Gastroenterology

Gastroenterology

Volume 157, Issue 5, November 2019, Pages 1383-1397.e11
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
An Immune Gene Expression Signature Associated With Development of Human Hepatocellular Carcinoma Identifies Mice That Respond to Chemopreventive Agents

https://doi.org/10.1053/j.gastro.2019.07.028Get rights and content

Background & Aims

Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis.

Methods

We analyzed gene expression profiles of nontumor liver tissues from 392 patients with early-stage HCC (training set, N = 167 and validation set, N = 225) and liver tissue from patients with cirrhosis without HCC (N = 216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up, 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of diethylnitrosamine (DEN) followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then orally given the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, orally given aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet.

Results

We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of nontumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor β signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% confidence interval, 1.21–4.80). Mice with chemically induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel down-regulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle.

Conclusions

We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that is associated with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and development of fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis.

Section snippets

Human Cohort

Gene expression data from a cohort of 167 surgically resected fresh-frozen samples (Heptromic data set, GSE63898) with matched tumor and adjacent nontumor tissue were analyzed. Samples were previously collected (1998–2008) in the setting of the HCC Genomic Consortium after institutional review board approval. Full descriptions of the cohort and RNA profiling data are available in previous publications.15, 16 Supplementary Table 1 provides a summary of the clinicopathologic variables of the

Identification of a Novel Immune-Mediated Cancer Field Effect in Nontumor Liver Tissue of Patients With Early Hepatocellular Carcinoma

To characterize the immune features governing the unresolved cancer field in which new cancers arise, transcriptome-based analysis of a compendium of approximately 5000 annotated immunology-specific gene sets18 was performed in the nontumor liver tissue of patients with early-stage HCC. This analysis showed the presence of an ICF in approximately 60% (98/167) of samples (Figure 1A and Supplementary Figure 1). Specifically, these samples were characterized by enrichment of several gene sets

Discussion

This study represents an in-depth analysis of the inflammatory milieu associated with the field cancerization in the chronically injured liver and investigates its clinical implications for the prediction and prevention of HCC occurrence in patients with cirrhosis.

The role of the cancer field effect in promoting neoplastic transformation has gained much interest in recent years, and currently an altered microenvironment is considered a promoter of cancer.8, 10 Although under physiologic

Acknowledgements

The authors thank Juan José Lozano for technical assistance in the normalization of the transcriptomic array of the animal model. This study was developed at the building of Centre Esther Koplowitz from IDIBAPS/CERCA Programme/Generalitat de Catalunya. The authors also acknowledge Angelo Sangiovanni and Massimo Colombo for providing the seminal cohort of patients with cirrhotic in our previous studies.12, 14

Author contributions: Study concept and design: Agrin Moeini, Victoria Tovar, Daniela

References (52)

  • S. Nakagawa et al.

    Molecular liver cancer prevention in cirrhosis by organ transcriptome analysis and lysophosphatidic acid pathway inhibition

    Cancer Cell

    (2016)
  • M.J. Wolf et al.

    Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes

    Cancer Cell

    (2014)
  • D. Hanahan et al.

    Hallmarks of cancer: the next generation

    Cell

    (2011)
  • O.V. Makarova-Rusher et al.

    The yin and yang of evasion and immune activation in HCC

    J Hepatol

    (2015)
  • G.V. Papatheodoridis et al.

    Incidence and predictors of hepatocellular carcinoma in Caucasian chronic hepatitis B patients receiving entecavir or tenofovir

    J Hepatol

    (2015)
  • S. Omenetti et al.

    Dysregulated intrahepatic CD4+ T-cell activation drives liver inflammation in ileitis-prone SAMP1/YitFc mice

    Cell Mol Gastroenterol Hepatol

    (2015)
  • F. Bray et al.

    Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries

    CA Cancer J Clin

    (2018)
  • J.M. Llovet et al.

    Hepatocellular carcinoma

    Nat Rev Dis Prim

    (2016)
  • J.M. Llovet et al.

    Molecular therapies and precision medicine for hepatocellular carcinoma

    Nat Rev Clin Oncol

    (2018)
  • P.R. Galle et al.

