Molecular pathogenesis of virus-related
hepatocellular carcinomas (HCCs)
Massimo Levrero
Department of Internal Medicine (DMISM) - Sapienza University – Rome
IIT – Sapienza Center for Life-Nanosciences
INSERM U1052 - CRCL - Lyon
Dipartimento di
medicina interna e specialitA’ mediche
Disclosures
Massimo Levrero
Advisory Committees or Review Panels:
- BMS
- Jansen
- Gilead
- Tekmira
- Galapagos
- Assembly Pharma
Speaking and Teaching:
- MSD
- Roche
- BMS
- Jansen
- Gilead
Outline
1. introduction to HCCs: clinical and biological heterogeneity
i.e. are HBV/HCV-related HCCs different
common vs etiology specific alterations
2. viruses, inflammation, disease progression and cancer
i.e. HBV/HCV “net” contribution to HCC development
3. HBx, chromatin, miRNAs and lncRNAs
Geographical heterogeneity
in hepatocellular carcinoma distribution
Hepatocellular carcinoma is the second leading
cause of death among cancer patients worldwide
Globocan, 2012
HCC biological and clinical heterogeneity
• HCC nodules ”doubling time” varies from ~3.5 to 20 months
• The range of survival reported for patients at BCLC B (from 45
months to 11 months) and C (from 11 months to 5 months) is quite
large.
• Similar considerations apply when the response to a given
treatment is analysed.
Hepatocarcinogenesis is a multistep process
Risk Factors
Pre-neoplastic Steps
Tumors
Inflammation > necrosis > regeneration
Oxydative stress > DDR > Senescence
Viruses
Toxic exposure
Obesity
Chronic
hepatitis
Cirrhosis
HCC
Genetic diseases
Monoclonality
Telomerase activation
Epigenetic changes
Gene Mutations
-catenin, TP53
(AXIN1, RB1, PTEN, PIK3CA, HNF1a,
SMAD2 and 4, Ras, IRF2, ARID1, ARID2)
Chromosomal
alterations
miRNAs
DNA: global hypomethylation, TSG hypermetghylation
histone modifications
Gene expression (mRNAs)
•
•
cirrhosis as independent factor of progression to HCC but not required
role of inflammation and inflammatory cytokines (low level metabolic inflammation)
Major pathways commonly altered in HCCs
(somatic mutations or homozygous gene deletions)
TP53
pathway
more
frequently altered in HBV
infected HCCs.
IRF2 inactivation only in
HBV-HCC. IRF2 and TP53
mutations mutually exclusive
in tumors
TP53 mutations associated
with (tumor related death) in
HBV-HCC but not in non
HBV-related HCC
HBV
HCV
NRF2 a key transcription
factor involved in oxidative
stress response, was only
mutated in non-HBV HCC
CTNNB1 activating
mutations less frequent in
HBV- compared to non-HBV
ALCOHOL
• exome sequencing (24): gray
• validation series (125):
red(activated);
blue (inactivated)
Guichard C et al., Nature Genetics, 44, 694-698, 2012
Molecular classification of HCC (ILCA 2013)
- Wnt subclass
- proliferation class or G3
[with two subclasses: S1-TGFbeta and S2-EpCAM positive]
- inflammation class
[Boyault, 2007; Yamashita, 2008; Hoshida,
2008; Chiang, 2008].
The Putative Role of Liver Stem Cells in
Hepatocellular Carcinoma
Cancer stem cells constitute a
reservoir of self-sustaining cells with
the exclusive ability to self-renew
and maintain the tumor”
CK19 expression as a diagnostic and prognostic marker
Mechanisms of liver carcinogenesis
Dual role of telomeres and telomerase
in liver carcinogenesis
Farazi and DePinho Nature Reviews Cancer 6, 674–687, 2006
Plentz et al, Hepatology, 2007
Dual role of telomeres and telomerase
in liver carcinogenesis
TERT promoter mutations are the most frequent
genetic alterations in HCC
less frequent In HBV-related HCC
Nault et al. Nature Communications 2013
Frequent HBV insertion
in TERT promoter activates telomerase
Ding et al, Plos Genet, 2012
TERT promoter mutation in hepatocarcinogenesis:
early or late event?
