Digital-resolution and highly sensitive detection of multiple exosomal small RNAs by DNA toehold probe-based photonic resonator absorption microscopy.

Small noncoding RNAs (snRNA) have been emerging as promising diagnostic biomarkers for detecting early stage cancer. Currently existing methods for snRNA detection, including northern blot, reverse transcription-polymerase chain reaction, microarrays and RNA-Seq, are limited to time-consuming, low sensitivity, expensive instrumentation or complex analysis of data. Herein, we present a rapid quantitative analysis of multiple liver cancer-associated exosomal snRNA by a nucleic acid toehold probe-based photonic resonator absorption microscopy (PRAM) assay, with digital resolution and high sensitivity. The assay relies on the use of three toehold probe-encoded gold nanoparticles (AuNPs) and addressable photonic crystal (PC) sensing chips. The presence of target snRNA will initiate toehold-mediated strand displacement reactions that trigger the capture of gold particles onto the PC surface, which is subsequently imaged by PRAM for digital counting of detected snRNA molecules. We achieved highly sensitive and selective detection of three snRNA targets in buffer with a 30 min assay protocol, with detection limits of 4.56 fM, 4.68 fM and 0.69 pM. Having confirmed our assay's performance for detection of snRNA targets spiked into exosomal RNA extracts, we demonstrated its capability for quantitative detection of the same targets from patient blood plasma samples. The approach offers a rapid, simple workflow that operates at room temperature with a single step without enzymatic amplification, while the detection instrument can be implemented as a low-cost portable system for point of care environments.

Unannotated small RNA clusters associated with circulating extracellular vesicles detect early stage liver cancer.

OBJECTIVE: Surveillance tools for early cancer detection are suboptimal, including hepatocellular carcinoma (HCC), and biomarkers are urgently needed. Extracellular vesicles (EVs) have gained increasing scientific interest due to their involvement in tumour initiation and metastasis; however, most extracellular RNA (exRNA) blood-based biomarker studies are limited to annotated genomic regions. DESIGN: EVs were isolated with differential ultracentrifugation and integrated nanoscale deterministic lateral displacement arrays (nanoDLD) and quality assessed by electron microscopy, immunoblotting, nanoparticle tracking and deconvolution analysis. Genome-wide sequencing of the largely unexplored small exRNA landscape, including unannotated transcripts, identified and reproducibly quantified small RNA clusters (smRCs). Their key genomic features were delineated across biospecimens and EV isolation techniques in prostate cancer and HCC. Three independent exRNA cancer datasets with a total of 479 samples from 375 patients, including longitudinal samples, were used for this study. RESULTS: ExRNA smRCs were dominated by uncharacterised, unannotated small RNA with a consensus sequence of 20 nt. An unannotated 3-smRC signature was significantly overexpressed in plasma exRNA of patients with HCC (p<0.01, n=157). An independent validation in a phase 2 biomarker case-control study revealed 86% sensitivity and 91% specificity for the detection of early HCC from controls at risk (n=209) (area under the receiver operating curve (AUC): 0.87). The 3-smRC signature was independent of alpha-fetoprotein (p<0.0001) and a composite model yielded an increased AUC of 0.93. CONCLUSION: These findings directly lead to the prospect of a minimally invasive, blood-only, operator-independent clinical tool for HCC surveillance, thus highlighting the potential of unannotated smRCs for biomarker research in cancer.

Transcriptomic characterization of cancer-testis antigens identifies MAGEA3 as a driver of tumor progression in hepatocellular carcinoma.

Cancer testis antigens (CTAs) are an extensive gene family with a unique expression pattern restricted to germ cells, but aberrantly reactivated in cancer tissues. Studies indicate that the expression (or re-expression) of CTAs within the MAGE-A family is common in hepatocellular carcinoma (HCC). However, no systematic characterization has yet been reported. The aim of this study is to perform a comprehensive profile of CTA de-regulation in HCC and experimentally evaluate the role of MAGEA3 as a driver of HCC progression. The transcriptomic analysis of 44 multi-regionally sampled HCCs from 12 patients identified high intra-tumor heterogeneity of CTAs. In addition, a subset of CTAs was significantly overexpressed in histologically poorly differentiated regions. Further analysis of CTAs in larger patient cohorts revealed high CTA expression related to worse overall survival and several other markers of poor prognosis. Functional analysis of MAGEA3 was performed in human HCC cell lines by gene silencing and in a genetic mouse model by overexpression of MAGEA3 in the liver. Knockdown of MAGEA3 decreased cell proliferation, colony formation and increased apoptosis. MAGEA3 overexpression was associated with more aggressive tumors in vivo. In conclusion MAGEA3 enhances tumor progression and should be considered as a novel therapeutic target in HCC.