    EASL clinical practice guidelines: management of hepatocellular carcinoma

    J Hepatol

    (2018)
  • V. Calvaruso et al.

    Incidence of hepatocellular carcinoma in patients with HCV-associated cirrhosis treated with direct-acting antiviral agents

    Gastroenterology

    (2018)
  • F. Kanwal et al.

    Risk of hepatocellular cancer in HCV patients treated with direct-acting antiviral agents

    Gastroenterology

    (2017)
  • Y. Hoshida et al.

    Gene expression in fixed tissues and outcome in hepatocellular carcinoma

    N Engl J Med

    (2008)
  • J. Ji et al.

    Hepatic stellate cell and monocyte interaction contributes to poor prognosis in hepatocellular carcinoma

    Hepatology

    (2015)
  • D.Y. Zhang et al.

    A hepatic stellate cell gene expression signature associated with outcomes in hepatitis C cirrhosis and hepatocellular carcinoma after curative resection

    Gut

    (2016)
  • J.H. Kim et al.

    Genomic predictors for recurrence patterns of hepatocellular carcinoma: model derivation and validation

    PLoS Med

    (2014)
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    Conflicts of interest These authors disclose the following: Part of the study was supported with an investigator-initiated research grant by Boehringer Ingelheim. Josep M. Llovet has been a consultant and advisory board member and has received research funding from Boehringer Ingelheim and is receiving research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb, and Ipsen and consulting fees from Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Eli Lilly, Exelixis, Merck, Ipsen, Glycotest, Navigant, Leerink Swann LLC, Midatech Ltd, Fortress Biotech, Sprink Pharmaceuticals, and Nucleix. Scott L. Friedman has been a consultant for Abide Therapeutics, Allergan Pharmaceuticals, Angion Biomedica, Blade Therapeutics, Can-Fite Biopharma, Enanta Pharmaceuticals, Escient Therapeutics, Forbion, Galmed, Genfit, Glycotest, Glympse Bio, Metacrine Inc, Mistral Biosciences, Morphic Rock Therapeutics, North Sea Therapeutics, Novartis, Novo Nordisk, Pfizer Pharmaceuticals, Salix Pharmaceuticals, Scholar Rock, Seal Rock Therapeutics, Second Genome, Surrozen, Symic Bio, Viking Therapeutics, and Kintai; has received research funding from Blade Therapeutics, Can-Fite Biopharma, Ferring Research Institute, Galmed; and has stock options from Intercept, Exalenz, Madrigal, Akarna Therapeutics, BirdRock Bio, Blade Therapeutics, Conatus, DeuteRx, Exalenz, Galectin, Galmed, Genfit, Glympse. The remaining authors disclose no conflicts.

    Funding Josep M. Llovet is supported by the European Commission (EC)/Horizon 2020 Program (HEPCAR, reference no. 667273-2); US Department of Defense (CA150272P3); an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER, reference no. C9380/A26813), NCI Cancer Center Support Grant, National Cancer Institute; Tisch Cancer Institute (P30-CA196521); Samuel Waxman Cancer Research Foundation; Spanish National Health Institute (SAF2016-76390); and the Generalitat de Catalunya/AGAUR (SGR-1358). Agrin Moeini is supported by Spanish National Health Institute. Sara Torrecilla and Judit Peix are funded by Centro de Investigación Biomedica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd-ISCIII). Carla Montironi is a recipient of Josep Font grant. Carmen Andreu-Oller is supported by “la Caixa” INPhINIT Fellowship Grant (LCF/BQ/IN17/11620024). Roser Pinyol is supported by HEPCAR and AECC. Daniela Sia is supported by the Gilead Sciences Research Scholar Program in Liver Disease. Scott L. Friedman is supported by the National Institutes of Health Research project grant (R01,DK5662) and US Department of Defense (CA150272P3). Mathias Heikenwälder was supported by an ERC Consolidator grant (HepatoMetaboPath), the SFBTR 209, 1335 and SFBTR179. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 667273 and the Helmholtz future topic (Zukunftsthema) Immunology and Inflammation.

    Author names in bold designate shared co-first authorship.

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