Cancers due to 5 infections correspond to 18.6% of total cancer incidence
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1 Cancers due to 5 infections correspond to 18.6% of total cancer incidence 25% of cancers of the oral cavity (HPV) HPV 27.9% EBV 10.3% Helicobacter pylori 37.0% Cancer of the cervix (HPV) Hepatocellular carcinoma 80% (HBV, HCV) HBV +HCV 24.8% This graph ignores anal and perianal cancers (HPV) vulvar, vaginal and penile cancers (HPV) adult T cell leukemia Kaposi s sarcomas and pleural. effusion lymphomas Merkel cell carcinomas cancers linked to parasitic infections Gastric cancer 80% (Helicobacter pylori) Gastric cancer 10% (EBV) Nasopharyngeal carcinoma (EBV) Non-Hodgkin s lymphoma 10% (EBV) Hodgkin s lymphoma 30% (EBV)
2 Infection and cancer 2000,world estimated 17.6% (1.9m) new cancer caused by (Hepatitis B and C viruses, the HPV, EBV, HTLV-I, HIV, the bacterium Helicobacter pylori, schistosomes or liver flukes) The percentage of cancer case caused by main 3 infection factors among total cancer:h. pylori (5.6%); HPV (5.1%);hepatitis viruses (4.9%) Prevention of infection can reduce new cancer cases in developing countries 26.1% (1.5m) and developed countries 7.5% (380,000) Parkin: May 2, 2005, Vancouver
3 HBV HCV EBV Virus Human cancer HPV KSHV HHV-8 HTLV
4 Epidemiology Public health impact Which markers? Pathogenesis: Inflammation Direct vs indirect Effects Impact of genetic variability Virus Human Cancer Viral persistence as a risk factor for human Cancers Impact of genetic variability Interactions Virus-Host Immune response Genetic, Epigenetic Environment
5 Mortality Rates Categorized by Age-Adjusted Mortality Rates Fifth cancer world-wide, Third cause of cancer mortality El Seragh Gastro 07
6 El Seragh Gastro 07 Trands In US Cancer Mortality Rates
7 HBV (DNA virus) HCV (RNA virus) 90% HCCs develop on cirrhotic livers (60% in Africa) H C C Alcohol Iron overload Chemical carcinogens Aflatoxin B1 Hormonal factors Genetic predisposition? Metabolic disorders: Steatosis Diabetes
8 HBV: Millions chronic carriers HCV: Millions chronic carriers Chronic infection HCV: 60-90% HBV:5-10% (40-80% newborns) years Cirrhosis 10-30% Antivirals Therapeutic vaccines HCC 1-3/% /year
9 Mortality Due to Cirrhosis and HCC in Italia Fattovich Gastroenterology 2005; 127: S 35-S 50
10 Cirrhotic liver HCC Which risk factors? Biomarkers: Viral- and host- derived
11 Low chromosomal instability HCV: HCV core, E2, NS3 NS5A Chromosomal instability: P53, Axin 1 LOH: 1p, 4q, 6q, 9p, 13q, 16p, 16q HBV HBx, PreS/S HBSP HBV DNA integration HBV HCV CHRONIC INFLAMMATION FIBROSIS (IMMUNE RESPONSE CYTOKINES) Cirrhosis H C C
12 Cell proliferation HBV and HCV proteins modulate cell signalling pathways Cell viability/apoptosis Antiviral machinery. Mitochondrial function Calcium homeostasis EMT Etc «Direct effects» Inflammation Fibrosis Transformation
13 Persistent, restricted, viral genome expression in tumour cells Viral replication capacity? In vivo analyses
14 RELATIONSHIP BETWEEN HBV DNA LEVEL AND HCC High level HBV viremia is a risk factor For HCC Chien-Jen Jama 2006; 295: 65-73