Signaling pathways in hepatocellular carcinoma.

Despite the recent introduction of new effective systemic agents, the survival of patients with hepatocellular carcinoma (HCC) at advanced stages remains dismal. This underscores the need for new therapies, which has spurred extensive research on the identification of the main drivers of pathway de-regulation as a source of novel therapeutic targets. Frequently altered pathways in HCC involve growth factor receptors (e.g., VEGFR, FGFR, TGFA, EGFR, IGFR) and/or its cytoplasmic intermediates (e.g., PI3K-AKT-mTOR, RAF/ERK/MAPK) as well as key pathways in cell differentiation (e.g., Wnt/ß-catenin, JAK/STAT, Hippo, Hedgehog, Notch). Somatic mutations, chromosomal aberrations and epigenetic changes are common mechanisms for pathway deregulation in HCC. Aberrant pathway activation has also been explored as a biomarker to predict response to specific therapies, but currently, these strategies are not implemented when deciding systemic therapies in HCC patients. Beyond the well-established molecular cascades, there are numerous emerging signaling pathways also deregulated in HCC (e.g., tumor microenvironment, non-coding RNA, intestinal microbiota), which have opened new avenues for therapeutic exploration.

DNA Methylation Profiling of Human Hepatocarcinogenesis.

BACKGROUND AND AIMS: Mutations in TERT (telomerase reverse transcriptase) promoter are established gatekeepers in early hepatocarcinogenesis, but little is known about other molecular alterations driving this process. Epigenetic deregulation is a critical event in early malignancies. Thus, we aimed to (1) analyze DNA methylation changes during the transition from preneoplastic lesions to early HCC (eHCC) and identify candidate epigenetic gatekeepers, and to (2) assess the prognostic potential of methylation changes in cirrhotic tissue. APPROACH AND RESULTS: Methylome profiling was performed using Illumina HumanMethylation450 (485,000 cytosine-phosphateguanine, 96% of known cytosine-phosphateguanine islands), with data available for a total of 390 samples: 16 healthy liver, 139 cirrhotic tissue, 8 dysplastic nodules, and 227 HCC samples, including 40 eHCC below 2cm. A phylo-epigenetic tree derived from the Euclidean distances between differentially DNA-methylated sites (n = 421,997) revealed a gradient of methylation changes spanning healthy liver, cirrhotic tissue, dysplastic nodules, and HCC with closest proximity of dysplasia to HCC. Focusing on promoter regions, we identified epigenetic gatekeeper candidates with an increasing proportion of hypermethylated samples (beta value > 0.5) from cirrhotic tissue (<1%), to dysplastic nodules (≥25%), to eHCC (≥50%), and confirmed inverse correlation between DNA methylation and gene expression for TSPYL5 (testis-specific Y-encoded-like protein 5), KCNA3 (potassium voltage-gated channel, shaker-related subfamily, member 3), LDHB (lactate dehydrogenase B), and SPINT2 (serine peptidase inhibitor, Kunitz type 2) (all P < 0.001). Unsupervised clustering of genome-wide methylation profiles of cirrhotic tissue identified two clusters, M1 and M2, with 42% and 58% of patients, respectively, which correlates with survival (P < 0.05), independent of etiology. CONCLUSIONS: Genome-wide DNA-methylation profiles accurately discriminate the different histological stages of human hepatocarcinogenesis. We report on epigenetic gatekeepers in the transition between dysplastic nodules and eHCC. DNA-methylation changes in cirrhotic tissue correlate with clinical outcomes.

Mutations in circulating tumor DNA predict primary resistance to systemic therapies in advanced hepatocellular carcinoma.

Little is known about the mutational landscape of advanced hepatocellular carcinoma (HCC), and predictive biomarkers of response to systemic therapies are lacking. We aimed to describe the mutational landscape of advanced HCC and to identify predictors of primary resistance to systemic therapies using circulating tumor DNA (ctDNA). We prospectively enrolled 121 patients between October 2015 and January 2019. We performed targeted ultra-deep sequencing of 25 genes and Digital Droplet PCR of TERT promoter, including sequential samples throughout treatment. Primary endpoint was progression-free survival (PFS) stratified by mutation profiles in ctDNA. Secondary endpoints were overall survival and objective response rate. The most frequent mutations in ctDNA of advanced HCC were TERT promoter (51%), TP53 (32%), CTNNB1 (17%), PTEN (8%), AXIN1, ARID2, KMT2D, and TSC2 (each 6%). TP53 and CTNNB1 mutations were mutually exclusive. Patients with mutations in the PI3K/MTOR pathway had significantly shorter PFS than those without these mutations after tyrosine kinase inhibitors (2.1 vs 3.7 months, p < 0.001), but not after immune checkpoint inhibition (CPI). WNT pathway mutations were not associated with PFS, overall survival, or objective response after CPI. Serial profiling of ctDNA in a subset correlated with treatment response. Mutation profiling of ctDNA in advanced HCC shows similar mutation frequencies for known HCC drivers compared to early stages and identifies predictive biomarkers of response to systemic therapies.