Outline
1. introduction to HCCs: clinical and biological heterogeneity
i.e. are HBV/HCV-related HCCs different
common vs etiology specific alterations
2. viruses, inflammation, disease progression and cancer
i.e. HBV/HCV “net” contribution to HCC development
3. HBx, miRNAs and lncRNAs
Direct and indirect roles of hepatitis viruses
HBV
(overt or occult)
Integration of HBV DNA
Into host chromosomes :
Insertional mutagenesis
of cellular genes
Genetic
instability
Prolonged
expression
of viral genes
HBx, LHBs, HBc
HCV
Host immune
responses
Inflammation
Oxydative stress
Modifications of
the epigenome
AP1, NFkB
-catenin
Cell proliferation
Apoptosis
Prolonged
expression
of viral genes
Core, NS3, NS5a
Genetic
instability
NFkB, Lymphotoxins and HCC development
Cytokine
receptors
HBx (HBV) and NS5a (HCV) activate NFkB signaling by
multiple mechanisms
Lymphotoxin (LT) a and  and their receptor (LTR) are
upregulated in HBV- or HCV-induced hepatitis and HCC.
Oxidative stress
ER-stress
cytoplasm
IKKg (NEMO)
IKK1
IKK2
P
IkB
p50
p65
P
nucleus
Transcription of PROINFLAMMATORY
and ANTIAPOPTOTIC genes
modified from F Marra, Gut 2008;57:570
Haybaeck et al, Cancer Cell 16, 296, 2009
March 2013 | Volume 9 | Issue 3
- initiation of the inflammatory processes triggered by HCV viral proteins and their possible
link with HCV-related HCC
- upregulation of the lymphotoxin signaling pathway and LT-b in FL-N/35 HCV-TG mice HCCs
- Lymphotoxin expression is accompanied by activation of NF-kB
- IKKb inactivation in FL-N/35 mice drastically reduces tumor incidence
- NS5B, the HCV RNA dependent RNA polymerase drives LT-b activation
2012 vol. 57 j 1021–1028
Expression of HCV–NS5A in primary hepatic precursors and in immortalized hepatocyte cell lines alters cell
polarity and leads to epithelial to mesenchymal transition (EMT). NS5A by activating Twist2, a transcriptional
regulator of EMT. NS5A effects of NS5A were additive to TGF-b. NS5A cooperates with oncogenic Ras to give
rise to transformed, invasive cells that are highly tumorigenic in vivo.
Journal of Hepatology 2013 vol. 59 j 1160–1168
- In vivo model of HCV core expressing transgenic mice.
- 50% of genes deregulated by HCV core are TGF- targets [shifts of TGF- responses from tumor
suppression to Epithelial-Mesenchymal Transition (EMT)]
- Active TGF- increased in HCV core TG mice livers
- Smad2/3 phosphorylation is induced in JFH-1 cells
- TGF-b dependent activation of HSCs by HCV core expressing cells (co-colture or CMs)
Direct and indirect roles of hepatitis viruses
HBV
(overt or occult)
Integration of HBV DNA
Into host chromosomes :
Insertional mutagenesis
of cellular genes
Genetic
instability
Prolonged
expression
of viral genes
HBx, LHBs, HBc
HCV
Host immune
responses
Inflammation
Oxydative stress
Modifications of
the epigenome
AP1, NFkB
-catenin
Cell proliferation
Apoptosis
Prolonged
expression
of viral genes
Core, NS3, NS5a
Genetic
instability
HBc protein / capsid
wt
moc
k
 HBc binds the cccDNA and modifies cccDNA nucleosome spacing
Input
aHBc
IgG
HBV capsid
(120 HBc dimers)
Arbitrary
Units
20
1
15
05
aHBc
HBc dimer
Bock, 2001
 HBc binds to cellular promoters and
regulates gene expression
(Guo, BMC genomics, 2013)
6
4
2
0
6
4
2
0
c-Src
FI % Input
ChIP anti-HBc
FI % Input
(Durantel D, AASLD 2013)
6
4
2
0
Ezh2
6
4
2
0
IL29
FI % Input
 HBc binds to (and represses) the IFN-b,
IL-29 and OAS1 cellular promoters
Belloni 2009
6
4
2
0
IL6
E2F2
HBx protein
 HBx binds to and is required for cccDNA transcription and viral replication
