Genomic Analysis of Hepatocellular Carcinoma With Active Hepatitis B Virus Replication

1. Genomic Analysis ofHepatocellular Carcinoma WithActive Hepatitis B Virus Replication Huat Chye Lim, MD, and John Gordan, MD, PhD Divisions of Hospital Medicine and Hematology/Oncology University of California, San Francisco

2. Introduction Synopsis HCC with active HBV replication represents a molecularly distinct subset of HCC associated with differences in mutations, gene expression and survival. Objectives for this talk Which genes are differentially mutated in HCC with active HBV replication? Which genes are differentially expressed in HCC with active HBV replication? How does HBV replication status affect survival in HCC? Background: HBV and HBV-related HCC • Small enveloped DNA virus with partially double stranded genome • Persists in nuclei of infected hepatocytes via episomal cccDNA • Rarely integrates into host genome • HBV-related HCC known to be associated with clinical and genomic differences

3. Methods • We used GATK PathSeq software, which performs sequential computational subtraction, to measure HBV RNA in HCC tumors • Tumor RNA-Seq data were obtained from two databases: • TCGA – n = 371 • ICGC – n = 68 (from LIRI-JP project) • We classified tumors as HBV RNA+ if more than 1 HBV RNA read was detected per million human reads • We investigated association between HBV RNA status and nonsynonymous somatic mutations, gene set expression and survival All tumor RNA-Seq reads Quality filtering and duplicate removal Filtered reads Low quality and duplicate reads Subtraction of human reads Low quality and duplicate reads Non-human reads Human reads Mapping to known microbes Other microbe reads Human reads Low quality and duplicate reads HBV reads Reference: Walker MA, et al. Bioinformatics. 2018;34(24):4287-4289.

4. Results: HBV RNA Status is Associated With Differences in Clinical Characteristics • HBV RNA+ status was associated with: • Male gender • Younger age • Higher grade • HBV history • No HCV history • There was no association with stage, vascular invasion or alcohol consumption • p values from χ2 (*) and Mann-Whitney (**) tests • Stage determined using either AJCC (for TCGA) or LCSGJ (for ICGC LIRI-JP) criteria

5. Results: HBV RNA Status is Associated With Differential Gene Mutation Rates • Figure shows the 94 genes where nonsynonymous mutation rate depended significantly on HBV RNA status • Most (82/94) were preferentially mutated in HBV RNA+ tumors • Bolded genes are among the 30 significantly-mutated genes identified in 2017 TCGA HCC Cell paper • TP53 and CDKN2A were more frequently mutated in HBV RNA+ • BAP1 was more frequently mutated in HBV RNA- • TP53 had substantially higher mutation rates than other genes Preferentially mutated in HBV RNA+ tumors Preferentially mutated in HBV RNA- tumors 0.35 • Circle size is inversely proportional to log(p) • Bolded genes are TCGA top 30 SMGs Reference: Cancer Genome Atlas Research Network. Cell. 2017;169(7):1327-1341.e23.

6. Results: HBV RNA+ Status is Associated With Differential Gene Set Expression Cell cycle regulation Chromatin modification • Figure shows GSEA enrichment map of Gene Ontology gene sets with FDR < 10% in TCGA dataset (n = 531 of 4,464) • All such gene sets were enriched only in HBV RNA+ tumors • Enriched gene sets included: • Multiple DNA damage repair pathways • Genes upregulated by MYC and mTORC1 • Genes upregulated in “proliferative” HCC: • Boyault subclass G1-G3 HCC • Hoshida subclass S2 HCC • Lee subclass A HCC • Chiang “proliferation” subclass HCC • We then evaluated association between HBV RNA status and several measures of genomic instability Mitotic spindle regulation DNA replication Methylation and chromatin modification DNA damage repair Nucleases Nuclear transport RNA processing Translation Enrichment in HBV RNA+ 0.0001 FDR 0.1 Circle size is proportional to gene set size RNA processing and splicing Transcription References: Boyault S, et al. Hepatology. 2007;45(1):42-52. Hoshida Y, et al. Cancer Res. 2009;69(18):7385-92. Lee JS, et al. Hepatology. 2004;40(3):667-76. Chiang DY, et al. Cancer Res. 2008;68(16):6779-88.

7. Results: HBV RNA+ Status is Associated With Increased Homologous Recombination Deficiency (HRD) Score p = 1e-6 • HRD score for TCGA dataset was calculated as the sum of three independent HRD measures: • Large-scale state transitions • Loss of heterozygosity • Telomeric allelic imbalance • HBV RNA+ status was associated with increased HRD score (22.19 for RNA+ vs. 15.97 for RNA-, p = 1e-6) • There was no association with tumor mutational burden (TMB) • Error bars show mean ± SD • p value from Mann-Whitney test Reference: Knijnenburg TA, et al. Cell Rep. 2018;23(1):239-254.e6.

8. Results: HBV RNA Status is Associated With Survival Differences BAP1 mutations were associated with increased survival in HBV RNA- patients • We used Cox multivariate regression to evaluate effect of mutation and HBV RNA status on overall survival • Covariates: HBV RNA status, mutation status, age, sex, grade, stage, cohort • Table shows Cox coefficients for the 30 SMGs identified in 2017 TCGA HCC Cell paper • Mutations associated with survival difference in HBV RNA-: • Increased survival:BAP1 • Decreased survival:TP53, EEF1A1, ARID1A, APOB • Mutations associated with survival difference in HBV RNA+: • Decreased survival:KEAP1, RB1, ARID2 BAP1 KEAP1 p = 0.008 KEAP1 mutations were associated with decreased survival in HBV RNA+ patients p = 0.01 Reference: Cancer Genome Atlas Research Network. Cell. 2017;169(7):1327-1341.e23.

9. Summary Which genes are differentially mutated in HCC with active HBV replication? • These included TP53 and CDKN2A in HBV RNA+ tumors and BAP1 in HBV RNA- tumors • Most differentially mutated genes were preferentially mutated in HBV RNA+ tumors • TP53 mutation rates were substantially higher than other differentially mutated genes Which genes are differentially expressed in HCC with active HBV replication? • All Gene Ontology gene sets with FDR < 10% were enriched only in HBV RNA+ tumors • These included multiple DNA damage repair gene sets and multiple gene sets upregulated in “proliferative” HCC • HBV RNA+ status was associated with increased homologous recombination deficiency score How does HBV replication status affect survival in HCC? • BAP1 mutations were associated with increased survival in HBV RNA- patients • KEAP1 mutations were associated with decreased survival in HBV RNA+ patients

10. Conclusions and Future Directions • HCC with active HBV replication represents a molecularly distinct subset of HCC associated with differences in mutations, gene expression and survival • HBV replication is a factor that could impact HCC prognosis and response to targeted therapies • HBV status is a factor that should be more rigorously defined and stratified in HCC clinical trials • Limitations of our study include that TCGA and ICGC comprise mainly early stage/resectable tumors and contain incomplete HBV serologic data • Questions for future investigation include: • Is this molecularly distinct subset of HCC amenable to targeted therapy? • Is there a role for direct measurement of tumor HBV replication in HCC? • How concordant is tumor HBV RNA status with serum HBV markers (e.g., serologies and viral load)? Acknowledgements • Mentors: John Gordan and Katie Kelley • For valuable feedback: Gary Chan, Mary Feng, Christina Hwang, David Quigley, Neil Mehta, Dom Mitchell and Rigney Turnham