Abstract A07: Gene analysis maps HCC heterogeneity and orientates personalized therapy
Authors: Critelli Rosina Maria , Milosa Fabiola , Lei Barbara , Marzi Luca , Condello Rosario , Turola Elena , Maria Nicola De , Maiorana Antonino , Giannelli Gianluigi , Villa Erica 
Published: May 2016

Hepatocellular carcinoma (HCC) has variable prognosis depending both on the severity of underlying liver disease and of its biological characteristic. We recently identified, in a prospective series of HCCs at first presentation, a 5-genes transcriptomic signature, which accurately and significantly predicts growth speed and survival (Gut. 2015 Feb 9. pii:gutjnl-2014-308483. doi: 10.1136/gutjnl-2014-308483). This signature encounters 5 up-regulated genes (ANGPT2, DLL4, NETO2, ESM1, NR4A1), all associated with neo-angiogenesis. Such gene analysis prompted us to characterize further the molecular pathways underlying tumor aggressiveness and consequently clinical outcome.

The cohort of 78 prospectively identified HCCs was further characterized. Transcriptomic analysis was performed by microarray experiments (Agilent Technologies, Palo Alto, CA; Genomics Service Department of Miltenyi Biotec GmbH Bergisch Gladbach, Germany). Circulating cytokines were measured using human Quantikine® ELISA kit (R&D Systems, Minneapolis, MN, USA). For PD-1 and PD-L1 immunohistochemistry, samples were incubated with mouse monoclonal antibody (Ventana), for 24 minutes at 37°C. Biological data were analyzed according to HCC growth speed and patients’ survival.

Genes down- or up-regulated affected prognosis and survival. In particular, the worst survival was correlated with down-regulation of CLEC4G and CLEC1B and up-regulation of MMP1, MMP10, MMP12 and WNT11. Next, we also measured a large panel of circulating cytokines tested, and among them TGF-β1 was the only significantly and positively associated with HCC aggressiveness. PD-1 and PD-L1 were strongly co-expressed especially at the boundary between tumoral and non-tumoral cirrhotic tissue but only in the most aggressive type. In slow-growing HCCs, PD-1/PD-L1 were only faintly expressed.

Bringing together all these results, the most aggressive and associated with a worst clinical outcome aggressively HCCs were characterized by genes regulating different molecular pathways, including neo-angiogenesis (ANGPT2, DLL4, NETO2, ESM1, NR4A1and, indirectly, CLEC4G and CLEC1B), tumoral spread (MMP1, MMP10, MMP12), tissue remodelling, loss of immune local control (hyperactivated PD-1 and PD-1L). Aberrant PD-L1 expression in cancers facilitates escape from immune attack. PD-1 ligation by PD-L1 down-modulates anti-tumor immune effector functions allowing the development of a subset of hyper-aggressive HCCs. All such different pathways contribute to explain HCC heterogeneity, leading to therapeutic failures, but at the same time, stimulate a better stratification of patients directing the options for personalized therapies.

Citation Format: Rosina Maria Critelli, Fabiola Milosa, Barbara Lei, Luca Marzi, Rosario Condello, Elena Turola, Nicola De Maria, Antonino Maiorana, Gianluigi Giannelli, Erica Villa. Gene analysis maps HCC heterogeneity and orientates personalized therapy. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A07.