HBV quantitative sag assay: Ready for Prime time? yes

HBV quantitative sag assay: Ready for Prime time? yes Robert G Gish MD Professor Consultant, Stanford University Medical Director: Hepatitis B Foundation 1

Relevant Disclosures Consultant: Abbott, Genentech (Roche), Gilead 2

Refining Current Treatments: Treatment Endpoints Long-term suppression of HBV DNA Ideally by achievement of HBsAg seroconversion HBeAg-positive Sustained HBeAg seroconversion If no HBeAg serconversion--> suppression of HBV DNA to low levels HBeAg-negative Sustained low level HBV DNA On treatment if nucleosides Off treatment if peg-ifn AASLD Guidelines 2009 EASL Guidelines 2012 APASL Guidelines 2012

Benefits of HBsAg Clearance Hepatic decompensation HCC Survival Levels of cccdna As close to cure as we can expect to achieve in chronic hepatitis B Fattovich G, et al. Am J Gastro 1998; 93:896-900. Werle-Lapostolle B, et al. Gastroenterology 2004; 126(7):1750-1758. Perrillo R. Hepatology 2009; 49:1063-1065.

HBsAg, HBeAg Levels for Prediction of Anti-HBe Seroconversion HBeAgpositive CHB Biomarker Level PPV NPV Ref Baseline HBsAg 5,000 50,000 HBeAg 31 PE > 1300 42% 54% 77% 76% Piratvisuth, HI 2011 Fried. Hepatology 2008 Week 12 HBsAg < 1500 55% Piratvisuth, HI 2011 Sonneveld, Hepatology 2011 No decline 97% Vs. 82/71% Sonneveld, Hepatology 2011 Piratvisuth, Hepatology 2011 >20,000 84% (93%) Piratvisuth, HI 2011 HBeAg < 10 53% Fried, Hepatology 2008 Week 24 HBsAg > 20,000 85% (95%, 100%) HBeAg > 100 Fried, Hepatology 2008

J Hepatology 2011;55:1121 1 IU/ml = 1-10 ng/ml HBsAg = 2 x 10 8 subviral particles HBsAg = 5 x 10 7 virions What is a Significant Change? > 4 fold change > 1.0 log IU/ml

HBeAg-Positive Chronic Hepatitis B On-treatment HBsAg levels can predict: PEG-IFN treatment response

On-Treatment HBsAg Levels Identify Good Responders* Week HBV genotype HBsAg Rule N identified PPV response Week 12 A (n=98) <1500 IU/mL 14 (14%) 86% 1500-20,000 42 (42%) 41% >20,000 IU/mL 42 (42%) 17% B (n=199) <1500 IU/mL 60 (30%) 42% 1500-20,000 101 (51%) 26% >20,000 IU/mL 38 (19%) 8% C (n=377) <1500 IU/mL 89 (24%) 42% 1500-20,000 221 (59%) 20% >20,000 IU/mL 67 (18%) 2% D (n=105) <1500 IU/mL 2 (2%) 0% 1500-20,000 28 (27%) 7% >20,000 IU/mL 75 (71%) 4% * Response = HBeAg loss + HBV DNA < 2,000 IU/mL Sonneveld et al. Hepatology 2013

On-Treatment HBsAg Levels Identify Futility: Stopping Rules Week HBV genotype HBsAg Rule N identified NPV response Week 12 A (n=55) No decline* 13 (24%) 100% B (n=120) >20,000 IU/mL 24 (20%) 92% C (n=225) >20,000 IU/mL 45 (20%) 98% D (n=54) No decline 33 (61%) 97% Week 24 A (n=55) >20,000 IU/mL 24 (44%) 96% B (n=122) >20,000 IU/mL 16 (13%) 100% C (n=224) >20,000 IU/mL 27 (12%) 100% D (n=53) >20,000 IU/mL 36 (68%) 100% * HBsAg decline < 10% Sonneveld et al. Hepatology 2013

HBeAg-Negative Chronic Hepatitis B On-treatment HBsAg levels can provide: Futility Rule

Combination of HBsAg and HBV DNA to Predict SVR in HBeAg-Negative CHB Validation cohorts (ph III / PegBeLiver) (n = 160, genotype D = 91) HBsAg decline Week 12 No N = 84 (53%) Yes N = 76 (48%) HBV DNA decline Week 12 < 2 logs N = 22 (14%) 2 logs N = 62 (39%) < 2 logs N = 22 (14%) 2 logs N = 54 (34%) SVR* 5% 37% 45% 43% STOP * HBV DNA < 2,000 IU/mL and normal ALT Rjickborst, J Hepatology 2012

Predicting HBeAg Seroconversion (HBV DNA compared with quantitative HBeAg) HBeAg level at Week 24 (Paul Ehrlich units) HBeAg seroconversion Week 72 All patients (n=263) > 100 52% NPV 96% 4% (3/72) HBV DNA level at Week 24 (copies/ml) All patients (n=263) > 9.0 log 10 21% NPV 86% 14% (8/59) NPV = negative predictive value. Fried MW, et al. Hepatology 2008;47:428-434.