15 HBV genome expression in tumour cells Decline in serum HBV DNA during progression from cirrhosis to HCC (specific patterns in Asia) Persistence of HBV genome, with a restricted expression, in the tumour cells: Low or undetectable HBV DNA replication (Southern Blots) (HBsAg detction, PreS1, not HBcAg) PCR: Integrated DNA + cccdna + free HBV DNA HBV RNA Truncated HBx (COOH-terminally deleted) Truncated PreS2/S
16 HBV Recurrence In Relation with HCC Recurrence Paul Brousse Multivariate analysis for HBV recurrence after OLT for HBsAg + cirrhosis HCC pre OLT is an independent risk factor (with HBV viremia and HBIG + antiviral Combination) Faria Gastroenterolgy 2008 HCC recurrence No HCC recurrence
17 HBV in metastatic cells HBV DNA: 5/6 (cccdna in 2/3 tested) PreS1 staining in 3/6 Sequence analysis
18 Persistence of the viral genomes in tumor cells The impact of the detecting experimental procedures
19 HBV and HCC HBV DNA integration Early event during HBV infection (Acute hepatitis, chronic carriers Contribute to persistence (Leads to COOH-terminally deleted HBx and deleted PreS synthesis) Induces genetic instability HBV DNA integration in cellular genes Effect on target genes transcriptional activity and/or function? Rare in human HCC vs Woodchucks HCC? PCR-based methodologies: frequent event Real impact?
20 Unique target genes: Growth Factors Insertional mutagenesis Meval. kinase RAR beta Cyclin A MCM8 SERCA1 TRAP150 FR7 EMX2-like MAPK1 IRAK2 TRUP NRTK2 Ras-REBP-1 Calmodulin 1 Recurrent target genes: htert IP3R MLL2 HBV NRTK 2 FR7 IP 3 Cytoplasm Ca 2+ IP 3 R Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Endoplasmic Reticulum Ca 2+ Ca 2+Ca2+ HBV Ca 2+ S1 Ca 2 + p21 Calmodulin Ca 2+ IRAK2 CycA HBV TRUP HBV RARb EMX1/2-like RAS MAPK1 TR MEVAL. KINASE Ras-REBP-1 TRAP150 RB E2F TRANSCRIPTION MCM8 HBV NMP htert Paterlini-Brechot oncogene 2003, Murakami Gut 2005,
21 HBV DNA integration sites Frequent insertion in cellular genes: 73/93 HCC tested Recurrent integration sites: - htert - Calcium signalling related genes - Mixed lineage leukemia genes: MLL2 and MLL4-60s ribosomal protein-like encoding genes Evidence for alterations in the target cellular gene expression Saigo Human mutation 2008, 0-6; Murakami Gut 2005; 54:1162; Tamori Clin Cancer Res 2005; 11: 5821
22 HBV DNA INSERTIONAL ACTIVATION NL T77 NT Exon (201 pb) htert HBV X HBV X hmcm Kb cellular sequence Hela T NT NL NT T NL Gozuacik et al. Oncogene, 2001 Tamori Clin Cancer Res 2005; 11: 5821
23 Cirrhotic liver HCC Random integration followed by selection of clonally expanding cells? HBV genome as a driver, a «passenger», both? Integration into cellular genes frequently present in a subset of tumor cells (rearrangements during tumor progression and/or emergence of new clones) insertion
24 Identification of cellular genes at HBV genome integration sites: Which impact? Screening tool, in the «sequencing era», for identification of cellular genes involved at different steps of liver carcinogenesis? A conditional transposon- based insertional mutagenesis screen for for genes associated with mouse hepatocelularcarcinoma. Keng et al Nature biotechnology 2009 Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Collier et al Nature 2005 Novel tool for early identification of at risk patients?
25 Impact of diagnostic tests evaluating the viral genetic variability on early detection of the cancer? Identification of «at risk» patients?
