GENOMIC INSTABILITY IN HCV INFECTION : MANIFESTATIONS AND MECHANISMS
O. Kalinina1, A. Marchio2, A. Dejean2, P. Pineau2
1 - Laboratory of Molecular Microbiology,
Saint-Petersburg Pasteur Institute, Saint Petersburg, Russian Federation
2 - Unité « Organisation Nucléaire et Oncogenèse », INSERM U993, Institut Pasteur, Paris, France
Riga, Feb 11th, 2013
Genomic instability is important in Clinics but poorly targeted by Drugs
From Dvorchik et al., Liver Transpl, 2007
FAL (Fractional Allelic Loss) measures the CIN :
Cumulative proportion of Chromosomes with Loss Of heterozygosity in a given tumor
NB : Other studies performed in Japan and Europehave shown that LOH is more useful than P53 mutation analysis to predict patients
survival
FAL>40%+invasion vasc.
FAL20-40% + vasc.inv.FAL>40% w/o vasc inv.
FAL<20%+ vasc invFAL20-40% w/o vasc inv.
FAL<20% w/o vasc inv.
N=155P<0.0001
100
50
0
SURVIVAL w/o Recurrence
(in %)
Time (in Days)0 1500 2500 4000
A-« Anticancer drugs routinely used :No positive association between cytotoxic profiles and aneuploid state »
B-« Epithelial cancers (as HCC) reside at the more karyotypically complex end of the cancer spectrum » Anna Roshcke, 2005
Ellipticine/Olivacinium group 10
Fuchsine group 3
Cytochalasin group 3
Propenamide group 6
Benzodithiophene-dione group 6
Combretastatin group 4
Antibiotics 13
Others 8
53 Compounds under investigation/ NCI60 Panel
8 classesw. more growth-inhibitory
Activity toward cancer cell lines with complex and/or unstable
Karyotypes.
Genomic (chromosome) instability in Hepatocellular Carcinoma
: TUMOR DNA : NORMAL DNA
Prominence of the Phenomenon
T N T NC025 C028
Alu+/399pb
Alu-/116pb
Loss Of Heterozygosity (LOH)at RIP_Alu_chr1_076_01,
Chrom 1p31.1T :tumor DNA, N : non tumor DNA
Comparative Genomic Hybridization
Magnitude of the PhenomenonGenomic Instability in Hepatocellular Carcinoma (HCC):
Recurrent Copy Number Changes affect selected chromosomes
Comparative genomic HybridizationN=90 (A. Marchio)
-60
-40
-20
0
20
40
60Loss gains
Allelotyping N=120 (P. Pineau)
1p
4q6q 8p9p
13q
16pq
17p
18q
1q2q
8q 17q
%
%
Mean copy number changes/tumour=8-9
TP53SMAD4AXIN1
CDH1
RB1PTEN
CDKN2A-ARF
APCVHLCDC4
WRNDLC&
ARID1a
from Cécile Guichard, 2012, Nature Genetics, vol.44n=125
Common mutation targets in European HCCExome analysis outcome
0 10 20 30 40 50 60
Wnt pathwayCTNNB1 (beta-catenin)
+AXIN1
TP53
ARID1 and 2(AT-rich interacting Domain,
SWI-SNF, ATPase-helicase)Chromatin remodeling
Mutation rate in %
Genomic Instability in HCC: Current View
Sandrine Boyault, Hepatology,2007263 citations in Google Scholar
0
10
20
30
40
50
60
70
1p 4q 6q 8p 9p 13q 16p 16q 17p
Italy n=90France n=80Romania n=48China n=52North-Africa n=42
* ** ***
**
**Allelic Loss in %
Chromosomes
Aspergillus
Aflatoxin B1
ChromosomeBreaks : J. Lily,Nature, 1965
Personal ViewP.Pineau, 200824 citations Gs
Hepatitis BVirus: the bad guy
but
HCV HBV nonBC Pn=70 n=70 n=70 value
Age (yo) 59.0±8 58.9±9 59.8±8 ns(39-76) (36-76) (36-77)
Sex Ratio M:F 62:8 62:8 62:8 ns
Comparative Study of HCC in European Patientsaccording to the Etiology-I
Clinico-pathological features
P=0,007 P=0,01
P=0,0001
Tumour Diameter (cm)Cirrhosis prevalence %
0
10
20
30
40
50
60
70
80
90
100
n=70 n=70 n=70HCV HBV nonBC
%
.009<.0001
.007
Comparative Study of HCC in European Patientsaccording to the Etiology-II
Genomic features
Fractional Allelic loss (on 14 chromosomes)
0
5
10
15
20
VHC VHB nonBC
beta-catenin p53% mutation
NS
NS
Loss of Heterozygosity (LOH) in %
0
5
10
15
20
25
30
35
40
1p (A
RID1A
)
2q (N
RF2)
4q (F
BXW7)
6q (P
ARK2)
8p (D
LC1)
9p (C
DKN2A
)
10q
(PTE
N)
13q
(BRCA2)
16p
(AXIN
1)
16q
(CADHE)
17p
(p53
)
19q
(BAX)
22q
(NF2
)
HCV+, n=70HBV+, n=70non Viral, n=70
.01 .04
Comparative Study of HCC in European Patientsaccording to the Etiology-III
Mutation spectrum
HCV HBV nonBnonC
3 subsets with c:g>t:a, major mutation type in European HCC.
