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GENOMIC INSTABILITY IN HCV INFECTION : MANIFESTATIONS AND MECHANISMS

GENOMIC INSTABILITY IN HCV INFECTION : MANIFESTATIONS AND MECHANISMS O. Kalinina 1 , A. Marchio 2 , A. Dejean 2 , P. Pineau 2 1 - Laboratory of Molecular Microbiology, Saint-Petersburg Pasteur Institute, Saint Petersburg, Russian Federation

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GENOMIC INSTABILITY IN HCV INFECTION : MANIFESTATIONS AND MECHANISMS

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  1. 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

  2. Genomic instability is important in Clinics but poorly targeted by Drugs 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 100 FAL<20% w/o vasc inv. SURVIVAL w/o Recurrence (in %) FAL<20%+ vasc inv FAL20-40%w/o vasc inv. 50 8 classes w. more growth-inhibitory Activity toward cancer cell lines with complex and/or unstable Karyotypes. FAL20-40% + vasc.inv. FAL>40% w/o vasc inv. N=155 P<0.0001 FAL>40%+invasion vasc. 0 53 Compounds under investigation/ NCI60 Panel 0 1500 2500 4000 Time (in Days) Ellipticine/Olivacinium group 10 Fuchsine group 3 Cytochalasin group 3 Propenamide group 6 Benzodithiophene-dione group 6 Combretastatin group 4 Antibiotics 13 Others 8 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 Europe have shown that LOH is more useful than P53 mutation analysis to predict patients survival

  3. C025 C028 T T N N Genomic (chromosome) instability in Hepatocellular Carcinoma Prominence of the Phenomenon Loss Of Heterozygosity (LOH) at RIP_Alu_chr1_076_01, Chrom 1p31.1 T :tumor DNA, N : non tumor DNA Comparative Genomic Hybridization : TUMOR DNA : NORMAL DNA Alu+/399pb Alu-/116pb

  4. % Loss gains 60 1q 2q 40 8q 17q 20 0 18q -20 17p 16pq 8p 9p -40 6q 1p 13q 4q -60 % Allelotyping N=120 (P. Pineau) Comparative genomic Hybridization N=90 (A. Marchio) Magnitude of the Phenomenon Genomic Instability in Hepatocellular Carcinoma (HCC): Recurrent Copy Number Changes affect selected chromosomes Mean copy number changes/tumour=8-9 ARID1a VHL APC WRN DLC& PTEN RB1 TP53 SMAD4 AXIN1 CDH1 CDC4 CDKN2A-ARF

  5. Common mutation targets in European HCC Exome analysis outcome from Cécile Guichard, 2012, Nature Genetics, vol.44 n=125 Mutation rate in % ARID1 and 2 (AT-rich interacting Domain, SWI-SNF, ATPase-helicase) Chromatin remodeling TP53 Wnt pathway CTNNB1 (beta-catenin) +AXIN1 0 10 20 30 40 50 60

  6. Italy n=90 Allelic Loss in % France n=80 Romania n=48 70 * China n=52 * * * North-Africa n=42 60 * * * * 50 * * 40 30 20 10 0 1p 4q 6q 8p 9p 13q 16p 16q 17p Chromosomes Genomic Instability in HCC: Current View Sandrine Boyault, Hepatology, 2007 263 citations in Google Scholar Hepatitis B Virus: the bad guy but Aspergillus Aflatoxin B1 Chromosome Breaks : J. Lily, Nature, 1965 P.Pineau, 2008 24 citations Gs Personal View

  7. Comparative Study of HCC in European Patients according to the Etiology-I Clinico-pathological features Cirrhosis prevalence % <.0001 Tumour Diameter (cm) .009 100 .007 90 80 70 60 % 50 40 30 20 10 0 n=70 n=70 n=70 HCV HBV nonBC P=0,0001 P=0,007 P=0,01

  8. % mutation NS beta-catenin p53 20 15 10 5 0 nonBC VHC VHB Loss of Heterozygosity (LOH) in % .04 .01 40 35 30 25 20 HCV+, n=70 HBV+, n=70 15 non Viral, n=70 10 5 0 19q (BAX) 2q (NRF2) 22q (NF2) 8p (DLC1) 17p (p53) 10q (PTEN) 6q (PARK2) 4q (FBXW7) 16p (AXIN1) 13q (BRCA2) 1p (ARID1A) 16q (CADHE) 9p (CDKN2A) Comparative Study of HCC in European Patients according to the Etiology-II Genomic features Fractional Allelic loss (on 14 chromosomes) NS

  9. Comparative Study of HCC in European Patients according 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 defect HBV -> c:g>a:t : sensitivity to mutagens nonBnonC -> indel : genetic defect

  10. Influence of HCV subtypes on Genomic alterations in HCC N=130 1b non-1b P value (n=82) (n=48) Fractional allelic loss (FAL) in % Age (y. S.D.) 63.59.1 65.89.6 ns ns 45 P=0.014 Sex Ratio M:F 64:18 (3.5) 40 29:14 (2.1) ns 35 Co-infection with HBV (%) 10 (12.1%) 5 (11.6%) ns 30 25 Presence of Cirrhosis (%) 75 (91.4%) 39 (90.7%) ns 20 15 Tumor diameter (cmS.D.) 4.02.6 4.63.8 ns 10 5 Europeans 80 (97.5%) 42 (97.6%) ns 0 non-1b 1b 4 3b 1a/1b 2a/3aa/3ab non-1b 1a Loss of heterozygosity 6q (LOH) in % 1b 2a

