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HBV-Related Insertional Mutagenesis in Hepatocellular Carcinomas: European and African Patients Study

This study aims to characterize the landscape of HBV integration leading to HBV-related hepatocellular carcinomas (HCC) in European and African populations. It investigates the role of cis-mediated insertional mutagenesis, clonality, and hotspot regions of HBV integration in HCC development.

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HBV-Related Insertional Mutagenesis in Hepatocellular Carcinomas: European and African Patients Study

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  1. Characterization of Hepatitis B Virus-related insertional mutagenesis in hepatocellular carcinomas from European and African patients ILCA 2019 – SEPTEMBER 20th 2019, CHICAGO CAMILLE PÉNEAU INSERM – U1138 (JESSICA ZUCMAN-ROSSI)

  2. Disclosures No conflict of interests

  3. Cis-mediated insertional mutagenesis can lead to carcinogenesis Modification of expression or function Cellsurvival Proliferation HBV Cancer gene Insertion near a cancer gene CIS EFFECT Random insertion in non-tumorliver tissues

  4. Cis-mediated insertional mutagenesis can lead to carcinogenesis Malignant transformation Selectiveadvantage Insertion near a cancer gene Random insertion in non-tumorliver tissues CLONALEXPANSION HCC

  5. HBV integrationmayoccur in preferentialgenomicloci • First HBV integration identified in cyclin A2 • (Wang et al., Nature 1990) • Studies on large number of HCC have enlighted preferential hotspots for HBV integration Sung et al., Nat Genet 2012 • What is the landscape of HBV integration leading toHBV-related HCC in European and African populations AIM OF THE STUDY Zhao LH, et al. Nat Commun. 2016

  6. Viral capture of 180 HCC fromEuropean and African patients Quantitative PCR VIRAL CAPTURE Fragmentation (1kb) Hybridizationwith HBV specific probes (all genotypes) Bead capture of DNA fragments containing HBV sequences Sequencing (2x250bp) Quantification of HBV DNA fromtumor and non-tumorlivertissues (785 patients) Asia Africa ANALYSIS HBV genotyping Identification of HBV insertions Normalizationof coveragewithhumangenes to defineclonality Europe Selection of highly positive samples Density Copy number/cell

  7. Clonal or sub-clonal HBV integrations are more frequentlyobserved in tumorsthan in adjacent liver tissues p < 0.001 p < 0.001 p < 0.001 • In tumor and non-tumorliver tissues, the majority of integrationbreakpointscomesfrom minor integrationevents (79%). • 79 % of tumors have clonal integrationsevents and only 18% of non-tumor tissues Non-tumorliver tissues Tumors N=175 N=180

  8. HBV insertions in driver genesinduce clonal selection of hepatocytes • Overall HBV integrations are enriched in early-replicatingregions and aroundhighly-expressedgenes. • In tumors HBV integrations are recurrentlyobserved in centromeric and telomericregions and around driver genes. (Furuta M, et al. Oncotarget. 2018) BREAKPOINTS IN TUMORS BREAKPOINTS IN ALL SAMPLES p < 0.001 p < 0.001 p < 0.001

  9. The TERTpromoter is the most recurrent hotspot of HBV integration Pangenomic plot of Clonal insertions TERT (n=50) CCNE1 (n=4) KMT2B (n=3) Centr (n=12) CCNA2 (n=1) Telomeric (n=5) Centr (n=9) Centr (n=4) Centr (n=4) TUMOR NON-TUMOR • Clonal integrations in the TERTpromoterare observed in 50 tumors (28%) and not in non-tumorliver tissues. • The otherrecurrenthotspots are known cancer-genes(mainlyCCNE1 and KMT2B) as in the Asian population.

  10. HBV integration in CCNA2 or CCNE1 as an oncogenic mechanism • Tumorswith HBV insertion in CCNA2 or CCNE1 belong to a homogenous group of HCC (CCN-HCC) presenting an activation of CCNA2 or CCNE1 and characterizedby specific structural rearragementsinduced by stress replication. Enhancerupstream CCNE1 CCN-HCC Truncated CCNA2 proteinwith no regulatorydomain Bayard Q, Meunier L, Péneau C et al. Nat Commun. 2018

  11. HBV integration can be complex events linked to chromosomal rearrangements amplifying or deleting cancer-related genes • The Copy NumberAlteration (CNA) wasassessed at eachintegrationbreakpoint for 20 tumorssequenced in WholeGenomeSequencingwiththeir non-tumorlivercounterpart. • One third of the breakpoints in thesetumorswereassociatedwith a copy numberalterationat the preciseintegration site and a large chromosomalrearrangement. Chr17p Chr5p 1,295,410 21,304,102 Large deletion (TP53) LogR Ratio HBV integration (631bp) HBV HBV Coverage (Capture) LogR Ratio Focal gain (TERT) HBV genome (3221 bp)