26 Genotypes Different levels of HBV, HCV genetic variability HBV Genotypes A,B,C,D,E,F HCV Genotypes 1 (1a, 1b),2,3,4,5,6. Individual genome mutations Quasi-species
27 HBV Genetic Variability Impact on chronic hepatitis severity and liver carcinogenesis? HBV Genotypes A: Africa B: Asia C: Asia D: Mediteranean area E: Middle Africa F: South America HBV genome mutations PreS EnhII Basal Core promoter (BCP) PreCore X
28 Association of HBV genotypes and mutations with severity of liver diseases? HBV genotype C: increased risk of liver cirrhosis and HCC? (in older age) HBV genotype B: increased risk of HCC in younger patients? (without cirrhosis?) Difficulties: multiple parameters: Age Sex Geographical origin (genetic and environmental factors) Interplay between genotypes and mutations Viral load etc..
29 Association of HBV genotypes and Mutations with severity of liver diseases? Meta-analysis (Liu et al, JNCI 2009, 101:1066) 43 studies: subjects (2801 with HCC) PreS mutations (C1653T, T1753V and A1762/G1764A) are independent factors for risk of HCC. Mutation frequencies increased from asymptomatic carriers to cirrhosis and HCC HBx mutations 1762T/1764A: HCC: 74% in liver tumor, 52.1% in serum Prospective study in serum: HBV mutations preceeds HCC (up to 8 years) Kuang et al PNAS 2004;101:3575
30 Current HCV Epidemic HCV Genotypes: HCV 1b frequently associated with HCC: Duration of infection Intrinsic properties of some isolates?? (Pybus et al., Infect Genet Evol 2005;5:131-9)
31 HCV core mutations and HCC HCV Genotype 1b full length core sequences (data bank analysis) Serum samples: HCC vs non-hcc Seven polymorphisms associated with HCC (one also linked to Interferon resistance) Fishman Clin Cancer Res 2009; 15 (9) 3205
32 Clin Cancer Res 2009;15:3205
33 Biological implications of Natural HBV and HCV variants Identified in patients with HCC Selection of some viral genome quasispecies in tumor cells :questions? -- adaptation (fitness)? - cellular growth-advantage due to some biological properties of the viral genomes? HBV COOH-truncated HBx: Iavarone et al. J Hepatol 2003 HCV core: Sobesky et al Hepatology2007
34 HCV HCV polyprotein and processing 5'NTR IRES Positif strand RNA, 9,6 kb ( aa) 3'NTR S NS U/UC AUG SPP Core Signal peptidase ***** E1 Capsid (21 kda) + alternative form(s) of core protein Ø *********** E2 E1-E2 envelope glycoproteins (30 and 70 k Da) Ø p7 Viroporin (?) (7 k Da) NS2 NS2/3 autoprotease (23 kda) NS3 Proteinase Helicase (70 kda) NS4B? (30 kda) NS4A NS3 cofactor (6 k Da) NS5A ER lumen NS5B Serine- phophoprotein (56-58 kda) RNA-dependent RNA polymerase (68 kda) (Penin F., Structural Biology of Hepatitis C Virus, Liver Clinics, 2003)
35 Annular fibrosis Inflammatory infiltrates Tumoral nodule Fibrous stroma Regenerative nodule T and NT full length Laser-based microdissection HCV cores HCV RNA (low level in HCC cells) Cloning (10 clones/sample) Sobesky et al., Hepatology2007
36 HCC tumor and non tumor core variants nucleotidic aa Aminoacids Serum 74 Non tumor Tumor
37 Modulation of TGF-beta -mediated biological responses by HCV core variants: consequences on fibrosis and HCC Cytoplasm Core P II Smad3 I Core P P Smad3 TGF-b P Smad 3 Smad 4 Smad 4 HCV core T and NT variants differentially bind Smad3 (T cores show increased affinity vs NT cores) Resistance to TGF-B dependent antiproliferative effects Induction of Epithelium- Mesenchymal transition Nucleus SBE p300 Core Smad3-dependent transcription Pavio oncogene 2005; Battaglia et al PLoS one 2009; Vol 4:1
38 HCV core proteins increase TGF-b-mediated EMT Control TGF-b TbRI inhibitor Control TGF-b TbRI inhibitor WT NT T SMA vimentin Battaglia et al PLoS one 2009 ; vol 4:1 TGF b TbRI inhibitor E-cadherin Fibronectin Actin CTL T NT
39 Normal liver Cirrhosis HCC Fibrosis Inflammation HCV chronic infection High levels of TGF-b HCV Core increases TGF-b expression: Fibrosis (Battaler R. et al Gastroenterology 2004; Taniguchi et al J Med Virol ) HCV Tumor- and non-tumor derived core variants Bind Smad3 Resistance to TGF-B dependent antiproliferative effects Epithelium- Mesenchymal transition Pavio oncogene 2005; Battaglia et al PLoS one 2009; vol4:1