Response Guided Therapy for Peg- Interferon in the Treatment of Hepatitis B Week 12 and 24 are the Key HBeAg - Positive HBeAg - Negative Week 12 Define Possible Non-Responders Criteria: 1) Absence of HBsAg decline OR 2) HBsAg > 20,000 IU/ml Criteria: 1) Absence of HBsAg decline AND 2) HBV DNA reduction < 2 log Week 24 Define the Level of Treatment Response High: HBsAg < 1,500 IU/ml Mid: HBsAg 1,500 to 20,000 IU/ml Low: HBsAg > 20,000 IU/ml High: HBsAg decline >10% Low: HBsAg decline < 10% Chan, HLY et al 2011. J Hepatol;55:1121

HBsAg (IU/mL) Change from baseline in HBV DNA log 10 copies/ml) HBsAg decline can predict HBsAg loss on tenofovir N=266 patients treated with tenofovir (or adefovir-tenofovir) for 2 years 0 HBsAg levels during treatment 0 HBV DNA levels during treatment -25000-50000 -2-75000 -4-100000 -125000-6 -150000 0 12 24 36 48 60 72 84 96 Weeks of study -8 0 12 24 36 48 60 72 84 96 Weeks of study N=266 overall N=16 cleared HBsAg Heathcote et al. EASL and AASLD 2009

HBsAg Serum Levels Vary in the Different Phases of HBV Infection Combined single-point quantification of serum HBsAg and HBV DNA levels Accurate diagnosis of the inactive carrier with immune control HBsAg quantification is a new, reliable diagnostic tool Likely to improve (in conjunction with viral load) the management of chronic HBV infection and disease

Summary #1 HBsAg in Natural History HBsAg is an old marker with new applications HBsAg clearance is the closest to cure in CHB: functional cure HBsAg quantification provides additional clinical guidance: Helps determine phase of infection The increasing access to quantification assay will foster learning about how to use it to manage both carriers and patients more effectively Threshold levels can be set which predict for inactive carrier status in HBeAg-negative patients Levels of 500 1000 should be evaluated further After S Locarnini 2014

Summary #2 HBsAg Seroclearance as an Endpoint HBsAg seroconversion is the hallmark of successful immunological control of HBV infection Associated with favourable long-term clinical outcomes including a lower incidence of cirrhosis and improved survival Can be induced by IFN-based therapy Less commonly achieved in patients treated with direct antiviral therapies, i.e. nucleos(t)ide analogues Most patients have persistently normal ALT levels Is usually associated with improvement of liver biochemistry Most patients have undetectable serum HBV DNA with 6-12 months Still harbour HBV DNA in the liver (cccdna), but transcriptionally inactive HCC still did develop in some patients, but only in those over 50 years of age qhbsag provides a great NPV: Can HBsAg quantification provide a predictive value? Yuen, M-F. et al 2008. Gastroenterology;135:1192-1199 After S Locarnini 2014

Summary #3 HBsAg in Response Guided Therapy On-treatment monitoring of HBsAg with or without HBV DNA offers the prospect of response-guided, individualized therapy in patients with CHB PEG-IFN NAs On-treatment HBsAg decline is associated with sustained immune control HBsAg monitoring may help identify patients who are able to stop treatment HBsAg monitoring has the potential to guide patient management in the future After S Locarnini 2014

Study GS-US-174-0149 Design Week Primary endpoint: HBsAg loss 0 16 24 48 72 120 n=186 TDF + PEG n=184 TDF+PEG TDF n=185 TDF n=185 PEG Start TDF during follow-up if prespecified safety criteria met Randomized, controlled, open-label study (N=740) Stratified by screening HBeAg status and HBV genotype Inclusion criteria HBeAg+ and HBV DNA 20,000 IU/mL; HBeAg- and HBV DNA 2,000 IU/mL ALT >54 and 400 U/L (men); ALT >36 and 300 U/L (women) No bridging fibrosis or cirrhosis on liver biopsy or by transient elastography H Chan, EASL 2015