 HBx binds to cellular promoters and modulates the epigenome by
relocating chromatin regulators
 HBx contributes to hepato-carcinogenesis
HBx protein
VDAC3
mitochondria
Ras
NF-kBI-kB
Ca++
MAPK
AP-1
Cytochrome C
Caspases
Apoptosis
TF
NF-kB
DDB1
Ac Ac
- Prevents silencing of cccDNA by
HDAC1 and PMRT1
cccDNA
proteasome
HMT
HBx
(Belloni 2009, Benhenda 2013)
- Relocates CBP and DNMT3 on
cellular genes
Me
Me Me
Ac
(Cougot, 2007, Zhang, 2009)
Protein stability
DNA repair
TFs activation
(Belloni 2009; Lucifora 2011)
Src
TF
Ac
- Required for cccDNA transcription
NF-AT
HBx
Epigenetics
Regulation of gene expression
Viral replication
HBx ChIP and ChIP-Seq assays
HepG2 cells
HBV
transient transfection of
linear full-length HBV monomers
HBxHBX
Pollicino Gastroenterology 2006
Belloni, PNAS 2009
ChIPed DNA
ChIP-Seq
Real-Time PCR
with gene specific
primers
 ChIP-Seq combines chromatin immuno-precipitation
(ChIP) with massive parallel DNA sequencing.
 ChIP-Seq can be used to:
- precisely map global binding sites for any protein of
interest
- to obtain a genome wide assessment of the histone
code.
Guerrieri et al. 2015 (submitted)
Guerrieri et al. 2015 (submitted)
HBx ChIP-Seq assays
Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources
Sofware DAVID (Wei Huang, Brad T Sherman & Richard A Lempick - Nature Protocols 2009)
Gene Ontology
Pathways
Wnt signaling pathway
regulation of Ras protein…
ABC transporters
calcium ion transport
MAPK signaling pathway
activation of protein kinase…
Calcium signaling pathway
extracellular structure…
Endocytosis
transmembrane receptor…
ErbB signaling pathway
regulation of small GTPase…
Pathways in cancer
phosphorus metabolic…
Signaling by Rho GTPases
intracellular signaling cascade
Signalling by NGF
protein amino acid…
0
Phosphatidylinositol signaling…
100 200 300 400 500 600
Focal adhesion
0
50
100
150
q-value<= 0.05 (after multiple hypothesis testing correction with Benjamini-Hochberg)
•
Enriched genes
cell metabolism and cell signaling
• Ras, GTPase
• calcium transport
Enriched pathways
Cancer related
MAPK and Wnt signaling
200
HBx ChIP-Seq
HBx and the RABs family: a role in endocytosis?
a)
MOCK
b) ChIP anti-HBx
15
HBV WT
Fold Induction
10
5
t
Rab2B
Rab5B
8
8
8
4
4
4
0
0
0
x
nGFP
HBx is recruited in vivo on the
promoters of RABs family members;
HBx (HBV replication) increases Rab
family members mRNA levels
and modulates endocytosis
ChIP anti-ACH4
Fold Induction
HB
c)
Rab1A
Rab2B
3
3
2
2
1
1
0
0
d)
mRNA levels
Tfn-594
Hoechst
m
V
V
HB
HBV mt HBx
HB
Ct
l
0
Rab1A
Rab2B
Rab1A
2
*
Rab5B
5
4
3
2
1
0
2
*
Rab5B
2
1
1
1
0
0
0
HBx ChIP-Seq
HBx deregulates direct target miRNA
liver
26b, 129.1, 138.2, 452,
liver cancer
21, 26a, 224, 378g, 495, 543, 555, 576, 596, 663a, 3692, 3914.1,
133a.2, 548a.2, 548p, 552, 626, 639, 640, 943, 944, 1539, 3139, 3197,
3617, 3622b, 3648, 3650, 3657, 3667, 3687, 4429, 4438, 4442, 4446,
4448, 4476, 4488, 4501, 4662b, 4664, 4666b, 4681, 4682, 4698, 4703,
4710, 4740, 4770, 5006, 5095, 5193, 5698
302, 551b, 584, 663b, 1244.2, 1913, 1973, 3170, 3909, 4276, 4286, 4309,
4317, 4321, 4425, 5588
cancer
na
Multiple TF mediate HBx
binding to chromatin
HBx bound
promoters
ChIP-Seq
Validated
ChIP
Validated
TF binding
sites
MIR552
X
X
CREB, NFKB, IRFF
MIR138-2
X
X
CREB, HNF4
MIR302-e
X
X
CREB, IRF, HNF
MIR129-1
X
X
AP1, HNF1
MIR26b
X
X
NFkB, E2F
MIR21
X
X
WNT
MIR224
X
X
NFKB
miRNA
Literature
21
Plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular
carcinoma.