40 Epidemiological studies Which markers?
41 HBV «occult» (HBsAg negative) infections Occult HBV infections exist: HBV DNA detection Prevalence greatly varies but is frequently high in patients with HCC Low level of HBV DNA: sensitivity and specificity are major issues Occult HBV infections can be detected in patients with HCC and chronic hepatitis but also in resolved acute HBV infections Occult HBV infections are frequently detected in HCV chronically infected patients Bréchot Hepatology 2001, Seminars Cancer Biology 2000 Torbenson Lancet 2002, I Chemin J Clin Virol 2005, Raimondo 2007, Kew J Gastroenterlology and Hepatology 2008; 23:1426
42 HBV DNA detection in serum and liver of HBsAg negative patients Sensitivity; Specificity Low HBV DNA levels: sensitivity is a major issue (Bréchot Hepatology 2001; 34:194) Analysis by regular PCR-based assay: very rare detection of serum HBV DNA (Toyoda Intervirology 2007; 50:241) Analysis with different sets of primers, in-depth optimisation of the assay: much more frequent detection of HBV DNA (Pollicino et al Gastroenterology 2004; 126: 102) (Kew Journal of Gastroenterology and Hepatology 2008; 23: 1426)
43 Persistent HBV infection as a risk factor for human cancer? Detection of the viral genome and its expression (RNAs, Proteins) in a limited percentage of tumor cells and/or at a low level Real impact of occult HBV infections? Co-infection with HCV Prospective studies are needed
44 Occult HBV infections and HCV Impact on Chronic hepatitis severity and response to IFN (Mranie et al J Med Virol 2007; 79:1075) Impact on the risk of HCC (Pollicino et al Gastroenterology 2004) Anti-HCV + Anti-HCV - HBV DNA HCC CLD HCC CLD 45 (61.6%) 56 (36.6%) 23 (67.6%) 7 (18%) cccdna, free HBV DNA and HBV RNA detected in T and NT cells Integrated, truncated, HBV DNA Multivariate analysis: association HBV-HCC, independently of age
45 Influence of Occult Hepatitis B Virus Coinfection on the Incidence of Fibrosis and Hepatocellular Carcinoma in Chronic Hepatitis C Prospective study HBV DNA + Vs HBV DNA Odds ratio: Matsuoka et al Intervirology 2008;51:
46 Hepatitis B Virus DNA in Liver Tissue and Risk for Hepatocarcinogenesis in Patients with Hepatitis C Virus-Related Chronic Liver Disease A Prospective Study High HBV Viremia Vs Low HBV Viremia: Significant difference Obika et al Intervirology 2008;51:59 68
47 HBV and HCV negative serologies HBV DNA + vs HBV DNA 8.25 fold increased Risk of HCC Ikeda J Viral Hepatitis 2009; 16: 437
48 HCC PREVENTION HBV (DNA virus) HCV (RNA virus) 90% HCCs develop on cirrhotic livers (60% in Africa) H C C Alcohol Chemical carcinogens Aflatoxin B1 Hormonal factors Metabolic disorders: Steatosis Diabetes Genetic predisposition? Iron
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