, by Grubb’s test for outlier detection) over all chromosomes (Fig. 4a), consistent with other studies [15,32,33,54,55]. The observed HBV DNA integration sites were clustered around the expected right-hand end of dslDNA at nt 1832 [43,56], mostly upstream of nt 1832 (Fig. 4b). These results are consistent with previous studies [15,43,54] and support the role of dslDNA as the precursor to HBV DNA integration [30,31].

DISCUSSION This study examined the clonal expansion of hepatocytes during chronic HBV infection. The results suggest that normal-appearing hepatocytes clonally expand at rates faster than expected if all cells were equally likely to die or undergo compensatory division. This clonal expansion begins prior to extensive fibrosis or other evidence of significant immune-mediated liver disease and is not restricted to late-stage liver disease. This is consistent with results from studies of clonal expansion in other cancers and other aetiologies of HCC [24–28,57]. The association of clonal expansion with age showed high patient-to-patient variability. This poor fit is not unexpected, as liver turnover is not linear with time; for example, accelerated liver turnover is seen during seroconversion from HBeAg-positive to HBeAg-negative, the timing of which varies from patientto-patient. We did not find any evidence that hepatocytes with altered or preneoplastic histology are a major source of the clonal hepatocyte expansion detected in our studies. While overexpression of HBsAg containing Pre-S mutations reportedly stimulates clonal hepatocyte expansion [36–39], we did not observe this in our patients. Our results also failed to support the notion that HBV DNA integration per se causes clonal expansion via effects on host genes, as suggested by studies of other tumour tissues [14,55,58– 67]. Even in larger clones, integration near cellular genes

was not beyond what would be expected due to random integration of HBV DNA. However, our data do not rule out the possibility that HBV DNA integration causes chromosomal instability or long-range transcriptional dysregulation of cellular genes [58,68–70]. Our data are consistent with selective clonal expansion of nontransformed, normal-appearing cells, which has been associated with carcinogenesis in gastrointestinal cancers [24,25], the chemically-induced HCC rat model [26,27] and with human genetic diseases predisposing to HCC [28]. In these nonviral models of HCC, selective clonal expansion is thought to involve altered hepatocytes that can evade the effects of a cytotoxic chemical or gene product. This leads to clonal hepatocyte expansion, a HCC risk factor [28]. It seems plausible that, during chronic HBV infection, clonal expansion of hepatocytes with a selective advantage (e.g. being more resistant to HBV infection) that enables them to escape antiviral immunity might have a major role in progression to HCC. Alternatively, clonal expansion may be driven by hepatocytes that proliferate at a higher rate than neighboring cells. Further studies are needed to identify the cellular phenotypes that confer a SA during chronic HBV infection. Extensive clonal expansion of hepatocytes also appears to be a feature of some patients (e.g. Patients P1-3) early in chronic HBV infection. Despite the idea that many young patients are immune tolerant, clonal expansion of hepatocytes might be ongoing in these patients via selective targeting of some hepatocytes by antiviral immunity and the selective survival and expansion of others, that is that the patients may not be completely immune tolerant. It will be important to extend our studies to further explore this concept. Future studies are also needed to determine whether hepatocyte expansion can predict HCC risk. This may be especially helpful in the up to 35% of patients with HBV-associated HCC in which cirrhosis (one of the strongest predictors of HCC) is absent [71]. Further, our results would imply that HCC is driven by host–virus interactions that begin early in HBV infection. While antiviral therapy has not been considered necessary during the early immune tolerant phase, inhibiting virus replication prior to significant fibrosis could indirectly reduce the ongoing immune response that may be driving clonal expansion of hepatocytes and facilitating the development of HCC [5,72].

ACKNOWLEDGEMENTS AND DISCLOSURES The authors thank Emeritus Professor Chris Burrell (University of Adelaide), Dr. Christoph Seeger and Dr. Samuel Litwin (Fox Chase Cancer Center), Dr Jesse Summers (University of New Mexico) and Professor Fabien Zoulim (INSERM, Lyon) for productive discussions and comments on the manuscript. The authors also wish to thank Ms. © 2015 John Wiley & Sons Ltd

Clonal expansion of hepatocytes in chronic HBV Libby John (Flinders Medical Centre) for helping to coordinate access to liver tissues and clinical information. This work was funded by Project Grant 453507 from the National Health and Medical Research Council of Australia. NHMRC website: www.nhmrc.gov.au/. TT was supported

15

by a Postgraduate Study Divisional Ph.D. Scholarship from the University of Adelaide. The funding body had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