26a-2
Down-regulation of microRNA-26a promotes mouse hepatocyte proliferation
during liver regeneration. MicroRNA-26a promotes cholangiocarcinoma growth
by activating β-catenin.
26b
MicroRNA expression, survival, and response to interferon in liver cancer.
129-1
A functional screening identifies five micrornas controlling glypican-3: role of
mir-1271 down-regulation in hepatocellular carcinoma.
138-2
MiR-138 induces cell cycle arrest by targeting cyclin D3 in hepatocellular
carcinoma
224
Transcriptional regulation of miR-224 upregulated in human HCCs by NFκB
inflammatory pathwaysJ
551b
[Effect of microRNA on proliferation caused by mutant HBx in human
hepatocytes].
570
Analysis of differentially expressed genes and microRNAs in alcoholic liver
disease.
1.5
1.0
log2
0.5
0.0
-0.5
-1.0
-1.5
Guerrieri et al. 2015 (submitted)
HBx recruitment results in miR-224, miR-138 and miR-596
promoter repression
HepG2 cells
ChIP anti-DNMT3A
ChIP anti-ACH4
Factor occupancy
20
15
10
5
0
1 HBV2wt
Mock
MIR 224 promoter
ChIP anti-DNMT3A
ChIP anti-ACH4
80
1,5
60
1
40
0,5
20
0
1
Mock
HBV2wt
0
1
Mock
HBV2 wt
ChIP anti-DNMT3A
4
1,5
3
1
2
0,5
1
0
1
Mock
HBV2 wt
0
1 HBV2wt
Mock
1
Mock
HBV2wt
 HBx binding is accompanied by the recruitment of the DNMT3a methyltransferase and
reduced H4 histone acetylation
MIR 138-2
MIR 224
ChIP anti-p65
Factor occupancy
Factor occupancy
ChIP anti-ACH4
1,2
1
0,8
0,6
0,4
0,2
0
MIR 596 promoter
Factor occupancy
MIR 138-2 promoter
2
ChIP anti-p65
miR-224
miR-138.2
miR-596
2
1,5
1
1
0,5
0
HBx
P65
nfkb
TF
p50
miR-Promoter
HBV1 wt
0
HBV1wt
Guerrieri et al. 2015 (submitted)
Guerrieri et al. 2015 (submitted)
miR-224 is transcriptionally regulated
by NFkB inflammatory pathways
MIR224
GABRE
Relative luciferase
activity
a-p65
IgG
INPUT
12
 Exogenously expressed p65/RelA activates
the miR-224 promoter
 p65/NFkB is recruited in vivo on the miR-224
promoter and its binding increases after
exposure to LPS, TNFa and Lta
 The
inflammatory
stimuli
potentiate
endogenous miR-224 expression
8
4
0
p65
p50
cat
p53
- +
- - - -
+
-
+
+
-
+
-
+
6
14
12
10
8
6
4
2
0
TNF a TNF  LPS -
2
1.5
1
0.5
0
+
-
+
-
+
0 2 16 24 48 h
TNFa
0 2 16 24 48 h
TNF
0 2 16 24 48 h
LPS
miR-224 expression
miR-224 expression
p65 occupancy
2.5
1.2
1
0.8
0.6
0.4
0.2
0
0 2 16 24 48 h
BMS
Scisciani C et al. J Hepatol 2012
miR-224 levels affect HCC cells migration.