Patients with HBsAg Loss, Kaplan-Meier Estimate (%) Results: HBsAg Loss Over Time (Week 72) 0.15 0.14 0.13 0.12 0.11 0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 48 weeks 72 weeks TDF + PEG 48 wk 9.1% p=0.003 PEG 48 wk 2.8% TDF + PEG 16 wk TDF 32 wk 2.8% p=ns p=ns TDF 120 wk 0% p<0.001 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 Week 7 patients had HBsAg seroreversion on or after Week 48 (4 [TDF + PEG 48 wk], 3 [TDF + PEG 16 wk TDF 32 wk]) 5/7 had 1 week of therapy after HBsAg loss H Chan, EASL 2015

On-Treatment Predictors of HBsAg Loss at Week 72 HBsAg decrease >1 log 10 IU/mL by Week 12 Univariate p-value HBsAg <100 IU/mL at Week 12 <0.001 Multivariate p-value Hazard Ratio* <0.001 <0.001 17.8 HBsAg <1000 IU/mL at Week 12 <0.001 HBsAb >10 miu/ml at Week 12 0.138 0.003 9.97 HBV DNA <15 IU/mL at Week 12 0.014 0.070 15.68 ALT >400 U/L (males) or >300 U/L (females) during first 12 weeks <0.001 0.031 2.85 *Multivariate H Chan, EASL 2015

FINITE CHB Stopping TDF After Long-Term Virologic Suppression in HBeAg-Negative CHB Randomized N=45 Withdrew consent n=3 TDF-Stop n=21 TDF-Continue n=21 Week 48 TDF-Restart n=3 Week 48 TDF-Stop n=18 Week 48 TDF-Continue n=21 86% of TDF-Stop subjects did not restart TDF by Week 48 Berg, EASL, 2015, O119 36

FINITE CHB Week 48 HBsAg log 10 Reduction (Individual Patients) TDF-Stop (n=21) TDF-Continue (n=20) HBsAg loss HBsAg loss * HBsAg Log 10 Reduction: Median 0.283 Mean 0.773 HBsAg loss n=2 HBsAg Log 10 Reduction: Median 0.088 Mean 0.109 HBsAg loss n=0 * * -1 1 3 5 HBsAg (log 10 reduction) * TDF-Restart Berg, EASL, 2015, O119-1 1 3 5 HBsAg (log 10 reduction) Stopping TDF was associated with a more profound decline in HBsAg levels compared to continuous TDF 37

HBsAg clearance after addition of 48 weeks of PEGIFN in HBeAg negative CHB patients on NUC therapy with undetectable HBV DNA for at least one year : Final results from ANRS-HB06 PEGAN study : a multicenter randomized controlled phase III trial. Marc Bourlière 1, Pascaline Rabiega 2, Nathalie Ganne-Carrie 3, Lawrence Serfaty 4, Patrick Marcellin 5, Noëlle Pouget 2, Dominique Guyader 6, Christophe Hezode 7, Magali Picon 8, Xavier Causse 9, Vincent Leroy 10, Jean Pierre Bronowicki 11, Ghassan Riachi 12, Isabelle Rosa 13, Pierre Attali 14, Jean Michel Molina 15, Yannick Bacq 16, Albert Tran 17, Jean Didier Grangé 18, Fabien Zoulim 19, Hélène Fontaine 20, Inga Bertucci 21, Magali Bouvier-Alias 22, Fabrice Carrat 2, Yves Benhamou 23 and the ANRS HB06 PEGAN Study Group. Immuno-Virologique 1 Hôpital Saint Joseph, Marseille, France, 2 INSERM UMR-S1136 Saint Antoine Paris, 3 Hôpital Jean Verdier, Bondy, 4 Hôpital Saint Antoine, Paris, 5 Hôpital Beaujon, Clichy, 6 Hôpital Pontchaillou, Rennes, 7 Hôpital Henri Mondor, Créteil, 8 Centre Hospitalier du Pays d Aix, Aix en provence, 9 Hôpital de la Source, Orléans, 10 CHU Grenoble, 11 Hôpital de Brabois, Nancy, 12 Hôpital Charles Nicolle, Rouen, 13 1 CHIC Créteil, 14 Hôpital Bicêtre, Le Kremlin Bicêtre, 15 Hôpital Saint Louis Paris, 16 Hôpital Trousseau, Tours, 17 Hôpital de Hôpital Saint Joseph, Marseilles, France, 2 INSERM UMR-S1136 Saint Antoine Paris,3 L Archet, Nice, 18 Hôpital Tenon, Paris, 19 Hôpital Hotel Dieu Lyon, 20 Hôpital Cochin, Paris, 21 Inserm-ANRS, 22 INSERM U635, 23 Hôpital La Pitié Salpétrière, Paris. 1 Hôpital Saint Joseph, Marseilles, France, 2 INSERM UMR-S1136 Saint Antoine Paris,3 EASL 2015 / April 22-26 - Vienna Titre document / Le 15-09 - 2013 38

