Key genetic elements in HBsAg significantly correlate with liver cancer onset by hampering HBsAg secretion and promoting cell proliferation in vitro.

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1 13th European Meeting on HIV & Hepatitis - Treatment Strategies & Antiviral Drug Resistance Key genetic elements in significantly correlate with liver cancer onset by hampering 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 *The author declare that there are no conflicts of interest HIRMA PROJECT Bandiera PB05 PROJECT

2 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. HBV-related deaths HBV infection <5% immunocompetent adults 30% of CHB CDC Division of Viral Hepatitis. Chronic hepatitis B: Information on testing % of CHB w/o cirrhosis Modified from Thornton K., et al A systematic characterization of viral genetic factors that can modulate the progression of chronic hepatitis toward HCC is still needed

3 HBV surface glycoprotein () plays an important role in modulating HBV oncogenetic potential. HBV genome The role of genetic variability in this region in the mechanisms related to HCC onset is not well characterized

4 Aims of the study The main goals of this study are: - To define the correlation of mutations with HCC onset in chronically HBV infected patients. - To characterize the impact of these mutations on the production/secretion and cell proliferation in in vitro models.

5 Materials and Methods This study includes 133 chronically HBV infected patients: 23 patients with HCC 26% 74% 110 patients without cirrhosis or HCC 27% 73% HBV Genotype A HBV Genotype D 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 Association of mutations with HCC was assessed by Fisher-exact test.

6 Materials and Methods HBV- wt / mut plasmids 1. mutations were introduced into a full genome HBV genotype D plasmid. 2. WT and mutated clones were then transfected into Huh7 and HepG2 cells. HuH7 and HepG2 cell lines HBV- wt / mut viral particles 3. Supernatants and cell lysates were harvested in triplicate daily until day 7 post-transfection. 4. amount (24 hours production) was quantified by Alexxis assay, cell proliferation and viability were evaluated by using FACS analisys.

7 Characteristics of patients with or without HCC 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 ( ) 2012 ( ) 0,01 HBeAg negative, N(%) 14 (60.9) 64 (58.2) 0,93 Median HBV-DNA [log IU/ml] (IQR) 4.0 ( ) 4.0 ( ) 0,20 Median ALT [IU/L] (IQR) 82 (28-112) 49 (29-103) 0,47

8 Details of HCC characteristics 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 ( ) 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)

9 P203Q and S210R in the C-terminus result significantly correlated with HCC 35 ** * p<0.05 ** p<0.001 Gender: , p-value Prevalence (%) * ** P203Q S210R P203Q+S210R Age ALT AST Male Female HCC (N=23) NON HCC (N=110) At least one mutation (P203Q+/-S210R) Two 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.

10 Both P203Q and S210R mutations were located in the C-terminus ( aa) that is known to play an important role in the secretion of HBV surface glycoproteins S210R P203Q Schematic representation of the Small Surface glycoprotein () encoded by the S region of HBV genome Jenna et al., Virology 1998; Jenna et al., J Virol 1999

11 The intracellular retention can increase intracellular oxidative stress and induce the neoplastic transformation of hepatocytes. mutant mutant mutant mutant mutant mutant mutant mutant Modified by Pollicino T. et al, J of Hepatology, 2014

12 P203Q, S210R and P203Q+S210R decrease secretion factor, suggesting their ability to hamper secretion The histogram reports the 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 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.

13 P203Q and P203Q+S210R significantly correlate with an increased percentage of cells in the S phase of cell cycle 26±13% p< ±14% p<0.01 % cells in S-Phase 15±6% 18±9% 10±5% 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.

14 UDPS analisys confirmed that HCC-related mutations occurred as major viral species in Intra-patient prevalence (IPP) of mutations associated with HCC (%) Intra-patient prevalence (IPP) of mutations associated with HCC (%) of 13 HCC patients with a median intrapatient prevalence of 70.0% Patients with HCC (N=13) 86,58 49,81 Limit of detection by standard population sequencing. IPP < 20% Minority species 18,80 11,46 4,80 17,31 100,00 61,96 53,54 65,46 99,99 73,56 70, Control patients (N=24) 1,04 10,59 2, ,80 Limit of detection by standard population sequencing. IPP < 20% Minority species P203Q S210R 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. In chronically infected patients used as control, the median (IQR) intrapatient prevalence of S- mutations associated with HCC was remarkably lower (11.5% [10.6%- 18.8%])

15 Conclusions Key mutations, residing in C-terminal domain, are highly correlated with HBV-induced HCC in CHB patients They affect secretion and stimulate cell proliferation in vitro, suggesting their potential involvement in HCC development.

16 The Clinicians: Policlinico di Tor Vergata Massimo Andreoni Cesare Sarrecchia Mario Angelico Simona Francioso Daniele Di Paolo Thanks to 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 HIRMA PROJECT 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: Bandiera PB05 PROJECT Stephen Locarnini Nadia Warner