www.nature.com/bjc BRIEF COMMUNICATION Clinical Study Molecular portrait of high alpha-fetoprotein in hepatocellular carcinoma: implications for biomarker-driven clinical trials Robert Montal1, Carmen Andreu-Oller1, Laia Bassaganyas1, Roger Esteban-Fabró1, Sebastián Moran2, Carla Montironi1, Agrin Moeini1, Roser Pinyol1, Judit Peix1, Laia Cabellos1, Augusto Villanueva3, Daniela Sia3, Vincenzo Mazzaferro4, Manel Esteller2,5,6,7,8 and Josep M. Llovet1,3,6 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. British Journal of Cancer (2019) 121:340–343; https://doi.org/10.1038/s41416-019-0513-7 BACKGROUND For surveillance and diagnostic purposes, AFP sensitivity and The global disease burden of hepatocellular carcinoma (HCC) is specificity depend on the established cut-off, with a global increasing worldwide, with an estimated 50% of cases receiving accuracy that is suboptimal for routine clinical practice.2 As a systemic treatments for advanced stage.1,2 In the last 2 years, prognostic factor, it has been clearly demonstrated that patients several compounds have shown clinical efficacy in the first- with AFP > 400 ng/ml have poor outcomes.2 (lenvatinib) or second-line (regorafenib, cabozantinib) setting and Considering the prognostic and predictive capabilities of AFP in joined the standard of care, sorafenib, ultimately leading to a HCC, our hypothesis is that the molecular profile of high AFP median survival of 2 years with sequential therapies.1 Ramucir- tumours differs from those with low AFP and might be associated umab, a monoclonal antibody against VEGFR2, is the first drug to with VEGF signalling. Herein, we describe the biological traits of demonstrate efficacy in a biomarker-driven phase III trial in HCC, HCC with high serum AFP levels through a comprehensive showing a survival benefit as second-line treatment in those molecular analysis that may provide the rationale for the design patients with alpha-fetoprotein (AFP) serum levels higher than of future biomarker-driven clinical trials. 400 ng/ml.3 Nevertheless, the rationale behind the use of AFP as a predictive biomarker is not fully understood. AFP is a protein transcribed from the albuminoid genes located METHODS on chromosome 4, with a known multifunctionality (i.e., binding For the purpose of the study, we analysed the molecular profiles of hydrophobic ligands, regulation of proliferation and immuno- of 520 HCC human samples with available baseline AFP serum modulation) provided by its multi-modular structure.4 AFP is concentrations, including an internal cohort of 244 surgically considered an oncofoetal protein due to its presence during foetal resected fresh frozen samples (HEPTROMIC data set),5 and an development and its association with some tumour types, such as external publicly available cohort of 276 primary HCC from The liver, testes and ovary.4 In HCC, AFP serum concentration may vary Cancer Genome Atlas (TCGA data set)6 (Supplementary Fig. 1). from normal (< 10 ng/ml) to extremely high (>100000 ng/ml).2,4 Differential molecular patterns of HCC patients based on serum For this reason, AFP has been extensively explored as a biomarker. AFP levels were obtained from whole-genome expression, DNA 1 Translational Research in Hepatic Oncology, Liver Unit, IDIBAPS, CIBERehd, Hospital Clínic, University of Barcelona, Barcelona, Catalonia, Spain; 2Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet, Barcelona, Catalonia, Spain; 3Liver Cancer Program, Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA; 4University of Milan and Gastrointestinal Surgery and Liver Transplantation Unit, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy; 5Centro de Investigación Biomédica en Red Cancer (CIBERONC), Madrid, Spain; 6Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain; 7 Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Catalonia, Spain and 8Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Catalonia, Spain Correspondence: Josep M. Llovet (jmllovet@clinic.cat) Received: 15 April 2019 Revised: 12 June 2019 Accepted: 14 June 2019 Published online: 9 July 2019 © Cancer Research UK 2019 Molecular portrait of high alpha-fetoprotein in hepatocellular carcinoma:. . . R Montal et al. 341 methylome profiling and whole-exome sequencing as described Based on the oncofoetal nature of AFP, we next analysed its in Supplementary Methods. DNA methylation