randomization Ptimary endpoint Study Design W-6 W0 W48 W96 W144 PEGIFN α2a 180 µg/wk N=185 CHB patients HBeAg negative N=92 N=90* NUC treatment N=93 N=93 * 1 patient withdrew consent and 1 patient excluded for major deviation inclusion criteria as pre-specified. Visits W144 are ongoing. Randomized, controlled, open-label study (N=185). Stratified by HBsAg titer (<2.25 log IU/ml or 2.25log IU/ml) Key inclusion criteria HBeAg negative Undetectable HBV DNA with NUC for at least 1 year Stable NUC regimens for at least 3 months (no Telbivudine) No decompensated cirrhosis Patients in the PEGIFN group were monitored monthly during the first 48 weeks, then every 3 months. Treatment discontinuation was allowed if HBsAg loss was sustained for 24 weeks. Bourliere EASL 2015 40

Efficacy : HBsAg loss and HBs seroconversion All patients included, N (%) NUCs Alone PEGIFN + NUCs Randomization stratification Log 10 (IU/ml) < 2.25 2.25 Total < 2.25 2.25 Total N=15 N=78 N=93 N=14 N=76 N=90 P-Value W48 Loss of HBsAg 0 (0) 0 (0) 0 (0) 3 (22) 4 (5) 7 (8) 0.0057 HBs seroconversion 0 (0) 0 (0) 0 (0) 2 (14) 2 (3) 4 (4) 0.0384 W96 Loss of HBsAg 2 (13) 1 (1) 3 (3) 4 (29) 3 (4) 7 (8) 0.1521 HBs seroconversion 0 (0) 1 (1) 1 (1) 3 (21) 3 (4) 6 (7) 0.0465 Patients Per Protocol, N(%) W48 W96 NUCs Alone PEGIFN + NUCs N=15 N=78 N=93 N=12 N=73 N=85 P-Value Loss of HBsAg 0 (0) 0 (0) 0 (0) 3 (25) 4 (6) 7 (8) 0.0038 HBs seroconversion 0 (0) 0 (0) 0 (0) 2 (17) 2 (3) 4 (5) 0.0296 Loss of HBsAg 2 (13) 1 (1) 3 (3) 4 (33) 3 (4) 7 (8) 0.1038 HBs seroconversion 0 (0) 1 (1) 1 (1) 3 (25) 3 (4) 6 (7) 0.0341 Patients Per Protocol full dose of PEGIFN, N (%) W48 W96 NUCs Alone PEGIFN + NUCs N=15 N=78 N=93 N=10 N=55 N=65 P-Value Loss of HBsAg 0 (0) 0 (0) 0 (0) 3 (30) 4 (7) 7 (11) 0.0011 HBs seroconversion 0 (0) 0 (0) 0 (0) 2 (20) 2 (4) 4 (6) 0.0145 Loss of HBsAg 2 (13) 1 (1) 3 (3) 4 (40) 3 (5) 7 (11) 0.0415 HBs seroconversion 0 (0) 1 (1) 1 (1) 3 (30) 3 (5) 6 (9) 0.0131 Bourliere EASL 2015 45

Baseline predictive factors at W96 in PEGIFN group Univariate analysis All patients included N=7/90 Patients Per Protocol N=7/85 Patients Per protocol full dose of PEGIFN N=7/65 OR IC 95% P-value OR IC 95% P-value OR IC 95% P-value HBeAg at diagnosis (- vs +) 2.77 0.37 18.27 0.3865 2.80 0.37 18.62 0.3814 2.74 0.35 18.99 0.4137 HBsAg at randomization by decreasing unit (log10) Related to HBsAg loss 3.43 1.09 12.47 0.0339 3.60 1.11 13.64 0.0300 4.00 1.17 16.41 0.0240 Fibrosis (F3-F4 vs F0-F2) 2.49 0.39 18.25 0.4319 2.30 0.36 16.86 0.4971 2.92 0.44 22.20 0.3368 Previous PEGIFN treatment (yes vs no) 2.33 0.37 17,00 0.4804 2.23 0.35 16.31 0.5190 2.32 0.36 17.37 0.4998 Univariate analysis Related to HBs seroconversion All patients included N=6/90 Patients Per Protocol N=6/85 Patients Per protocol full dose of PEGIFN N=6/65 OR IC 95% P-value OR IC 95% P-value OR IC 95% P-value HBeAg at diagnosis (- vs +) 3.74 0.46 30.64 0.2594 3.78 0.46 31.22 0.2555 3.71 0.44 31.77 0.2774 HBsAg at randomization by decreasing unit (log10) 3.50 1.02 14.29 0.0465 3.68 1.04 15.59 0.0419 4.03 1.09 18.22 0.0349 Fibrosis (F3-F4 vs F0-F2) 1.81 0.23 14.42 0.7611 1.67 0.21 13.33 0.8374 2.08 0.25 17.51 0.6498 Previous PEGIFN treatment (yes vs no) 3.55 0.48 41.37 0.2838 3.40 0.46 39.73 0.3096 3.54 0.47 42.28 0.2947 Age, sex, origin, IL-28B profil, duration of undetectable HBV DNA from first undetectability and from latest undetectability were tested and were not significant. Bourliere EASL 2015 52

