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