status. The AFP promoter is a low-density CpG region that was found hypermethylated in the non-tumour- adjacent tissues and in low AFP tumours, but hypomethylated in RESULTS AFP-high tumours (p < 0.001) (Supplementary Fig. 2D). The AFP serum concentrations followed a logarithmic distribution in inverse correlation observed between AFP promoter methyla- our internal HEPTROMIC cohort, with values ranging from 0 to tion and AFP expression (HEPTROMIC/TCGA: R = −0.56/−0.49; 71770 ng/ml (Supplementary Fig. 2A). According to the well- p < 0.001/< 0.001) suggests that this mechanism may play a key established 400 ng/ml cut-off,2 only 12% (29/244) of patients with role in the aberrant overexpression of AFP in HCC. Indeed, TET1, early HCC presented high serum levels of AFP, which was an enzyme able to reverse the DNA methylation status,7 was one accompanied by aberrant overexpression of the gene in the of the top genes whose expression was found significantly tumour (FC = 40; p < 0.001) compared with the adjacent non- associated with hypomethylation of the AFP promoter (Supple- tumoral tissue (Supplementary Fig. 2B). In accordance with mentary Table 3). Whether this correlation means causation is to previous reports,2 high AFP serum concentration was found be determined. significantly associated with aggressive clinical–pathological In order to determine unique somatic derangements associated features, poor differentiation (Supplementary Tables 1–2) and with AFP-high tumours, HCC samples were analysed by poor overall survival (Supplementary Fig. 2C). whole-exome sequencing (Fig. 1a; Supplementary Fig. 3A, a >400 ≤400 AFP serum concentration AFP RNA expression p < 0.001 AFP promoter methylation p < 0.001 Somatic CTNNB1 mutation p = 0.014 alterations BAP1 mutation p = 0.031 1234567890();,: Molecular classes (Chiang) p < 0.001 Molecular Molecular classes (Hoshida) p < 0.001 classes Immune classes (Sia) p < 0.001 G1 class (Boyault) p < 0.001 Progenitor Hepatoblastoma (Cairo) p < 0.001 features CK19 (Villanueva) p < 0.001 EPCAM (Yamashita) p < 0.001 IGF1R (Tovar) p < 0.001 Signalling RB1 loss of function (Bollard) p < 0.001 pathways NOTCH (Villanueva) p < 0.001 mTOR (Villanueva) p < 0.001 Gene expression Chiang classes Hoshida Sia Mutations Non available Proliferation classes classes SNP c Low High CTNNB1 S1 Immune WT VEGFB VEGFA IFN-response S2 Intermediate PGF Methylation Poly-7 S3 Excluded Gene signatures AFP AFP AFP AFP Unnanotated Presence low high low high Hypo Hyper Absence b AFP high AFP low AFP serum concentration VEGF KEGG pathway (ssGSEA) p < 0.001 VEGF signalling Ramucirumab VEGFA RNA expression p = 0.114 pathway VEGFB RNA expression p < 0.001 VEGFC RNA expression p = 0.044 VEGFD RNA expression p = 0.445 VEGFR1 VEGFR2 PGF RNA expression p < 0.001 –1 0 1 Z score Survival No activity Angiogenesis Fig. 1 AFP-high HCCs show a distinct molecular profile. a Heatmap representation of the most relevant molecular features of AFP-high tumours (> 400 ng/ml) in comparison with AFP-low tumours in the TCGA cohort. AFP-high HCCs show higher AFP RNA expression and AFP promoter (TSS1500) hypomethylation. In terms of somatic alterations, AFP-high tumours are associated with less CTNNB1 mutations and a higher rate of BAP1 mutations. High AFP tumours are predicted to belong to the proliferation (Chiang) and S2 (Hoshida) classes and show a significant enrichment of signatures of HCC with progenitor features (G1 Boyault, Hepatoblastoma Cairo, CK19 Villanueva and EPCAM Yamashita). Finally, AFP-high tumours do not present the immune-excluded phenotype (Sia) and present overexpression of HCC signalling pathways (IGF1R Tovar, RB1 loss-of-function Bollard, NOTCH Villanueva and mTOR Villanueva). Continuous variables (AFP RNA expression and AFP promoter methylation) and categorical variables (the rest) were analysed by T test and Fisher’s exact test, respectively. b Heatmap representation of the VEGF KEGG pathway activation (inferred by single sample Gene Set Enrichment Analysis) and VEGF ligands RNA expression according to AFP serum concentration in the TCGA cohort. AFP-high tumours show higher enrichment of VEGF signalling and overexpression of VEGFB and PGF. The mean values of each phenotype (AFP high and low) have been normalised and represented as a Z score. c Schematic representation of the VEGF pathway in HCC according to AFP serum