  12. Replicative HBV DNA canbedetected in tumor and non-tumor tissues EPISOMAL TEST withPlasmid-SafeDNAse Quantitative PCR Quantification of HBV DNA fromtumor and non-tumorlivertissues (785 patients) 1. Selection of HBV positive samples Viral DNA digestion 3. qPCRanalysis 2. PS-Dnase digestion HBV insertion in gDNA Viral DNA protection Episomal HBV La Bella et al. Gut, 2019 HBV mRNA EXPRESSION DNAsetreatment of extracted RNA RT-quantitative PCR with probes specific to HBV transcripts Selection of highly positive samples Samples positive for HBV episomalform and expressing all the HBV transcriptscontainsHBV replicativeforms. Density Copy number/cell

  13. Replicative HBV DNA isassociatedwith more HBV integrations • Replicative HBV DNA isdetected in 19% of non-tumorliver tissues and 9% of tumors . The sampleswithreplicative HBV DNA all have integrations and they have a largernumber of integrations. • Patients withnegativeAgHBs do not have replicative HBV in tumor or non-tumortissues, but they have integrated HBV DNA in the liver. AgHBs - AgHBs + p < 0.001 p < 0.001 N=92 N=8 N=101 N=8 N=144 N=140

  14. Conclusions • HBV integrationinduces a selection pressure as clonal insertions in tumors are mainlyobservedaround driver genes. The overallconsequences of HBV clonal insertions remain to bebetterunderstood but HBV integration in CCNA2 or CCNE1 acts as the sameoncogenicmechanism as anyotheralterationinducing an activation of one of thesetwogenes. • Frequentrearrangements are observedaround insertion breakpoints in the humangenome as in the integrated viral sequences. HBV insertion isthen a complexeventwhich may cause or derive from chromosomal instability. • SeveralHBV DNA forms are coexistingin livertissues and mightplay a role in hepato- carcinogenesis. As AgHBs-negative patients without active infection all have integrated HBV DNA in the liver, they may have a high risk for HCC development.

  15. Inserm U1138 Eq28 – Functional genomis of solid tumors Jessica ZUCMAN-ROSSI Sandrine IMBEAUD Tiziana LA BELLA Iadh MAMI Jean-Charles NAULT EricLETOUZE Sandra REBOUISSOU Quentin BAYARD Fidjie BORDES Anna-Line CALATAYUD Stefano CARUSO Gabrielle COUCHY Alix DEMORY Barkha GUPTA Théo HIRSCH Yannick LADEIRO Léa MEUNIER Olivier MSIKA Guillaume MORCRETTE Ana NEGULESCU Massih NINGARHARI Bénédicte NOBLET Jill PILET Samantha SCHAEFFER Eric TREPO Jie YANG

  16. Definition of Clonality Insertions detected in WGS: > 0.05 copy/cell Clonal or Sub-clonal 0.35 copy/cell Sub-clonal Clonal

  17. HBV insertion in CCNA2induces the overexpression of fusion and truncated proteins lacking N-terminal regulatory domains Cyclin A2 protein: 49 kDa Fusion protein M-HBS - Cyclin A2: 49 kDa D-box Ub Cyclin folds Cyclin folds Cyclin folds Cyclin folds S S preS2 N-ter C-ter C-ter C-ter C-ter 1 47 72 123 157 208 208 208 208 399 399 399 399 432 432 432 432 1 Fusion protein S-HBS - Cyclin A2: 153 158 153 44 kDa 101 156 1 55 Truncated Cyclin A2 protein: 32 kDa  The fusion HBs-CCNA2 and truncated CCNA2 proteins lack the N-terminal regulatory domains required for the protein degradation and a rigid cell cycle progression.

  18. The same stable truncated protein is induced by gene fusions and viral insertions in CCNA2 Western blot analysis:

  19. Cyclin A2/E1 activation defines a homogenous HCC subclass  CCN-HCC are large and highly proliferative tumors of poor prognosis

  20. CCN-HCC display a unique rearrangement signature (RS1) FR-3798T (HBV-insertion in CCNA2) • Signature RS1 is characterized by • specific structural rearrangements • Induced by replication stress and • frequenlyactivating TERT

  21. The natural history of CCN-HCC 7% of HCC 3% of HBV-HCC CCNA2/E1 Aberrant proliferation with premature S phase entry CCNA2/E1 activation HBV or otheralteration DNA replication stress Cell proliferation with accumulation of structural rearrangements TERT promoter activation Large, highly proliferative tumors of poor prognosis Bayard Q, Meunier L, Péneau C et al. Nat Commun. 2018

  22. Over 50% of HCC cases worldwide are attributed to HBV infection Japan/Egypt France HCV > 50% alcohol United States NASH associated with diabetes and/or obesity China/Sub-Saharan Africa Rare Hémochromatose HBV AflatoxinB1 Globocan 2018

  23. HBV inducescarcinogenesis via direct and indirect mechanisms Chronichepatitis • 70% Cirrhosis Early HCC Metabolic syndrome Inflammation Necrosis and regeneration Oxydative stress Telomere shortening Hepatitis B Telomerase reactivation Advanced HCC • 30% Viral oncoprotein Insertional mutagenesis Neuveut C, et al. J Hepatol 2010 Risk factors

  24. NGS-basedapproach to detect HBV insertions in HCC

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