REFERENCES 1 WHO. Hepatitis B: World Health Organization fact sheet 204. In: WHO, ed. Fact Sheets, Vol. 2007. World Health Organization, 2000: http://www.who.int/mediacentre/ factsheets/fs204/en/ Last accessed 29 December 2014 2 Lai CL, Wong DKH. Discovery and classification. In: Lai CL, Locarnini S, eds. Hepatitis B Virus, 2nd edn. London: International Medical Press, 2008: 1.1–1.10. 3 Yim HJ, Lok AS. Natural history of chronic hepatitis B virus infection: what we knew in 1981 and what we know in 2005. Hepatology 2006; 43(2 Suppl 1): S173–S181. 4 EASL clinical practice guidelines: Management of chronic hepatitis B virus infection. Journal of Hepatology 2012; 57: 167–185. 5 Mekky MA. To treat or not to treat the “immunotolerant phase” of hepatitis B infection: a tunnel of controversy. World J Hepatol 2014; 6(4): 226–229. 6 Kennedy PT, Sandalova E, Jo J et al. Preserved T-cell function in children and young adults with immune-tolerant chronic hepatitis B. Gastroenterology 2012; 143(3): 637–645. 7 Wang HY, Chien MH, Huang HP et al. Distinct hepatitis B virus dynamics in the immunotolerant and early immunoclearance phases. J Virol 2010; 84(7): 3454–3463. 8 Chu CM, Karayiannis P, Fowler MJ, Monjardino J, Liaw YF, Thomas HC. Natural history of chronic hepatitis B virus infection in Taiwan: studies of hepatitis B virus DNA in serum. Hepatology 1985; 5(3): 431–434. 9 Lin X, Robinson NJ, Thursz M et al. Chronic hepatitis B virus infection in the Asia-Pacific region and Africa: review of disease progression. J Gastroenterol Hepatol 2005; 20(6): 833–843. 10 Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus. A prospective

© 2015 John Wiley & Sons Ltd

11

12

13

14

15

16

17

18

19

study of 22 707 men in Taiwan. Lancet 1981; 2(8256): 1129–1133. Mancini R, Marucci L, Benedetti A, Jezequel AM, Orlandi F. Immunohistochemical analysis of S-phase cells in normal human and rat liver by PC10 monoclonal antibody. Liver 1994; 14(2): 57–64. Malato Y, Naqvi S, Schurmann N et al. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. J Clin Invest 2011; 121(12): 4850–4860. Grompe, M. 2014. Liver stem cells, where art thou? Cell Stem Cell. 15: 257–258. Hino O, Kitagawa T, Koike K et al. Detection of hepatitis B virus DNA in hepatocellular carcinomas in Japan. Hepatology 1984; 4(1): 90–95. Mason WS, Liu C, Aldrich CE, Litwin S, Yeh MM. Clonal expansion of normal-appearing human hepatocytes during chronic hepatitis B virus infection. J Virol 2010; 84 (16): 8308–8315. Tanaka Y, Esumi M, Shikata T. Frequent integration of hepatitis B virus DNA in noncancerous liver tissue from hepatocellular carcinoma patients. J Med Virol 1988; 26(1): 7–14. Fowler MJ, Greenfield C, Chu CM et al. Integration of HBV-DNA may not be a prerequisite for the maintenance of the state of malignant transformation. An analysis of 110 liver biopsies. J Hepatol 1986; 2(2): 218–229. Yasui H, Hino O, Ohtake K, Machinami R, Kitagawa T. Clonal growth of hepatitis B virus-integrated hepatocytes in cirrhotic liver nodules. Cancer Res 1992; 52(24): 6810– 6814. Traulsen A, Lenaerts T, Pacheco JM, Dingli D. On the dynamics of neutral mutations in a mathematical model for a homogeneous stem cell population. J R Soc Interface 2013; 10(79): 20120810.

20 Gowans EJ, Burrell CJ. Widespread presence of cytoplasmic HBcAg in hepatitis B infected liver detected by improved immunochemical methods. J Clin Pathol 1985; 38(4): 393–398. 21 Gowans EJ, Burrell CJ, Jilbert AR, Marmion BP. Cytoplasmic (but not nuclear) hepatitis B virus (HBV) core antigen reflects HBV DNA synthesis at the level of the infected hepatocyte. Intervirology 1985; 24 (4): 220–225. 22 Murray JM, Wieland SF, Purcell RH, Chisari FV. Dynamics of hepatitis B virus clearance in chimpanzees. Proc Natl Acad Sci USA 2005; 102(49): 17780–17785. 23 Berquist KR, Peterson JM, Murphy BL, Ebert JW, Maynard JE, Purcell RH. Hepatitis B antigens in serum and liver of chimpanzees acutely infected with hepatitis B virus. Infect Immun 1975; 12(3): 602– 605. 24 Chen R, Rabinovitch PS, Crispin DA, Emond MJ, Bronner MP, Brentnall TA. The initiation of colon cancer in a chronic inflammatory setting. Carcinogenesis 2005; 26(9): 1513–1519. 25 Merlo LM, Shah NA, Li X et al. A comprehensive survey of clonal diversity measures in Barrett’s esophagus as biomarkers of progression to esophageal adenocarcinoma. Cancer Prev Res 2010; 3(11): 1388–1397. 26 Bralet MP, Calise D, Brechot C, Ferry N. In vivo cell lineage analysis during chemical hepatocarcinogenesis using retroviral-mediated gene transfer. Lab Invest 1996; 74(5): 871–881. 27 Cameron RG. Identification of the putative first cellular step of chemical hepatocarcinogenesis. Cancer Lett 1989; 47(3): 163–167. 28 Marongiu F, Doratiotto S, Montisci S, Pani P, Laconi E. Liver repopulation and carcinogenesis: two sides