W96 HBsAg loss according to HBsAg titer at inclusion All patients included HBsAg titer at W0 (log10 IU/ml), N Patients Per Protocol HBsAg titer (log 10 IU/ml) Patients Per Protocol full dose of PEGIFN NUCs Alone N=93 Trend test PEGIFN + NUCs N=90 <2 2/12 2/8 2-3 1/22 0.0038 3/26 3 0/59 2/56 NUCs Alone N=93 PEGIFN + NUCs N=85 <2 2/12 2/7 2-3 1/22 0.0038 3/25 3 0/59 2/53 NUCs Alone N=93 NUCs Alone Trend N=93 test Trend test PEGIFN + NUCs N=65 <2 2/12 2/6 2-3 1/22 0.0038 3/16 3 0/59 2/43 PEGIFN+NUCs N=65 < 2 log 10 IU/ml 16% 33% < 3 log 10 IU/ml 8% 23% > 3 log 10 IU/ml 0% 4% HBsAg loss is associated with low baseline HBsAg titer. Trend test 0.0245 Trend test 0.0200 Trend test 0.0150 Bourliere EASL 2015 53

Conclusions of ANRS HB06 PEGAN Addition of a 48 weeks of PEGIFN α2a to NUCs therapy in HBeAg negative CHB patients with undetectable HBV DNA for at least one year, results in higher rates of HBsAg loss and HBs seroconversion. Low baseline qhbsag levels increase HBsAg loss and HBs seroconversion at week 96. However, acceptability of such regimen in patients treated with NUCs is poor and discontinuation due to adverse events occurred in 20% of the patients. Therefore, this combination should be proposed in a selected HBeAg negative CHB patients population. Bourliere EASL 2015 56

Cumulative probability Cumulative probability Multivariate analysis:factors associated with HBsAg clearance Variable HR IC 95% p HBV DNA negative 2.37 1.34 4.19 0.03 HBsAg <1000 IU/ml 49.4 6.76 361.02 <0.001 HBV DNA HBs Ag Negative Positive < 1000 IU/ml 1000 IU/ml González-Diéguez EASL 2015

Groups of patients with a significant different probability of HBsAg loss at fifth year Based on HBV-DNA and HBsAg Based on HBsAg P < 0.001 53.5% n= 45 HBV DNA negative and HBsAg <1000 IU/mll P < 0.001 53 % n= 79 HBsAg < 100 IU/mll 27.2% n= 101 HBV DNA negative or HBsAg <1000 IU/mll 12.2 % n= 55 HBsAg 100-1000 IU/ml 0% n= 151 HBV DNA positive and HBsAg >1000 IU/mll 0% n= 160 HBsAg > 1.000 IU/ml González-Diéguez EASL 2015

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Conclusions part II Discovery of HBsAg has revolutionized the diagnosis and management of HBV infection A highly sensitive HBsAg test that is able to detect most surface region mutant is important to avoid false negative results HBsAg quantification Combined with viral load, improve the management of chronic HBV infection Serum HBsAg quantification can provide additional information on the transcriptional activity and amount of cccdna inside the liver Low HBsAg level can identify inactive carriers and predict lower risk of HCC On-treatment HBsAg quantification can predict response to peginterferon Monitoring of serum HBsAg level can potentially time the cessation of oral antiviral therapy Over 3 Million qhbsag assays are used worldwide each year = Prime time is here!

Thank you to my sag Gurus Henry Chan Harry Janssen Steve Locarnini Pietro Lampertico Seng-Gee Lim and the post- EASL Singapore Viral Hepatitis Meeting 79