  11. Sex and CTNNB1 status % LOH 16q in % 50 P= 0.006 45 P=0.018 F 40 35 30 P= 0.008 25 M M 20 P=0.02 15 10 5 Men Women NT liver status % 100% 90% : Cirrhosis 80% 70% 60% 100% P= 0.02 50% 90% 40% : Not Cirrhotic 80% 30% 20% 70% 10% 60% CTNNB1 mutated CTNNB1 wt 50% 100% 40% NT liver status % 90% 30% 80% 70% 20% 60% 10% 50% 40% CTNNB1 mutated CTNNB1 wt 30% 20% 10% TP53 mutated TP53 wt Genetic Analysis of Hepatitis C virus subtype 1b-associated HCC, N=82 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

  12. HCV-associated genetic Instability in liver Cancer: Conclusions –I Chromosome Alterations and point Mutations At least in the west-european context, genomic instability of HCCdoes 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 least25% 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 appears an important driver in a significant subset of HCV-associated HCC cases (t:a>c:g, >20% of cases)

  13. HCV-associated Hepatocellular Carcinoma Other forms of instability affecting: - the epigenome - the transcriptome including microtranscriptome

  14. Chr. 11 11q13 P<0.0001 11p15 P=0.01 11q12 P=0.01 Chr. 9 9q34 p<0.0001 9p13 p=0.002 Chr. 1 1q21 p<0.0001 1q24 p=0.002 1q p<0.0001, 20% of overexpressed genes Molecular Epidemiology of HCC and Functional Genomics Hierarchical Clustering 12 Asian HCC vs 43 European HCC Differential signature in HCC from Asian HBV+ patients GO:0006396 : RNA processing n=20, P=0.0009 GO:0007049 : Cell Cycle n=31, P=0.005 GO:0015031 : Protein Transport n=29, P < 0.0001 GO:0051169 : Nuclear Transport n=9, P=0.002 GO:0007275 : Development n=21, P=0.002

  15. Transcriptome analysis of HCV-associated HCC N=23 HCC vs 6 Healthy Livers Pools, Affymetrix HG-133a N Genes N Genes 30 0.02 60 0.02 0.02 0.006 0.005 0.0007 <0.0001 0.002 0.009 0.0003 <0.0001 25 78 Activated Genes P<0.01 50 20 40 15 30 MAPK signaling pathway 10 20 5 10 Transcription Factors Binding Sites 0 0 Response to biotic stimulus TNF IFNA1 IRF8- IRF7 SOX5 FOXO4 ELK1 FOXJ2 CREL CSBSP 90 0.02 0.0008 0.004 0.007 0.01 0.0002 0.006 0.0003 80 50 70 103 Repressed Genes P<0.01 40 60 50 30 40 30 20 20 10 primary metabolism 0.0002 10 0 DEAF1 CREB MAZR CREB MAZ CDX2 NKX2.5 SREBP

  16. Different transcriptome for different HCV subtypes Affymetrix Hg133a, n=23 1b 1b 2a 1a 1b 1b 1a 2a 2a 1a Hierarchical clustering n=150 differentially expressed genes

  17. Significance (Log) GO : Fatty acid metabolism SS18 (TF) E2F (TF) ELK (TF) -5 GO : Lipid metabolism NFY (TF) -4 Thrombospondin (binding) TITF1 (TF) GO: glucose catabolism PPARG (binding) -3 ANXA11 (binding) FOXO4 (TF) LEPTIN (binding) STAT3 (Lit) ESR (binding) IL6 (Lit) -2 ERRB3 (Lit) -1 Underexpressed n=54 Overexpressed n=96 Bioinformatic analysis of Differentially Expressed Genes according to HCV subtypes (1b vs non 1b) GATHER Software : Gene Annotation Tool to Help Explain Relationships

  18. DNA methylation according to HCV Genotype Methylation-specific PCR technique non-1b. n=8 1b. n=13 1 0.8 0.6 Methylation 0.4 0.2 NS NS NS NS NS NS 0.0066 NS 1p36 3p21.3 5q21 6q24 9p21 13q14 16p13 16q22.1 metRIZ1 metAPC metp16 metSOCS1 metRASSF1 metER metRB1 metE-Cadh Genes

  19. Microtranscriptome of HCC : not only miR-122 OSU-CCC_ hsa-miRNA-chip n=398 elements 90 paired HCC/NTL 31 HCC cell lines No changes According to P53 mut, FAL, Geography 40 50 200 10 miR-221 miR-222 miR-224 let-7c 1 20 25 100 0.1 0 0 0.01 0 NT T RTQPCR validation on a second subset of samples 14 microRNAs participate to Liver Cancer Progression SAM analysis

  20. Differential expression of microRNA in HCV-associated HCC OSU-CCC_ hsa-miRNA-chip n=398 elements Subtypes 1a 1b 2 3 4

  21. 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 be an 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 significantly Disease 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

  22. Bruno Turlin Abdellah E. El Feydi Acknowledgements Mikhail Generalov Dmitri Gradov CRIRR Saint Petersburg Anna-Maria Tabnase Irinel Popescu Simona Dima Traian Dumitrescu Vincenzo Mazzaferro Lun-Xiu Qin Gabriela Oprisan Soumaya Benjelloun, Sayeh Ezzikouri Benoît Terris

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