HBsAg quantitativo: impiego nella diagnosi dello

stato di portatore inattivo e nelle stopping rules in

corso di terapia con PEGIFN

G Missale

UO Malattie Infettive ed Epatologia

Azienda Ospedaliero-Universitaria di Parma

Conventional HBV Markers

Direct markers

- HBs antigen - HBe antigen

POL

HBcAg

LHBs

MHBs

SHBs

Partially double- stranded DNA

Indirect markers

- Anti-HBs

- Anti-HBc - Anti-HBc IgM - Anti-HBe

Molecular marker

HBV DNA

Novel diagnostic tools

Molecular HBV genotype cccDNA Assessment of virus mutations -Precore and core promoter

mutations -HBV envelope mutations -Antiviral resistance mutations

Serological

• QUANTITATIVE HBsAg

• QUANTITATIVE HBeAg

HBV ccc DNA

RC primer

CCC primer

RC

primer

CCC primer

Commercial tests

Elecsys II – Roche

Architect – Abbott

ADVIA Centaur HBsAg Assay – Bayer

Hepanostika HBsAg – Biomerieux

In-house assays widely used in some countries

HBsAg measuring

Dynamic Range: 0 – 250.0 IU/ml (WHO) Specimens with values exceeding 250 IU/ml are flagged and may be diluted with the Manual Dilution Procedure. Operator enters the dilution factor in the Patient or Control order screen. System uses this dilution factor to automatically calculate the concentration of the sample before dilution and report the result. Dual epitope capture format, detects all known HBsAg mutants

Quantitative HBsAg measurement with some assays after dilution

Architect HBsAg (QT)

Qualitative screening assay for HBsAg universally available

nucleus cytoplasm

repair

mRNA

transcription

Core particle plus

strand synthesis

recycling

mRNA

transport

translation Ribosome

Transport to cell

membrane

HBV

preCore

HBX

Secreted HBeAg

ER

Spheres and Filaments

containing HBsAg

Small HBs Medium HBs

Large HBs

Envelope

Subgenomic and

Genomic RNA

Pre-genomic RNA Core

Pol

cccDNA

Golgi

complex

Entry of HBV

into cell and

uncoating

Core particle minus

strand synthesis

Core assembly and

RNA packaging

HBV Life Cycle

Raimondo et al, J Hepatol 2007

Hepatic HBV cccDNA Levels in Different Patient Populations

Werle-Lapostolle, Petersen, Locarnini, Zoulim Gastroenterology 2004

HBeAg+ patients had significantly higher cccDNA (90-fold) and total HBV DNA (147- fold) levels compared to HBeAg- patients (p<0.001, Wilcoxon tests)

Tota

l HB

V D

NA

(c

op

ies/

cell)

10 -3

10 -2

10 -1

10 0

10 1

10 2

10 3

10 4

nucleus cytoplasm

repair

mRNA

transcription

Core particle plus

strand synthesis

recycling

mRNA

transport

translation Ribosome

Transport to cell

membrane

HBV

preCore

HBX

Secreted HBeAg

ER

Spheres and Filaments

containing HBsAg

Small HBs Medium HBs

Large HBs

Envelope

Subgenomic and

Genomic RNA

Pre-genomic RNA Core

Pol

cccDNA

Golgi

complex

Entry of HBV

into cell and

uncoating

Core particle minus

strand synthesis

Core assembly and

RNA packaging

HBV Life Cycle

Raimondo et al, J Hepatol 2007

NUCs

Changes in Serum HBsAg Levels are Positively Correlated with Changes in cccDNA Levels

p<0.01

-3 -2 -1 0 1

-3

-2

-1

0 ADV+PEG

Change in Serum HBsAg (log 10 ng/mL)

Ch

ange

in c

ccD

NA

(l

og

10

co

pie

s/ce

ll)

Petersen J et al Hepatology 2006

HBsAgsl decline predicts SVR of Peginterferon treated HBeAg pos CH-B patients

Sonneveld MJ et al, Hepatology 2010;52:1251-57

HBsAgsl <1500 UI/ml at week 12 is associated with SVR of Peginterferon treated HBeAg pos CH-B patients

Piratvisuth T et al, Hepatol Int 2011

NPV 84%

HBsAg levels week 12 HBsAg levels week 24

Neptune study confirms prediction of response at week 12 for HBsAgsl decline <1500 UI/ml

Gane E et al, J Hepatol 2011 Abst 69

0.5 Log10 IU/ml HBsAg decline at week 12 has high predictive value of SVR in Peginterferon treated HBeAg-neg patients

Moucari R, et al. Hepatology 2009; 49:1151-57

Stopping rule for in Peginterferon treatment of HBeAg-neg patients by combining on-treatment HBsAg and HBV-DNA

Rijckborst V, et al. Hepatology 2010; 52:454-61

PARC trial

Validation of topping rule for in Peginterferon treatment of HBeAg-neg patients combining HBsAg and HBV-DNA

Rijckborst V, et al. J Hepatol 2012; 56:1006-11

PreS/S mutations impact on HBsAg and cccDNA levels

Pollicino T et al, Hepatology 2012; 56:434-43

Conclusions: HBeAg-pos patients

• In HBeAg-positive CHB, no decline of HBsAg levels at 12 weeks is associated with a very low probability of subsequent anti-HBe seroconversion

• In HBeAg-positive CHB, decline of HBsAg levels below 1500 IU/ ml at 12 weeks is a strong predictor of anti-HBe seroconversion, while HBsAg levels >20,000 UI/ml are associated with a very low probability of anti-HBe seroconversion

Conclusions: HBeAg-neg patients

• In HBeAg-negative CHB, a combination of no HBsAg decline and <2 log10 IU/ml decline of HBV DNA seems to be a predictor of nonresponse in European HBeAg-negative patients with genotype D

Kinetics of HBsAg during 3 years of Telbivudine

in HBeAg-pos patients

Wursthorn K, Hepatology 2010; 52:1611-20

Prediction of treatment response to ETV by

HBsAg serum levels

Lee JM, Hepatology 2011; 53:1486-93

86.8% sensitivity 78.9% specificity

Conclusions: NUC treated patients

• A decline of HBsAg during NA treatment in HBeAg-positive patients may identify cases with subsequent HBeAg or HBsAg loss

HBsAgsl helps to distinguish Active from Inactive HBsAg Gen D carriers

Brunetto MR et al, Gastroenterology 2010;139:483-90

>

HBsAgsl helps to distinguish Active from Inactive HBsAg Gen D carriers

Brunetto MR et al, Gastroenterology 2010;139:483-90

HBsAgsl helps to distinguish Active from Inactive HBsAg Gen D carriers

Brunetto MR et al, Gastroenterology 2010;139:483-90

>

at baselineHBsAgsl: 1709 vs 62.12 IU/ML

Conclusions

Inactive carriers may be identified by single determination of serum HBsAg levels <1000 IU/ml and HBV-DNA <2000 UI/ml but such levels may occasionally be detected in CHB patients as well