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. 2021 Oct;75(4):865-878.
doi: 10.1016/j.jhep.2021.04.049. Epub 2021 May 13.

Molecular characterisation of hepatocellular carcinoma in patients with non-alcoholic steatohepatitis

Roser Pinyol  1 , Sara Torrecilla  1 , Huan Wang  2 , Carla Montironi  1 , Marta Piqué-Gili  1 , Miguel Torres-Martin  3 , Leow Wei-Qiang  4 , Catherine E Willoughby  1 , Pierluigi Ramadori  5 , Carmen Andreu-Oller  3 , Patricia Taik  2 , Youngmin A Lee  6 , Agrin Moeini  1 , Judit Peix  1 , Suzanne Faure-Dupuy  5 , Tobias Riedl  5 , Svenja Schuehle  5 , Claudia P Oliveira  7 , Venancio A Alves  7 , Paolo Boffetta  8 , Anja Lachenmayer  9 , Stephanie Roessler  10 , Beatriz Minguez  11 , Peter Schirmacher  10 , Jean-François Dufour  9 , Swan N Thung  12 , Helen L Reeves  13 , Flair J Carrilho  7 , Charissa Chang  12 , Andrew V Uzilov  14 , Mathias Heikenwalder  5 , Arun Sanyal  15 , Scott L Friedman  12 , Daniela Sia  12 , Josep M Llovet  16
Affiliations

Molecular characterisation of hepatocellular carcinoma in patients with non-alcoholic steatohepatitis

Roser Pinyol et al. J Hepatol. 2021 Oct.

Erratum in

Abstract

Background and aims: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies.

Methods: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays.

Results: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-β proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved.

Conclusions: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature.

Lay summary: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC.

Keywords: animal model; liver cancer; metabolic syndrome; molecular class; mutational signature; obesity.

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Conflict of interest statement

Conflict of interest J.M.L. receives research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb, Boehringer-Ingelheim and Ipsen, and consulting fees from Eli Lilly, Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Exelixis, Merck, Ipsen, Genentech, Roche, Glycotest, Leerink Swann LLC, Fortress Biotech, Nucleix, Can-Fite Biopharma, Sirtex, Mina Alpha Ltd and AstraZeneca. S.L.F consults for the following companies: 89 Bio, Amgen, Axcella Health, Blade Therapeutics, Bristol Myers Squibb, Can-Fite Biopharma, ChemomAb, Escient Pharmaceuticals, Forbion, Foresite laboratories, Galmed, Gordian Biotechnology, Glycotest, Glympse Bio, Hepgene, In sitro, Morphic Therapeutics, North Sea Therapeutics, Novartis, Ono Pharmaceuticals, Pfizer Pharmaceuticals, Scholar Rock Surrozen. He has stock options in the following companies: Blade Therapeutics, Escient, Galectin, Galmed, Genfit, Glympse, Hepgene, Lifemax, Metacrine, Morphic Therapeutics, Nimbus, North Sea Therapeutics, Scholar Rock, Surrozen. He receives research support from Morphic Therapeutics, Novo Nordisk, and Galmed, and has an SBIR grant with Abalone Bio. A.L. is a consultant for Neuwave and Histosonics. C.P.M.S.O. consults for Bayer, Novartis, Novonordisk, Allergan, Pfizer, Roche and Zambon. P.S. is receiving research grants from BMS, Roche, Incyte, Chugai and consulting fees from BMS, MSD, Incyte, Janssen, Roche, AstraZeneca and Amgen. H.W., P.T., and A.V.U. are or were salaried employees of Sema4 at the time of the study. H.W., P.T., and A.V.U. hold Sema4 stock options. B.M. received consultancy fees from Bayer-Shering Pharma, and speaker fees from Eisai and MSD. The rest of authors have nothing to disclose. Please refer to the accompanying ICMJE disclosure forms for further details.

