M.L. Martínez-Chantar

Microenvironment inflammatory infiltrate drives growth speed and outcome of hepatocellular carcinoma: a prospective clinical study

In HCC, tumor microenvironment, heavily influenced by the underlying chronic liver disease, etiology and stage of the tissue damage, affects tumor progression and determines the high heterogeneity of the tumor. Aim of this study was to identify the circulating and tissue components of the microenvironment immune-mediated response affecting the aggressiveness and the ensuing clinical outcome. We analyzed the baseline paired HCC and the surrounding tissue biopsies from a prospective cohort of 132 patients at the first diagnosis of HCC for immunolocalization of PD-1/PD-L1, FoxP3, E-cadherin, CLEC2 and for a panel of 82 microRNA associated with regulation of angiogenesis, cell proliferation, cell signaling, immune control and autophagy. Original microarray data were also explored. Serum samples were analyzed for a panel of 19 cytokines. Data were associated with biochemical data, histopathology and survival. Patients with a more aggressive disease and shorter survival, who we named fast-growing accordingly to the tumor doubling time, at presentation had significantly higher AFP levels, TGF-β1 and Cyphra 21-1 levels. Transcriptomic analysis evidenced a significant downregulation of CLEC2 and upregulation of several metalloproteinases. A marked local upregulation of both PD-1 and PD-L1, a concomitant FoxP3-positive lymphocytic infiltrate, a loss of E-cadherin, gain of epithelial–mesenchymal transition (EMT) phenotype and extreme poor differentiation at histology were also present. Upregulated microRNA in fast-growing HCCs are associated with TGF-β signaling, angiogenesis and inflammation. Our data show that fast HCCs are characterized not only by redundant neo-angiogenesis but also by unique features of distinctively immunosuppressed microenvironment, prominent EMT, and clear-cut activation of TGFβ1 signaling in a general background of long-standing and permanent inflammatory state.

A morphological method for ammonia detection in liver

Hyperammonemia is a metabolic condition characterized by elevated levels of ammonia and a common event in acute liver injury/failure and chronic liver disease. Even though hepatic ammonia levels are potential predictive factors of patient outcome, easy and inexpensive methods aiming at the detection of liver ammonia accumulation in the clinical setting remain unavailable. Thus, herein we have developed a morphological method, based on the utilization of Nessler´s reagent, to accurately and precisely detect the accumulation of ammonia in biological tissue. We have validated our method against a commercially available kit in mouse tissue samples and, by using this modified method, we have confirmed the hepatic accumulation of ammonia in clinical and animal models of acute and chronic advanced liver injury as well as in the progression of fatty liver disease. Overall, we propose a morphological method for ammonia detection in liver that correlates well with the degree of liver disease severity and therefore can be potentially used to predict patient outcome.

Liver Angiopoietin‐2 Is a Key Predictor of De Novo or Recurrent Hepatocellular Cancer After Hepatitis C Virus Direct‐Acting Antivirals

Recent reports suggested that direct acting antivirals (DAAs) might favor hepatocellular carcinoma (HCC). In study 1, we studied the proangiogenic liver microenvironment in 242 DAA‐treated chronic hepatitis C patients with advanced fibrosis. Angiopoietin‐2 (ANGPT2) expression was studied in tissue (cirrhotic and/or neoplastic) from recurrent, de novo, nonrecurrent HCC, or patients never developing HCC. Circulating ANGPT2,vascular endothelial growth factor (VEGF), and C‐reactive protein (CRP) were also measured. In study 2, we searched for factors associated with de novo HCC in 257 patients with cirrhosis of different etiologies enrolled in a dedicated prospective study. Thorough biochemical, clinical, hemodynamic, endoscopic, elastographic, and echo‐Doppler work‐up was performed in both studies. In study 1, no patients without cirrhosis developed HCC. Of 183 patients with cirrhosis, 14 of 28 (50.0%) with previous HCC recurred whereas 21 of 155 (13.5%) developed de novo HCC. Patients with recurrent and de novo HCCs had significantly higher liver fibrosis (LF) scores, portal pressure, and systemic inflammation than nonrecurrent HCC or patients never developing HCC. In recurrent/de novo HCC patients, tumor and nontumor ANGPT2 showed an inverse relationship with portal vein velocity (PVv; r = –0.412, P = 0.037 and r = –0.409, P = 0.047 respectively) and a positive relationship with liver stiffness (r = 0.526, P = 0.007; r = 0.525, P = 0.003 respectively). Baseline circulating VEGF and cirrhotic liver ANGPT2 were significantly related (r = 0.414, P = 0.044). VEGF increased during DAAs, remaining stably elevated at 3‐month follow‐up, when it significantly related with serum ANGPT2 (r = 0.531, P = 0.005). ANGPT2 expression in the primary tumor or in cirrhotic tissue before DAAs was independently related with risk of HCC recurrence (odds ratio [OR], 1.137; 95% confidence interval [CI], 1.044‐1.137; P = 0.003) or occurrence (OR, 1.604; 95% CI, 1.080‐2.382; P = 0.019). In study 2, DAA treatment (OR, 4.770; 95% CI, 1.395‐16.316; P = 0.013) and large varices (OR, 3.857; 95% CI, 1.127‐13.203; P = 0.032) were independent predictors of de novo HCC. Conclusion: Our study indicates that DAA‐mediated increase of VEGF favors HCC recurrence/occurrence in susceptible patients, that is, those with more severe fibrosis and splanchnic collateralization, who already have abnormal activation in liver tissues of neo‐angiogenetic pathways, as shown by increased ANGPT2. (Hepatology 2018; 00:000‐000).

Neddylation, a novel paradigm in liver cancer

Liver cancer is the sixth most prevailing cancer worldwide. Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, has a rather heterogeneous pathogenesis making it highly refractive to current therapeutic approaches. Hence, HCC patients have a poor and gloomy prognosis making liver cancer the second leading cause of global cancer-related deaths. On this basis, a more global mechanism, such as post-translational modifications (PTMs) of proteins, may provide a valuable therapeutic approach for HCC clinical management by simultaneously regulating multiple disrupted signaling pathways. In the last years, the ubiquitin-like molecule NEDD8 (Neural precursor cell-expressed developmentally downregulated-8) conjugation pathway, neddylation, was shown to be aberrant in HCC patients with a significant positive correlation found among global levels of neddylation and poorer prognosis. Even though the best-established role for NEDD8 is the activation of ubiquitin E3 ligase family of cullin-RING ligases, the putative role for other NEDD8 substrates has been explored in recent years leading to the identification of novel neddylation targets in HCC. Importantly, treatment with the small pharmacological inhibitor Pevonedistat (MLN4924) (Millennium Pharmaceuticals, Takeda Pharmaceutical), currently in clinical trials for the treatment of some types of leukemias and other advanced solid tumors, was shown to suppress the outgrowth of hepatoma cells and liver cancer in pre-clinical mouse models. Overall, considering that the neddylation inhibitor Pevonedistat was well-tolerated and displayed a significant antitumor effect in pre-clinical models, combinatory pharmacological treatment based on Pevonedistat are highly recommended to enter clinical trials targeting advanced HCC.