2nd target differs between tumor types :
HCV -> t:a>c:g : oxidative stress or base excision repair defectHBV -> c:g>a:t : sensitivity to mutagensnonBnonC -> indel : genetic defect
Influence of HCV subtypes on Genomic alterations in HCCN=130
1b non-1b P value(n=82) (n=48)
Age (y. S.D.) 63.59.1 65.89.6 ns
Sex Ratio M:F 64:18 (3.5) 29:14 (2.1) ns
Co-infection with HBV (%) 10 (12.1%) 5 (11.6%) ns
Presence of Cirrhosis (%) 75 (91.4%) 39 (90.7%) ns
Tumor diameter (cmS.D.) 4.02.6 4.63.8 ns
Europeans 80 (97.5%) 42 (97.6%) ns
1b
non-1b 1a
2a
2a/3aa/3ab
3b 4 1a/1b
ns
Fractional allelic loss (FAL) in %
05
1015202530354045
non-1b 1b
P=0.014
Loss of heterozygosity 6q (LOH) in %
Genes Nom développé ChromosomesSNORD50 small nucleolar RNA, C/D box 50A 6q14.3CCNC cyclin C 6q16FYN FYN oncogene related to SRC, FGR, YES (FYN) 6q21FRK fyn-related kinase, protects PTEN 6q22.1PDSS2 prenyl (decaprenyl) diphosphate synthase, subunit 2 6q21BCLAF BCL2-associated transcription factor 1 6q23TNFAIP3 tumor necrosis factor, alpha-induced protein 3 6q23PLAGL1 pleiomorphic adenoma gene-like 1 6q24.2LATS1 large tumor suppressor, homolog 1 6q25.1IGF2R insulin-like growth factor 2 receptor 6q25.3
Genetic Analysis of Hepatitis C virus subtype 1b-associated HCC, N=82
P=0.02
5101520253035404550
Men Women
LOH 16q in %
P=0.018
10%20%30%40%50%60%70%80%90%100%
CTNNB1 mutated
CTNNB1 wt
Sex and CTNNB1 status %
M M
FP=
0.006
NT liver status %
10%20%30%40%50%60%70%80%90%100%
CTNNB1 mutated
CTNNB1 wt
: Cirrhosis
: Not Cirrhotic
TP53 mutated
TP53 wt
10%20%30%40%50%60%70%80%90%100%
NT liver status %
P=0.008
P=0.02
P=0.01
HCC and HCV 1b, 2 major subsets identified:
A-Males, almost always cirrhotic w. Large tumors, CTNNB1 mutated
B- TP53 mutated, Genomically instable, Significantly associated with a non-cirrhotic liver
At least in the west-european context, genomic instability of HCC does not vary drastically according to Grossly defined major risk factors (viruses, alcohol, dysmetabolic conditions).
HCV-associated HCCs do not display, therefore, a particularly strong genomic instability.
However, the mean value of the FAL (proportion of altered chromosomes) in the 4th quartile is quite high : 50% (meaning that at least 25% of the HCV-associated HCC are genomically instable).
The cause(s) of instability in these latter samples is/are unknown (lifestyle or genetic cofactors, viral subtypes, viral variants/quasispecies ?).
