Key genetic elements in HBsAg significantly correlate with liver cancer onset by hampering HBsAg secretion and

promoting cell proliferation in vitro.

Matteo Surdo*, Romina Salpini, Nadia Warner, Maria Francesca Cortese, Carmen Mirabelli, Danni Colledge, Sally Soppe, Michela Pollicita, Roberta Longo, Sara Romano, Giuseppina

Cappiello, Alberto Spanò, Pascale Trimoulet, Henry Fleury, Jacopo Vecchiet, Nerio Iapadre, Angelo Barlattani, Ada Bertoli, Terenzio Mari, Caterina Pasquazzi, Gabriele Missale, Cesare Sarrecchia, Elisa Orecchini, Alessandro Michienzi, Massimo Andreoni, Simona Francioso, Mario Angelico, Francesca Ceccherini-Silberstein, Stephen Locarnini, Carlo-Federico Perno, Valentina Svicher

Session 4: Determinants of HBV Pathogenesis - June 03, 2015 Abst#_13

Bandiera PB05

PROJECT

HIRMA PROJECT

*The author declare that there are no conflicts of interest

13th European Meeting on HIV & Hepatitis - Treatment Strategies &

Antiviral Drug Resistance

A systematic characterization of viral genetic factors that can modulate the progression of chronic hepatitis toward HCC is still needed

Hepatocellular carcinoma (HCC) is one of the most frequent solid tumors and the third cause of cancer death worldwide.

Recent estimates attribute to hepatitis B virus (HBV) over 50% of HCC cases worldwide.

15-20% of CHB w/o cirrhosis

HBV infection

<5% immunocompetent

adults 30% of CHB

Modified from Thornton K., et al

HBV-related deaths

CDC Division of Viral Hepatitis. Chronic hepatitis B: Information on testing.

HBV genome

HBsAg

HBV surface glycoprotein (HBsAg) plays an important role in modulating HBV oncogenetic potential.

The role of genetic variability in this region in the mechanisms related to HCC onset is not well characterized

The main goals of this study are:

- To define the correlation of HBsAg mutations with HCC onset in chronically HBV infected patients.

- To characterize the impact of these mutations on the HBsAg production/secretion and cell proliferation in in vitro models.

Aims of the study

This study includes 133 chronically HBV infected patients:

Materials and Methods

23 patients with HCC 110 patients without cirrhosis or HCC

74% 26%

73% 27%

Association of HBsAg mutations with HCC was assessed by Fisher-exact test.

The distribution of HBV genotypes in HCC and control population was comparable thus minimizing the possible influence of HBV genotypes

in the analysis of the two population groups

HBV Genotype D HBV Genotype A

1. HBsAg mutations were introduced into a full genome HBV genotype D plasmid.

2. WT and mutated clones were then transfected into Huh7 and HepG2 cells.

3. Supernatants and cell lysates were harvested in triplicate daily until day 7 post-transfection.

4. HBsAg amount (24 hours production) was quantified by Alexxis assay, cell proliferation and viability were evaluated by using FACS analisys.

Materials and Methods

HBV-HBsAgwt/mut plasmids

HuH7 and HepG2 cell lines

HBV-HBsAgwt/mut viral particles

Patients' characteristics (N=23) HCC pts (n=23) (%)

Control pts (n=110) (%) P value

Male, N(%) 21 (91.7) 70 (63.6) 0,03

Italian Nationality, N(%) 16 (69.6) 65 (59.1) 0,48

Median Age [Years] (IQR) 63 (53-70) 44 (35-58) < 0,001

Median Collection Date [Year] (IQR) 2010 (2006-2011) 2012 (2008-2013) 0,01

HBeAg negative, N(%) 14 (60.9) 64 (58.2) 0,93

Median HBV-DNA [log IU/ml] (IQR) 4.0 (2.1-6.5) 4.0 (3.4-6.2) 0,20

Median ALT [IU/L] (IQR) 82 (28-112) 49 (29-103) 0,47

Characteristics of patients with or without HCC

HCC characteristics N (%) Single Nodule HCC 14 (60.9) Multifocal-HCC 7 (30.4) Unknown 2 (8.7) Tumor size Median size of each HCC nodule (IQR) [cm] 5 (2.5-7.6) Median alfa-feto protein at HCC diagnosis (IQR) [ng/ml] 519 (6.2-13,012) HCC treatment No 3 (13.0) Transcatheter arterial Chemoembolisation (TACE) 8 (34.8) Surgical resection 4 (17.4) Thermoablation 2 (8.7) Unknown 6 (26.1) Liver status Diagnosis of cirrhosis 15 (65.2) Child-Pugh Classification A 11 (47.8) B 6 (26.1) C 2 (8.7) Unknown 4 (17.4) Therapy at HCC diagnosis No 9 (39.1) LMV 7 (30.4) ADV 1 (4.3) ETV 3 (13.0) TDF 3 (13.0) LMV+ADV 1 (4.3)

Details of HCC characteristics

P203Q and S210R in the C-terminus HBsAg result significantly correlated with HCC

Two HBsAg mutations significantly correlated with HCC:

- P203Q (17.4% [4/23] in HCC vs 0.8% [1/110] in non-HCC, p<0.05);

- S210R (34.8% [8/23] in HCC vs 3.6% [4/110] in non-HCC, p<0.001;

- P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, p<0.001.