concentration. The overexpression of VEGFB and PGF ligands observed in AFP-high tumours might result in an enhanced activation of VEGFR1, and at the same time, prevent VEGFA from binding VEGFR1. The competition of VEGFA with the other ligands could favour its binding to VEGFR2, ultimately leading to its subsequent activation and release of pro-angiogenic signals. The administration of ramucirumab (a monoclonal antibody against VEGFR2) might misbalance VEGFA signalling towards a preferential binding of VEGFR1, where it has limited biological activity. Purple and orange lines represent VEGFB/PGF and VEGFA signalling, respectively Molecular portrait of high alpha-fetoprotein in hepatocellular carcinoma:. . . R Montal et al. 342 Supplementary Table 4). AFP-high tumours had fewer non-silent 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 CTNNB1 mutations (high = 14.1%, low = 29.9%; p = 0.009), a the paper for important intellectual content: R.P., D.S., V.M., M.E. and J.M.L. feature that has been associated with T-cell priming failure8 and resistance to immune checkpoint inhibitors.9 The lower rate of CTNNB1 mutations is in line with the observation that AFP-high ADDITIONAL INFORMATION tumours fall outside of the recently described Immune Exclusion Supplementary information is available for this paper at https://doi.org/10.1038/ class of HCC.8 Mutations statistically more prevalent in the AFP- s41416-019-0513-7. high group included the driver gene BAP1 (high = 8.5%, low = Competing interests: J.M.L. is receiving research support from Bayer HealthCare 1.6%; p = 0.009), a member of the polycomb-group proteins, Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb and Ipsen, and consulting fees from required for long-term silencing of genes that regulate the cell Bayer HealthCare Pharmaceuticals, Eli Lilly, Bristol-Myers Squibb, Eisai Inc, Celsion cycle and cellular differentiation.10 Corporation, Exelixis, Merck, Ipsen, Glycotest, Navigant, Leerink Swann LLC, Midatech Aiming to explore the putative link between high AFP levels Ltd, Fortress Biotech, Sprink Pharmaceuticals, Nucleix and Can-Fite Biopharma. Manel and targetable phenotypic traits, we evaluated the enrichment of Esteller reports grants from Ferrer International SA and Quimatryx. Augusto signalling pathways and previously reported molecular classes of Villanueva reports personal fees from Exelixis, Health Advances LLC, GroupH, Gerson HCCs1 (Fig. 1a; Supplementary Fig. 3A). High AFP tumours were Lehrman Group and Exact Sciences. particularly associated with the proliferation and the S2 classes, with a consistent enrichment of gene signatures defining Ethics approval and consent to participate: The institutional review boards of the progenitor features and overexpression of the known epidriver participating centres (IRCCS Istituto Nazionale Tumori [Milan] and Hospital Clínic IGF211 (Supplementary Table 5) when compared with AFP-low [Barcelona]) approved the study. Patients provided written informed consent. The study was conducted in accordance with International Standards of Good Clinical tumours. Moreover, the targetable signalling pathways IGF1R, Practice and the Declaration of Helsinki. NOTCH and mTOR were upregulated in AFP-high tumours. On the other hand, the RB1 loss-of-function signature (designed to Funding: Robert Montal is supported by a FSEOM-Boehringer Ingelheim Grant. predict the absence of benefit to CDK4/6 inhibitors) was also a Carmen Andreu-Oller has received financial support through the “la Caixa” INPhINIT key characteristic of AFP-high tumours. Finally, we identified VEGF Fellowship Grant for Doctoral studies at Spanish Research Centres of Excellence, from pathway enrichment in AFP-high tumours (Fig. 1b; Supplementary “la Caixa” Banking Foundation (ID 100010434), fellowship code (LCF/BQ/IN17/ Fig. 3B). While analysing the RNA expression of VEGF receptor 11620024). Laia Bassaganyas was supported by Beatriu de Pinós grant from Agència ligands, we observed overexpression of VEGFB and PGF, but not de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, Generalitat de Catalunya). Roger VEGFA. As previously reported,12,13 VEGFB and PGF compete with Esteban-Fabró is supported by MICINN/MINECO (BES-2017-081286). Carla Montironi VEGFA for the binding of VEGFR1. 