Figures

Fig. 1.
Fig. 1.. Genomic landscape of NASH-HCC and in vitro evidence supporting a tumour suppressor role of ACVR2A in HCC.
(A) Mutations and focal copy number alterations in driver genes altered in ≥4% of the NASH-HCC cohort. (B) Mutational frequency of the most commonly altered genes in the NASH-HCC cohort (n = 50) and in the viral/alcohol-HCC cohort (n = 624).,, Statistical test: Fisher. (C) Genomic and clinicopathological features of NASH-HCC according to cirrhosis. Statistical test: Fisher and Mann-Whitney. (D) ACVR2A mutations identified in the NASH-HCC cohort. (E, F) Cell viability rate (E) and colony formation quantification (F) of Hep3B cells stably transfected with ACVR2A- or control-shRNA. Error bars represent mean ± SEM of ≥ 3 experiments performed in triplicate. Statistical test: t test. (G) Representative image of the colony formation assay. HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; shRNA, short hairpin RNA.
Fig. 2.
Fig. 2.. Mutational signatures in NASH-HCC and in viral/alcohol-HCC.
(A) Unsupervised hierarchical clustering of the mutational signatures obtained for 43 NASH-HCCs and 43 viral/alcohol-HCCs. Red asterisks mark samples where MutSig-NASH-HCC presented an exposure >20% when setting the confidence at 90%. (B) Heatmap with clinicopathological data, mutational status of CTNNB1 and TP53, and mutational signatures (confidence >90%, exposure >20%). Statistical test: Fisher and Mann-Whitney. HCC, hepatocellular carcinoma; MutSig, mutational signature; NASH, non-alcoholic steatohepatitis.
Fig. 3.
Fig. 3.. Signalling pathways altered in NASH-HCC.
(A) Driver genomic alterations identified by WES grouped according to signalling pathways. (B) Heatmap displaying differentially enriched pathways in NASH-HCCs (n = 53) compared to viral/alcohol-HCCs (n = 184). Statistical test: t test. (C) Molecular classes and activated signalling pathways in the NASH-HCC cohort. Samples were classified into proliferative (S1/S2) and non-proliferative tumours (S3). Statistical test: t test and Fisher. Displayed p values were obtained comparing proliferation and non-proliferation HCCs. Gene signatures were obtained from MSigDB or other sources (see supplementary information). HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; WES, whole exome sequencing.
Fig. 4.
Fig. 4.. Characterisation of the NASH-HCCs according to HCC immune classes and signalling pathways differentiating cirrhotic from non-cirrhotic NASH-HCC.
(A) Heatmap displaying NASH-HCC tumours classified according to the HCC immune classes., Gene signatures used are referenced in the supplementary information. Statistical test: t test. (B, C) Pre-ranked GSEA enrichment plots of representative signalling pathways or molecular classes enriched in non-cirrhotic (B, n = 16) and cirrhotic NASH tumours (C, n = 37). GSEA, gene set enrichment analysis; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; n.s., not significant.
Fig. 5.
Fig. 5.. Characterisation of the NASH cancer field.
(A) Heatmap characterising the cancer field in NASH livers and NASH-HCC adjacent tissues. Plotted are ssGSEA scores for NASH-related gene sets. t test p values report differences between cirrhotic and non-cirrhotic samples. Healthy liver (H). Cirrhotic liver (Ci). NASH liver from patients with no HCC (NASH). Non-tumorous tissue adjacent to NASH-HCC (NASH-HCC adjacent). (B) Heatmap displaying ssGSEA scores of immune signatures capturing different immune cell populations. Gene signatures are referenced in the supplementary information. Statistical test: t test. HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; n.s., not significant; ssGSEA, single sample gene set enrichment analysis.
Fig. 6.
Fig. 6.. NASH-HCC murine models recapitulate key molecular and immune features of human NASH-HCC.
(A) Submap analysis displaying the molecular similarity between human and murine NASH-HCC and adjacent tissue samples. Numbers on heatmap indicate FDR values for transcriptome similarity. (B) Heatmap displaying enrichment of fatty and bile acid metabolism, oxidative stress and inflammation-related gene signatures in NASH-HCC vs. non-NASH-HCC. Statistical test: t test. (C) NASH-HCC murine and human samples classified according to the HCC molecular and immune classes. FDR, false discovery rate; HCC, hepatocellular carcinoma; NASH, non-alcoholic steatohepatitis; n.s., not significant.
See this image and copyright information in PMC

Comment in

  • Molecular landscape of NASH-HCC.
    Hindson J. Hindson J. Nat Rev Gastroenterol Hepatol. 2021 Jul;18(7):456. doi: 10.1038/s41575-021-00478-6. Nat Rev Gastroenterol Hepatol. 2021. PMID: 34103708 No abstract available.

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