ARHGAP
(ARHGAP9
ARHGAP21)
miR-224
ARHGDI
ARHGEF
(FAK)
GTP
GDP
RHO
RAC
CDC42
RHO
RAC
CDC42
INACTIVE
ACTIVE
MIGRATION
AND INVASION
- A miR224 specific antagomir blocks LPS- and LTa
stimulated HepG2 (and Heo3B) cells migration.
- The IKK inhibitor BMS-345541 blocks pre-miR224
induced cellular migration.
How to reconcile these results with the original observation that miR224
expression in increased in HCC ?
Scisciani C et al. J Hepatol 2012
How to reconcile these results with the
original observation that miR224 expression
is increased in HCC ?
A model for miR224 expression
in HBV infection and HBV-related HCCs
cccDNA
cccDNA
pgRNA
HBx
HBx
TNFα
LTβ
TNFα
LTβ
p65 p50
p65 p50
miR-224
miR-224 Pr
High Replication
in acute hepatitis and the
early chronic phase HBx
contributes to high viral
replication both by targeting
the cccDNA (Belloni, 2009), to
increase pgRNA production,
and by repressing miR224
expression to shield pgRNA
and relieves the negative
effects of miR-224 on HBV
replication.
pgRNA
HBeAg +
miR-224
miR-224 Pr
Low Replication
HCC (?)
anti-HBe Ab +
1000
100
10
1
0,1
0,01
0,001
DNA
in late phases of infection
and HCC HBV replication is
often
low
and
HBx
disrupted (Amadeo, 2014)
If the HBx regulatory is lost
TNF/LT-induced NFkB is
free to activate miR-224
that acts both on viral
pgRNA and cellular targets.
tolerance chronic hepatitis inactive carrier pre-core mt occult HBV
• Our results are compatible with the recent observation that miR224 are low in acute HBV
infection as compared to chronic infection and HCC (Zhang, 2011)
Scisciani C et al. J Hepatol 2012
HBV (HBx)
HCC
MIR21
HBV
HCV
HBV
METABOLIC GENES
OLEATE
P-STAT3
S3I
Anti-STAT3
Steatosis
HCC
IL6
?
P-STAT3
MIR21
AMPK
Genetic and Dietary Obesity Promote
DEN-Induced Hepatocarcinogenesis
Enhanced IL-6 Production Is Required
for Obesity-Induced Tumor Promotion
Park et al., Cell 140, 197, 2010
Metformin reverts oleate-induced deregulation of selected
miRNAs in HepaRG cells exposed to lipid overload
b)
Metformin
a)
5000
ctrl
MFI Bodipy
4000
-
+
+
-
Oleate
3000
Oleate
2000
Oleate+Metformin
STAT3
Metformin
Actin
P-STAT3
1000
0
d)
e)
miR21 promoter
Relative luciferase activity
c)
1,2
1
0,8
0,6
0,4
0,2
0
1
UNT
2
Oleate
Oleate
3
+ Met
miR21 promoter
HBV
HCV
HBV
METABOLIC GENES
OLEATE
P-STAT3
S3I
Steatosis
HCC
Anti-STAT3
IL6
?
P-STAT3
MIR21
?
?
METFORMIN
AMPK
Gut 2013;62:606–615
-
97 430 HCC patients and 194 860 age-, gender- and physician visit date-matched controls.
OR of diabetes in HCC patients was 2.29 (p<0.001).
each incremental year in metformin reduced by 7% HCC risk (p<0.0001).
