Clinical Study

Molecular portrait of high alpha-fetoprotein in hepatocellular carcinoma: implications for biomarker-driven clinical trials

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The clinical utility of serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC) is widely recognised. However, a clear understanding of the mechanisms of AFP overexpression and the molecular traits of patients with AFP-high tumours are not known. We assessed transcriptome data, whole-exome sequencing data and DNA methylome profiling of 520 HCC patients from two independent cohorts to identify distinct molecular traits of patients with AFP-high tumours (serum concentration > 400 ng/ml), which represents an accepted prognostic cut-off and a predictor of response to ramucirumab. Those AFP-high tumours (18% of resected cases) were characterised by significantly lower AFP promoter methylation (p < 0.001), significant enrichment of progenitor-cell features (CK19, EPCAM), higher incidence of BAP1 oncogene mutations (8.5% vs 1.6%) and lower mutational rates of CTNNB1 (14% vs 30%). Specifically, AFP-high tumours displayed significant activation of VEGF signalling (p < 0.001), which might provide the rationale for the reported benefit of ramucirumab in this subgroup of patients.

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  1. 1.

    Llovet, J. M., Montal, R., Sia, D. & Finn, R. S. Molecular therapies and precision medicine for hepatocellular carcinoma. Nat. Rev. Clin. Oncol. 15, 599–616 (2018).

  2. 2.

    EASL Clinical Practice Guidelines. Management of hepatocellular carcinoma. J Hepatol. 69, 182–236 (2018).

  3. 3.

    Zhu, A. X., Kang, Y., Yen, C., Finn, R. S., Galle, P. R., Llovet, J. M. et al. Ramucirumab after sorafenib in patients with advanced hepatocellular carcinoma and increased α-fetoprotein concentrations (REACH-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol. 2045, 1–15 (2019).

  4. 4.

    Mizejewski, G. J. Biological role of alpha-fetoprotein in cancer: prospects for anticancer therapy. Expert Rev. Anticancer Ther. 2, 709–735 (2002).

  5. 5.

    Villanueva, A., Portela, A., Sayols, S., Battiston, C., Hoshida, Y., Méndez-González, J. et al. DNA methylation-based prognosis and epidrivers in hepatocellular carcinoma. Hepatology. 61, 1945–1956 (2015).

  6. 6.

    Ally, A., Balasundaram, M., Carlsen, R., Chuah, E., Clarke, A., Dhalla, N. et al. Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell. 169, 1327–1341.e23 (2017).

  7. 7.

    Pastor, W. A., Aravind, L. & Rao, A. TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nat. Rev. Mol. Cell Biol. 14, 341–356 (2013).

  8. 8.

    Sia, D., Jiao, Y., Martinez-Quetglas, I., Kuchuk, O., Villacorta-Martin, C., Castro de Moura, M. et al. Identification of an immune-specific class of hepatocellular carcinoma, based on molecular features. Gastroenterology. 153, 812–826 (2017).

  9. 9.

    Harding, J. J., Nandakumar, S., Armenia, J., Khalil, D. N., Albano, M., Ly, M. et al. Prospective genotyping of hepatocellular carcinoma: clinical implications of next generation sequencing for matching patients to targeted and immune therapies. Clin. Cancer Res. 25, 2116–2126 (2019).

  10. 10.

    Carbone, M., Yang, H., Pass, H. I., Krausz, T., Testa, J. R. & Gaudino, G. BAP1 and cancer. Nat. Rev. Cancer. 13, 153–159 (2013).

  11. 11.

    Martinez-Quetglas, I., Pinyol, R., Dauch, D., Torrecilla, S., Tovar, V., Moeini, A. et al. IGF2 is up-regulated by epigenetic mechanisms in hepatocellular carcinomas and is an actionable oncogene product in experimental models. Gastroenterology. 151, 1192–1205 (2016).

  12. 12.

    Lal, N., Puri, K. & Rodrigues, B. Vascular endothelial growth factor B and its signaling. Front. Cardiovasc Med. 5, 39 (2018).

  13. 13.

    Fischer, C., Mazzone, M., Jonckx, B. & Carmeliet, P. FLT1 and its ligands VEGFB and PlGF: drug targets for anti-angiogenic therapy? Nat. Rev. Cancer. 8, 942–956 (2008).

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This study has been in part developed at the building Centre Esther Koplowitz from IDIBAPS/CERCA Programme/Generalitat de Catalunya.