16

29

30

31

32

33

34

35

36

37

38

T. Tu et al. of the same coin? Am J Pathol 2008; 172(4): 857–864. Esumi M, Tanaka Y, Tozuka S, Shikata T. Clonal state of human hepatocellular carcinoma and nontumorous hepatocytes. Cancer Chemother Pharmacol 1989; 23(Suppl): S1–S3. Bill CA, Summers J. Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration. Proc Natl Acad Sci USA 2004; 101(30): 11135–11140. Petersen J, Dandri M, Burkle A, Zhang L, Rogler CE. Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair. J Virol 1997; 71(7): 5455–5463. Jiang S, Yang Z, Li W et al. Re-evaluation of the carcinogenic significance of hepatitis B virus integration in hepatocarcinogenesis. PLoS One 2012; 7(9): e40363. Sung WK, Zheng H, Li S et al. Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma. Nat Genet 2012; 44(7): 765– 769. Fujimoto A, Totoki Y, Abe T et al. Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators. Nat Genet 2012; 44(7): 760–764. Miller RH, Lee SC, Liaw YF, Robinson WS. Hepatitis B viral DNA in infected human liver and in hepatocellular carcinoma. J Infect Dis 1985; 151(6): 1081–1092. Fan YF, Lu CC, Chen WC et al. Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection. Hepatology 2001; 33(1): 277–286. Wang HC, Wu HC, Chen CF, Fausto N, Lei HY, Su IJ. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. Am J Pathol 2003; 163(6): 2441–2449. Wang LH, Huang W, Lai MD, Su IJ. Aberrant cyclin A expression and centrosome overduplication induced

39

40

41

42

43

44

45

46

47

by hepatitis B virus pre-S2 mutants and its implication in hepatocarcinogenesis. Carcinogenesis 2012; 33 (2): 466–472. Su IJ, Wang HC, Wu HC, Huang WY. Ground glass hepatocytes contain pre-S mutants and represent preneoplastic lesions in chronic hepatitis B virus infection. J Gastroenterol Hepatol 2008; 23(8 Pt 1): 1169– 1174. Mendy ME, Welzel T, Lesi OA et al. Hepatitis B viral load and risk for liver cirrhosis and hepatocellular carcinoma in The Gambia, West Africa. J Viral Hepat 2010; 17(2): 115–122. Vandegraaff N, Kumar R, Burrell CJ, Li P. Kinetics of human immunodeficiency virus type 1 (HIV) DNA integration in acutely infected cells as determined using a novel assay for detection of integrated HIV DNA. J Virol 2001; 75(22): 11253–11260. Delaney WE, Isom HC. Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus. Hepatology 1998; 28(4): 1134–1146. Yang W, Summers J. Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor. J Virol 1999; 73(12): 9710–9717. Farinati F, Cardin R, D’Errico A et al. Hepatocyte proliferative activity in chronic liver damage as assessed by the monoclonal antibody MIB1 Ki67 in archival material: the role of etiology, disease activity, iron, and lipid peroxidation. Hepatology 1996; 23(6): 1468–1475. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferationassociated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984; 133(4): 1710–1715. Liaw YF, Tai DI, Chu CM, Pao CC, Chen TJ. Acute exacerbation in chronic type B hepatitis: comparison between HBeAg and antibodypositive patients. Hepatology 1987; 7(1): 20–23. Kawakita N, Seki S, Yanai A et al. Immunocytochemical identification of proliferative hepatocytes using