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 DISCUSSION supported by U.S. Department of Defense (CA150272P3) and Tisch Cancer Institute In this study, we confirm the aberrant tumour overexpression of (Cancer Center Grant P30-CA196521). Daniela Sia is supported by the Gilead Sciences AFP in those patients with serum concentrations above 400 ng/ml Research Scholar Program in Liver Disease. Vincenzo Mazzaferro is supported by and propose DNA methylation of its promoter as the driving grants from Associazione Italiana per la Ricerca sul Cancro and the Oncology mechanism of such overexpression. AFP-high tumours show a Research Project of the Italian Ministry of Health. Manel Esteller is supported by the distinct phenotype characterised by poor differentiation, enrich- Department of Health PERIS project SLT/002/16/00374 and AGAUR projects ment of progenitor features and enhanced proliferation. All these 2017SGR1080, 2014SGR633 and 2009SGR1315 of the Catalan Government (General- aggressive characteristics are in line with its known prognostic itat de Catalunya); the Spanish Institute of Health Carlos III (ISCIII) with project DTS16/ capacity and explain why the percentage of AFP > 400 ng/ml 00153 and the Integrated Project of Excellence PIE13/00022 (ONCOPROFILE), and the Ministerio de Economía y Competitividad (MINECO) grant SAF2014-55000-R, co- tumours increases with disease progression (from 9% in BCLC-A to financed by the European Development Regional Fund “A way to achieve Europe” 42% in BCLC-C) (Supplementary Table 1). This is relevant since (ERDF); CIBER 2016 CB16/12/00312 (CIBERONC); the Cellex Foundation; “la Caixa” patients at advanced stages are the ones treated with systemic Banking Foundation (LCF/PR/PR15/11100003). Josep M. Llovet is supported by therapies. In this regard, the inclusion in this study of mostly early- National Cancer Institute (P30-CA196521), U.S. Department of Defense (CA150272P3), stage HCCs treated with surgical resection may partially hamper to European Commission/Horizon 2020 Program (HEPCAR, Ref. 667273-2), EIT Health understand the complex biological properties of advanced (CRISH2, Ref. 18053), Accelerator Award (CRUCK, AECC and AIRC) (HUNTER, Ref. HCC. Nevertheless, we propose the VEGF ligands/receptors C9380/A26813), Samuel Waxman Cancer Research Foundation, Centro de Investiga- interplay12,13 (unbalanced in AFP-high tumours due to VEGFB/ cion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd)—ISCIII, PGF overexpression) as a rationale for the enhanced activation of Spanish National Health Institute (SAF2016-76390) and the Generalitat de Catalunya/ AGAUR (SGR-1358). the VEGF pathway and thus the efficacy of ramucirumab in AFP- high HCC3 (Fig. 1c). Other signalling pathways significantly deregulated in AFP-high tumours and worthy of further analysis Data availability: Data from the internal HEPTROMIC cohort are stored in the Gene Expression Omnibus (GEO) repository: whole-genome expression (GSE63898) and include IGF2–IGFR, mTOR, NOTCH and BAP1. DNA methylation (GSE56588). Whole-exome sequencing was deposited in the EGA In conclusion, the aberrant overexpression of targetable database (accessions EGAS00001000217, EGAS00001000679 and EGAS00001001002) molecular signalling pathways in HCC patients with high AFP and the ICGC data portal. Data from the external publicly available TCGA cohort were suggests that the measurement of its serum level might serve as a downloaded from www.cbioportal.org. noninvasive predictive tool for biomarker-based clinical trials with targeted therapies. Note: This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0). ACKNOWLEDGEMENTS This study has been in part developed at the building Centre Esther Koplowitz from Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims IDIBAPS/CERCA Programme/Generalitat de Catalunya. in published maps and institutional affiliations. AUTHOR CONTRIBUTIONS REFERENCES Study concept and design: R.M., R.P., D.S. and J.M.L. Acquisition, analysis or 1. Llovet, J. M., Montal, R., Sia, D. & Finn, R. S. Molecular therapies and precision 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 medicine for hepatocellular carcinoma. Nat. Rev. Clin. Oncol. 15, 599–616 (2018). Molecular portrait of high alpha-fetoprotein in hepatocellular carcinoma:. . . R Montal et al. 343 2. EASL Clinical Practice Guidelines. Management of hepatocellular carcinoma. J 8. 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