metformin reduced risk of HCC in all subgroup of diabetic patients in multivariate analysis
HBx is recruited in vivo several LncRNAs
Long non coding RNAs
Long intergenic non coding RNAs
C1orf126, CASC2, CHODLAS1, DGKK, DLEU2,
GPHB5, HCG18, KIAA1456, MIR210HG,
MIR31HG, MIR4321, MIR4500HG, PIK3R6,
PLK1S1, PVT1, RBM26-AS1, SKINTL, SNHG12,
SOX2-OT, SRD5A2, TMEM72-AS1, ZNF518A
LINC00271, LINC00277, LINC00299,
LINC00303, LINC00305, LINC00330,
LINC00340, LINC00441, LINC00473,
LINC00476, LINC00486, LINC00487,
LINC00511, LINC00526, LINC00535,
LINC00536
ChIP anti-HBx
LINC00277 promoter
0.04
DLEU2 promoter
6
4
2
0
0.00
Mock
HBV WT
Mock
HBV WT
HBx binds DLEU2 promoter and modifies its
expression profile
DLEU2 promoter
ChIP anti-HBx
ChIP anti-ACH4
6
3
4
2
2
1
0
0
Mock
HBV WT
DLEU2 splicing profile
ctl
HBV
HepG2 cells
Exon 2-7
18S
Exon 2-7
AD38 cells
18S
Mock HBV WT HBV X-mt
ctl
HepG2 cells
HBV
Exon 9-11
18S
HBx occupancy results in a different DLEU2
splicing profile
AD38 cells
Exon 9-11
18S
HBx-mediated co-regulation of TRIM13 expression
TRIM13 promoter
ChIP anti-HBx
ChIP anti-ACH4
TRIM13 mRNA
4
3
TRIM13 protein
1.5
2
2
1
1
0.5
0
0
0.0
Mock HBV WT HBV X-mt
TRIM13
1.0
Mock
HBV WT
ACTIN
Mock
HBV WT
DLEU2 and the antisense gene TRIM13 are co-regulated by HBx.
Importantly, HBx does not bind the TRIM13 promoter in ChIP experiments.
HBx binds DLEU2 lncRNA
?
HBx
HBx H3K4 met
DLEU2
DLEU2 RNA
TRIM13
Fatica & Bozzoni. Nature reviews
2014
RIP ANTI-HBx
ctl
HBV
RIP ANTI-HBx
DLEU2 primers (exon 2)
5
4
3
2
1
0
Mock NoA HBV
Primers DLEU2
In silico analysis indicates that DLEU2 RNA potentially binds HBx. Using a RIP (RNA
Immuno Precipitation) approach we confirmed HBx-DLEU2 interaction.
Conclusions
HBx is recruited to several genomic loci to modulate the
epigenetic control of genes and ncRNAs transcription
HBx binds to 208 miRNAs [75 putative miRNA promoters and 133
mirtrons]. Functional analysis shows that HBx can both
upregulate and repress the expression of miRNAs involved both in
HBV replication (i.e, miR224) and the control of cellular functions
(i.e. miR21, miR26b).
HBx binds to 39 lncRNAs promoters. HBx binding to the DLEU2
promoter region affects its epigenetic status and expression by
inducing a different DLEU2 splicing profile. HBx also directly
binds DLEU2 and affects TRIM13 expression
IL6/STAT3 signaling and miR-21 At the cross-road of HBV, HCV
and metabolic HCCs
To translate molecular knowledge in clinical
practive, we need new biomarkers
TERT promoter mutation in hepatocarcinogenesis:
translation in clinical practice?
Nault, Calderaro et al, Hepatology 2014
2012 vol. 56 j 167–175
•
•
•
•
•
Plasma miR-21 levels significantly diminished after surgery (p = 0.0125).
Plasma miR-21 level significantly higher in HCC vs CH vs HC (p <0.0001, p <0.0001)
ROC analysis AUC of 0.773 with 61.1% sensitivity and 83.3% specificity (HCC VS CH)
AUC of 0.953 with 87.3% sensitivity and 92.0% specificity (HCC VS HC)
Both values superior to a-fetoprotein and improved for the combination.
Molecular classification of HCC:
prognostic signatures
•
Several biomarkers (mir 26, AFP, Ang2) or gene signatures from tumour (5-gene, EpCAM) or
adjacent tissue (poor prognosis signature) have been reported as predictors of survival.
HN1, RAN, RAMP3, KRT19, TAF9
Nault JC, Gastroenterology 2013, 145, 176.