monoclonal antibody to proliferating cell nuclear antigen (PCNA/cyclin). Comparison with immunocytochemical staining for DNA polymerase-alpha. Am J Clin Pathol 1992; 97(5 Suppl. 1): S14– S20. 48 Wolf HK, Michalopoulos GK. Hepatocyte regeneration in acute fulminant and nonfulminant hepatitis: a study of proliferating cell nuclear antigen expression. Hepatology 1992; 15(4): 707–713. 49 Liaw YF, Yang SS, Chen TJ, Chu CM. Acute exacerbation in hepatitis B e antigen positive chronic type B hepatitis. A clinicopathological study. J Hepatol 1985; 1(3): 227– 233. 50 Robinson WS, Klote L, Aoki N. Hepadnaviruses in cirrhotic liver and hepatocellular carcinoma. J Med Virol 1990; 31(1): 18–32. 51 Heymsfield SB, Gallagher D, Mayer L, Beetsch J, Pietrobelli A. Scaling of human body composition to stature: new insights into body mass index. Am J Clin Nutr 2007; 86(1): 82–91. 52 Shepard TH, Shi M, Fellingham GW et al. Organ weight standards for human fetuses. Pediatr Pathol 1988; 8(5): 513–524. 53 Park YN. Update on precursor and early lesions of hepatocellular carcinomas. Arch Pathol Lab Med 2011; 135(6): 704–715. 54 Mason WS, Low HC, Xu C et al. Detection of clonally expanded hepatocytes in chimpanzees with chronic hepatitis B virus infection. J Virol 2009; 83(17): 8396–8408. 55 Nagaya T, Nakamura T, Tokino T et al. The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma. Genes Dev 1987; 1(8): 773–782. 56 Staprans S, Loeb DD, Ganem D. Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA. J Virol 1991; 65(3): 1255–1262. 57 Kvittingen EA, Rootwelt H, Berger R, Brandtzaeg P. Self-induced correction of the genetic defect in tyrosinemia type I. J Clin Invest 1994; 94(4): 1657–1661.

© 2015 John Wiley & Sons Ltd

Clonal expansion of hepatocytes in chronic HBV 58 Brechot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 2004; 127(5 Suppl 1): S56–S61. 59 Brechot C, Gozuacik D, Murakami Y, Paterlini-Brechot P. Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Semin Cancer Biol 2000; 10(3): 211–231. 60 Chen JY, Harrison TJ, Lee CS, Chen DS, Zuckerman AJ. Detection of hepatitis B virus DNA in hepatocellular carcinoma: analysis by hybridization with subgenomic DNA fragments. Hepatology 1988; 8(3): 518–523. 61 Cougot D, Neuveut C, Buendia MA. HBV induced carcinogenesis. J Clin Virol 2005; 34(Suppl 1): S75–S78. 62 Murakami Y, Saigo K, Takashima H et al. Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut 2005; 54(8): 1162– 1168.

63 Pineau P, Marchio A, Mattei MG et al. Extensive analysis of duplicated-inverted hepatitis B virus integrations in human hepatocellular carcinoma. J Gen Virol 1998; 79(Pt 3): 591–600. 64 Rogler CE, Sherman M, Su CY et al. Deletion in chromosome 11p associated with a hepatitis B integration site in hepatocellular carcinoma. Science 1985; 230(4723): 319–322. 65 Shafritz DA, Shouval D, Sherman HI, Hadziyannis SJ, Kew MC. Integration of hepatitis B virus DNA into the genome of liver cells in chronic liver disease and hepatocellular carcinoma. Studies in percutaneous liver biopsies and postmortem tissue specimens. N Engl J Med 1981; 305(18): 1067–1073. 66 Wang Y, Lau SH, Sham JS, Wu MC, Wang T, Guan XY. Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellular carcinogenesis. Oncogene 2004; 23(1): 142–148. 67 Yaginuma K, Kobayashi M, Yoshida E, Koike K. Hepatitis B virus inte-

68

69

70

71

72

17

gration in hepatocellular carcinoma DNA: duplication of cellular flanking sequences at the integration site. Proc Natl Acad Sci USA 1985; 82(13): 4458–4462. Feitelson MA, Lee J. Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis. Cancer Lett 2007; 252(2): 157–170. Levy L, Renard CA, Wei Y, Buendia MA. Genetic alterations and oncogenic pathways in hepatocellular carcinoma. Ann N Y Acad Sci 2002; 963: 21–36. Sanyal A, Lajoie BR, Jain G, Dekker J. The long-range interaction landscape of gene promoters. Nature 2012; 489(7414): 109–113. Kew MC. Hepatocellular carcinoma with and without cirrhosis. A comparison in southern African blacks. Gastroenterology 1989; 97(1): 136– 139. Zoulim F, Mason WS. Reasons to consider earlier treatment of chronic HBV infections. Gut 2012; 61(3): 333–336.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Text S1: Estimate of minimum 3dimensional (3D) hepatocyte clone size. Data set S1: Sequence of detected

© 2015 John Wiley & Sons Ltd

VCJs. Table S1: (A) InvPCR designs and nested PCR primers. (B) Nested PCR primers. Figure S1: InvPCR strategy. Figure S2: InvPCR assay for repeated VCJ.

Figure S3: Virus-cell junctions (VCJs) found in hepatocyte foci isolated by laser microdissection (LMD) of 5-lm sections of liver tissue from Patients P6 (A), P17 (B), P19 (C), P20 (D) and P21 (E).