**

** *

0

5

10

15

20

25

30

35

P203Q S210R P203Q+S210R

HCC (N=23) NON HCC (N=110)

Prev

alen

ce (%

)

* p<0.05 ** p<0.001

0,01 1 100

Gender:

Female

Age

ALT

AST

At least one mutation(P203Q+/-S210R)

Male

0.01 1001

0.177

0.177

0.384

0.334

0.020

-

p-value

Schematic representation of the Small Surface glycoprotein (HBsAg) encoded by the S region of HBV genome

P203Q S210R

Both P203Q and S210R mutations were located in the HBsAg C-terminus (170-226aa) that is known to play an important role in the

secretion of HBV surface glycoproteins

Jenna et al., Virology 1998; Jenna et al., J Virol 1999

Modified by Pollicino T. et al, J of Hepatology, 2014

The intracellular HBsAg retention can increase intracellular oxidative stress and induce the neoplastic transformation of hepatocytes.

HBsAg mutant HBsAg

mutant

HBsAg mutant

HBsAg mutant

HBsAg mutant

HBsAg mutant

HBsAg mutant

HBsAg mutant

P203Q, S210R and P203Q+S210R decrease HBsAg secretion factor, suggesting their ability to hamper HBsAg secretion

The histogram reports the HBsAg secretion factor for the wt and the viruses carrying the P203Q, S210R and P203Q+S210R. Secretion factor is defined as the ratio of extracellular to intracellular HBsAg amount determined by the Alexxis assay. Average of 3 experiments +/- SEM are shown. The significance between wt and each mutant at each timepoint was determined using 2-tailed unpaired T-test. *P<0.05 **P<0.01 ***P<0.005.

P203Q and P203Q+S210R significantly correlate with an increased percentage of cells in the S phase of cell cycle

The histogram reports the percentage of cells in S-phase of cell cycle for wt and mutants at 7 days post transfection determined by FACS analysis, selecting GFP+ HepG2 cells. Statistical analysis was perfomed with chi-square test.

26±13% p<0.01

29±14% p<0.01

18±9%

10±5% 15±6%

% c

ells

in S

-Pha

se

In chronically infected patients used

as control, the median (IQR)

intrapatient prevalence of S-HBsAg

mutations associated with HCC was

remarkably lower (11.5% [10.6%-

18.8%]) 18,80 11,46

73,56

1,04

10,59 2,60

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Limit of detection by standard population sequencing. IPP < 20% Minority species

86,58

49,81

4,80

17,31

53,54

100,00

61,96

99,99

65,46

70,60

0,80 0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Intr

a-pa

tient

pre

vale

nce

(IPP)

of H

BsAg

m

utat

ions

ass

ocia

ted

with

HCC

(%)

Intr

a-pa

tient

pre

vale

nce

(IPP)

of H

BsAg

m

utat

ions

ass

ocia

ted

with

HCC

(%)

Patients with HCC (N=13)

Control patients (N=24)

P203Q S210R

Limit of detection by standard population sequencing. IPP < 20% Minority species

UDPS analisys confirmed that HCC-related mutations occurred as major viral species in

6 of 13 HCC patients with a median intrapatient prevalence of 70.0%

Two additional HCC patients

presented the mutation S210R

as minor species with an

intrapatient prevalence of 0.8%

and 4.8%, respectively

compared to bulk sequencing.

Conclusions

Key mutations, residing in C-terminal HBsAg domain, are highly correlated with HBV-induced

HCC in CHB patients

They affect HBsAg secretion and stimulate cell proliferation in vitro, suggesting their potential

involvement in HCC development.

Thanks to… The Clinicians: Policlinico di Tor Vergata Massimo Andreoni Cesare Sarrecchia Mario Angelico Simona Francioso Daniele Di Paolo Angelo Barlattani, Polimabulatorio San Giacomo, Roma Nerio Iapadre, Osp. L'Aquila Alberto Spanò, Osp. Pertini Caterina Pasquazzi, Osp. Sant’Andrea, Roma Maurizio Koch, Osp.San Filippo Neri, Roma Jacopo Vecchiet, Osp. SS. Annunziata Chieti Terenzio Mari, Osp. Nuovo Regina Margherita Gabriele Missale, UO Malattie Infettive ed Epatologia, Azienda Ospedaliero-Universitaria di Parma

Virology Group: University of Tor Vergata

Carlo Federico Perno Valentina Svicher

Romina Salpini Maria Francesca Cortese

Carmen Mirabelli Michela Pollicita

Ada Bertoli Roberta Sbrocchi

Velia Chiara Di Maio Francesca Ceccherini-Silberstein

Victorian Infectious Diseases Reference

Laboratory, Melbourne: Stephen Locarnini

Nadia Warner

Bandiera PB05

PROJECT

HIRMA PROJECT