Collaborations:
University Medical Hospital Hamburg
Jorg Petersen
Maura Dandri
Laboratory of Gene Expression
Dept. of Internal Medicine - University of Messina
Giovanni Raimondo
Teresa Pollicino
Massimo Levrero
Gianna Aurora Palumbo
Laura Belloni
TUM - Helmholtz Zentrum München
Ulrike Protzer
Julie Lucifora
Natalia Pediconi
Francesca Guerrieri
Ludovica Calvo
Debora Salerno
Silvia Di Cocco
Leonardo Lupacchini
Safaa Jeddari
INSERM U761 -Lyon
Fabien Zoulim
Barbara Testoni
Dept of Molecular Virology – Heidelberg Univ Hospital
Stephan Urban
Jessika Sonnabend
Dept of Molecular & Cellular Biochemistry
Indiana University
Adam Zlotnick
Collaborations at Sapienza:
Antonello Mai
Daniela Secci
Dante Rotili
Sergio Valente
Anna Tramontano
Loredana Lepera
Daniel D’Andrea
Assembly Biosciences
Uri Lopatin
With the support of
Fondazione
Andrea
Cesalpino
Hoshida et al. J Hepatol 2014
Reversibility of liver cirrhosis: physiopathology
Baseline biopsy : F4
6 Years Post-Tx biopsy: F1
3 years of antiviral treatment
Liver cell regeneration
Remodelling of
portal tract
Thinning of fibrous septa
1. Thinning of fibrous septa: enzymatic degradation of fibrous septa; early or «young»
cirrhosis.
2. Remodeling of portal tracts: disappearance of fibrous septa is associated with vanishing
of shunting neovessels; reversal to a normal blood inflow through residual portal veins and
arterial vessels; - absence of extensive vascular thrombosis
3. Hepatocyte regeneration: decreasing regenerative capacity in advanced cirrhosis
s1topping necroinflammation - Virus eradication or supression
HCC rates in NUC (ETV, TDF) treated CHB patients
Papatheodoridis G, Lampertico P AASLD 2013
Are all HBVs oncogenic ?
i.e. the role of HBV genotypes, naturally occurring and
drug-selected mutations
Mutations affecting the HBV preS/S region selected
under the pressure of host immunity,
immunoprophylaxis, and antiviral therapy
Pollicino T et al J Hepatol, 2014
Distribution of preS2defective variants in
HBsAg+ patients (pts)
Prevalence of pre-S mutants
in patients infected with
genotype B, C, or mixed
genotypes
Raimondo et al, J Hepatol 2004
Chen F-B et al, Gastroenterology 2006
Diagnosis
N° pts
N° pS2 variants
Inactive Carriers°
15
2
Chronic hepatitis°*
50
25
Cirrhosis°*
26
13
HCC°*
19
16
°p=0.002
*p=0.02
All pts infected with genotype D
cc : chronic (inactive) carriers
CH : chronic hepatitis
LC : liver cirrhosis
Deletion
Wild-type + deletion
Wild-type
Comparison of cumulative incidence of cirrhosis
between patients with and without preS mutants
Chen Gastroenterology 2007
Cancer Sci. 2006 Aug;97(8):683-8.
Schematic representation of the proposed models for
hepato-carcinogenesis associated with preS/S mutant proteins
Pollicino T et al J Hepatol, 2014
HBV DNA Polymerase-Hepatitis B Surface
Antigen Link
entecavir
telbivudine
adefovir
lamivudine
tenofovir
X
Terminal
Protein
1
Spacer
183
GFA
X XX X
BCDE
349 (rt)
PreS1 PreS2
RNaseH
692 (rt 344)
S
X
The HBV surface gene
overlaps completely with
the
polymerase
gene;
hence
NA-selected
changes in the polymerase
gene
can
affect
the
overlapping surface gene.
Adapted from Locarnini S, et al. Hepatol Int 2008:2;147–154.
X X
845 a.a
The rtA181T / sW172* mutant has a dominant
negative secretion defect
intracellular
WT
extracellular
rtA181T
Impact of polymerase mutations on the overlapping Surface gene
a) Impact on virus infectivity and fitness
b) Impact on virion release (intracellular retention) and virologic monitoring of breakthrough
c) Impact on vaccine prophylaxis efficacy
Warner et al, Hepatology 2008
2011
A181T correlates with the onset of HCC in HBV infected humans
Factors associated with occurrence of HCC. The cumulative incidence of HCC was depicted
according to the presence of the rtA181T mutation (A), use of rescue therapy (B), the presence of liver
cirrhosis (C), and age > 50 years (D).