Author information

Study concept and design: R.M., R.P., D.S. and J.M.L. Acquisition, analysis or interpretation of data: R.M., C.A.O., L.B., R.E.F., S.M., C.M., A.M., R.P., J.P., L.C., A.V. and D.S. Drafting of the paper: R.M., C.A.O., L.B., R.E.F., A.M., R.P. and D.S. Critical revision of the paper for important intellectual content: R.P., D.S., V.M., M.E. and J.M.L.

Correspondence to Josep M. Llovet.

Ethics declarations

Competing interests

J.M.L. is receiving research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb and Ipsen, and consulting fees from Bayer HealthCare Pharmaceuticals, Eli Lilly, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Exelixis, Merck, Ipsen, Glycotest, Navigant, Leerink Swann LLC, Midatech Ltd, Fortress Biotech, Sprink Pharmaceuticals, Nucleix and Can-Fite Biopharma. Manel Esteller reports grants from Ferrer International SA and Quimatryx. Augusto Villanueva reports personal fees from Exelixis, Health Advances LLC, GroupH, Gerson Lehrman Group and Exact Sciences.

Ethics approval and consent to participate

The institutional review boards of the participating centres (IRCCS Istituto Nazionale Tumori [Milan] and Hospital Clínic [Barcelona]) approved the study. Patients provided written informed consent. The study was conducted in accordance with International Standards of Good Clinical Practice and the Declaration of Helsinki.


Robert Montal is supported by a FSEOM-Boehringer Ingelheim Grant. Carmen Andreu-Oller has received financial support through the “la Caixa” INPhINIT Fellowship Grant for Doctoral studies at Spanish Research Centres of Excellence, from “la Caixa” Banking Foundation (ID 100010434), fellowship code (LCF/BQ/IN17/11620024). Laia Bassaganyas was supported by Beatriu de Pinós grant from Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, Generalitat de Catalunya). Roger Esteban-Fabró is supported by MICINN/MINECO (BES-2017-081286). Carla Montironi is a recipient of Josep Font grant from Hospital Clinic de Barcelona. Roser Pinyol is funded by European Commission/Horizon 2020 Program (HEPCAR, Ref. 667273-2). Judit Peix is supported by Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd)—ISCIII. Augusto Villanueva is supported by U.S. Department of Defense (CA150272P3) and Tisch Cancer Institute (Cancer Center Grant P30-CA196521). Daniela Sia is supported by the Gilead Sciences Research Scholar Program in Liver Disease. Vincenzo Mazzaferro is supported by grants from Associazione Italiana per la Ricerca sul Cancro and the Oncology Research Project of the Italian Ministry of Health. Manel Esteller is supported by the Department of Health PERIS project SLT/002/16/00374 and AGAUR projects 2017SGR1080, 2014SGR633 and 2009SGR1315 of the Catalan Government (Generalitat de Catalunya); the Spanish Institute of Health Carlos III (ISCIII) with project DTS16/00153 and the Integrated Project of Excellence PIE13/00022 (ONCOPROFILE), and the Ministerio de Economía y Competitividad (MINECO) grant SAF2014-55000-R, co-financed by the European Development Regional Fund “A way to achieve Europe” (ERDF); CIBER 2016 CB16/12/00312 (CIBERONC); the Cellex Foundation; “la Caixa” Banking Foundation (LCF/PR/PR15/11100003). Josep M. Llovet is supported by National Cancer Institute (P30-CA196521), U.S. Department of Defense (CA150272P3), European Commission/Horizon 2020 Program (HEPCAR, Ref. 667273-2), EIT Health (CRISH2, Ref. 18053), Accelerator Award (CRUCK, AECC and AIRC) (HUNTER, Ref. C9380/A26813), Samuel Waxman Cancer Research Foundation, Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd)—ISCIII, Spanish National Health Institute (SAF2016-76390) and the Generalitat de Catalunya/AGAUR (SGR-1358).

Data availability

Data from the internal HEPTROMIC cohort are stored in the Gene Expression Omnibus (GEO) repository: whole-genome expression (GSE63898) and DNA methylation (GSE56588). Whole-exome sequencing was deposited in the EGA database (accessions EGAS00001000217, EGAS00001000679 and EGAS00001001002) and the ICGC data portal. Data from the external publicly available TCGA cohort were downloaded from


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