With regards to the mutagenic process, oxidative stress appearsan important driver in a significant subset of HCV-associated HCC cases (t:a>c:g, >20% of cases)
HCV-associated genetic Instability in liver Cancer:Conclusions –I
Chromosome Alterations and point Mutations
HCV-associated Hepatocellular Carcinoma
Other forms of instability affecting:
- the epigenome
- the transcriptome including microtranscriptome
Molecular Epidemiology of HCC and Functional Genomics
Hierarchical Clustering 12 Asian HCC vs 43 European HCC
Chr. 11q21 p<0.00011q24 p=0.0021q p<0.0001,
20% of overexpressed
genes
Chr. 99q34 p<0.00019p13 p=0.002
Chr. 1111q13 P<0.0001
11p15 P=0.0111q12 P=0.01
GO:0015031 : Protein Transport
n=29, P < 0.0001
GO:0051169 :Nuclear Transport
n=9, P=0.002
GO:0006396 : RNA processing n=20, P=0.0009
GO:0007049 : Cell Cycle
n=31, P=0.005
GO:0007275 : Development n=21, P=0.002
Differential signature in HCC from Asian HBV+ patients
Transcriptome analysis of HCV-associated HCCN=23 HCC vs 6 Healthy Livers Pools, Affymetrix HG-133a
0
5
10
15
20
25
30
0.002 0.009 0.0003<0.0001
IFNA1 TNF
MAPKsignalingpathway
Responseto bioticstimulus
N Genes
0
10
20
30
40
50
60
<0
.00
01
0.0
00
7
0.0
05
0.0
06
0.0
2
0.0
2
0.0
2
IRF8-CSBSP
IRF7 SOX5 FOXO4 ELK1 FOXJ2 CREL
N Genes
78 Activated
GenesP<0.01
103 Repressed
GenesP<0.01
0
10
20
30
40
50
60
70
80
90
0.00020.0008
0.00030.004
0.0060.007
0.01
DEAF1CDX2
CREBNKX2.5
MAZRMAZ
CREBSREBP
0.02
10
20
30
40
50
primary metabolism 0.0002
TranscriptionFactorsBinding
Sites
1b1b1b1b 1a1a1a 2a 2a 2a
Different transcriptome for different HCV subtypesAffymetrix Hg133a, n=23
Hierarchical clusteringn=150 differentially expressed genes
E2F(TF)
ELK(TF)
NFY(TF)
-1
-2
-3
-4
-5
Significance (Log)
Underexpressedn=54
Overexpressedn=96
GO : Fatty acid metabolism
GO : Lipid metabolism
Thrombospondin(binding)
ESR(binding)
PPARG(binding)
LEPTIN(binding)
STAT3(Lit)
ERRB3(Lit)
IL6(Lit)
TITF1(TF)
FOXO4(TF)
SS18(TF)
GO: glucosecatabolism
ANXA11(binding)
Bioinformatic analysis of Differentially ExpressedGenes according to HCV subtypes (1b vs non 1b)
GATHER Software :Gene Annotation Tool to Help Explain Relationships
DNA methylation according to HCV GenotypeMethylation-specific PCR technique
0.2
0.4
0.6
0.8
1
NS NS NS NS NS NS 0.0066 NS1p36 3p21.3 5q21 6q24 9p21 13q14 16p13 16q22.1
metRIZ1metRASSF1
metAPCmetER
metp16
metRB1metSOCS1
metE-Cadh
Genes
Meth
yla
tion
non-1b. n=8
1b. n=13
Microtranscriptome of HCC : not only miR-122
14 microRNAs participate to Liver Cancer Progression
SAM analysis
miR-222
0
25
50
NT T
miR-221
0
20
40
miR-224
0
100
200let-7c
0.01
0.1
1
10
RTQPCR validation on a second subset of samples
OSU-CCC_hsa-miRNA-chipn=398 elements
90 paired HCC/NTL31 HCC cell lines
No changesAccording toP53 mut,FAL, Geography
Differential expression of microRNA in HCV-associated HCC
1a1b234
Subtypes
OSU-CCC_hsa-miRNA-chipn=398 elements
Conclusions
-genomic instability: not an universal phenomenon during the course of HCV-triggered liver tumorigenesis
-the causes of its occurrence are unknown
-epigenomic and transcriptomic changes represent potentially more rewarding tracks to be followed than genomic alterations studies
-looking for viral (subtypes, variants/quasispecies) or hosts (IL28, etc…) Specificities capable to interact and modulate instability may bean option for future investigations
-given the well known low HCV genome copy number in liver tumors,an integrative approach of initial events taking place in preneoplastic foci may appears as reasonable
-assessement of tumor microenvironment known to modulate significantlyDisease outcome may provide explanation to instability
-more evidence from liver tissues and tumors (especially in Europeans) are warranted before production of a reasonably plausible model
Acknowledgements
Vincenzo MazzaferroLun-Xiu Qin
Benoît TerrisBruno Turlin
Soumaya Benjelloun,Sayeh Ezzikouri
Abdellah E. El Feydi
Gabriela Oprisan
Anna-Maria TabnaseIrinel PopescuSimona DimaTraian Dumitrescu
Mikhail GeneralovDmitri Gradov
CRIRR Saint Petersburg