Liquid biopsy in the clinical management of hepatocellular carcinoma.

With increasing knowledge on molecular tumour information, precision oncology has revolutionised the medical field over the past years. Liquid biopsy entails the analysis of circulating tumour components, such as circulating tumour DNA, tumour cells or tumour-derived extracellular vesicles, and has thus come as a handy tool for personalised medicine in many cancer entities. Clinical applications under investigation include early cancer detection, prediction of treatment response and molecular monitoring of the disease, for example, to comprehend resistance patterns and clonal tumour evolution. In fact, several tests for blood-based mutation profiling are already commercially available and have entered the clinical field.In the context of hepatocellular carcinoma, where access to tissue specimens remains mostly limited to patients with early stage tumours, liquid biopsy approaches might be particularly helpful. A variety of translational liquid biopsy studies have been carried out to address clinical needs, such as early hepatocellular carcinoma detection and prediction of treatment response. To this regard, methylation profiling of circulating tumour DNA has evolved as a promising surveillance tool for early hepatocellular carcinoma detection in populations at risk, which might soon transform the way surveillance programmes are implemented. This review summarises recent developments in the liquid biopsy oncological space and, in more detail, the potential implications in the clinical management of hepatocellular carcinoma. It further outlines technical peculiarities across liquid biopsy technologies, which might be helpful for interpretation by non-experts.

Steatosis as main determinant of portal hypertension through a restriction of hepatic sinusoidal area in a dietary rat nash model.

BACKGROUND & AIMS: Portal hypertension (PH) can be present in pre-cirrhotic stages, even in absence of fibrosis in non-alcoholic steatohepatitis (NASH) patients. Liver endothelial dysfunction (ED) has been shown as responsible for this effect in short-term dietary animal models. We evaluated the persistence of PH and underlying mechanisms in a long-term rat model of NASH. METHODS: Sprague-Dawley rats were fed 8 or 36 weeks with control diet or high-fat high-glucose/fructose diet. Metabolic parameters, histology, ED and haemodynamics were characterized. Structural characteristics of liver sections were analysed using image analysis. RESULTS: Both interventions reproduced NASH histological hallmarks (with steatosis being particularly increased at 36 weeks), but neither induced fibrosis. The 36-week intervention induced a significant increase in portal pressure (PP) compared to controls (12.1 vs 8.7 mmHg, P < .001) and the 8-week model (10.7 mmHg, P = .006), but all features of ED were normalized at 36 weeks. Image analysis revealed that the increased steatosis at 36-week was associated to an increase in hepatocyte area and a significant decrease in the sinusoidal area, which was inversely correlated with PP. The analysis provided a critical sinusoidal area above which animals were protected from developing PH and below which sinusoidal flux was compromised and PP started to increase. CONCLUSION: Liver steatosis per se (in absence of fibrosis) can induce PH through a decrease in the sinusoidal area secondary to the increase in hepatocyte area in a long-term diet-induced rat model of NASH. Image analysis of the sinusoidal area might predict the presence of PH.

Intratumoral heterogeneity and clonal evolution in liver cancer.

Clonal evolution of a tumor ecosystem depends on different selection pressures that are principally immune and treatment mediated. We integrate RNA-seq, DNA sequencing, TCR-seq and SNP array data across multiple regions of liver cancer specimens to map spatio-temporal interactions between cancer and immune cells. We investigate how these interactions reflect intra-tumor heterogeneity (ITH) by correlating regional neo-epitope and viral antigen burden with the regional adaptive immune response. Regional expression of passenger mutations dominantly recruits adaptive responses as opposed to hepatitis B virus and cancer-testis antigens. We detect different clonal expansion of the adaptive immune system in distant regions of the same tumor. An ITH-based gene signature improves single-biopsy patient survival predictions and an expression survey of 38,553 single cells across 7 regions of 2 patients further reveals heterogeneity in liver cancer. These data quantify transcriptomic ITH and how the different components of the HCC ecosystem interact during cancer evolution.

Tumour evolution in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, typically develops on the background of chronic liver disease and is an aggressive disease with dismal prognosis. Studies using next-generation sequencing of multiple regions of the same tumour nodule suggest different patterns of HCC evolution and confirm the high molecular heterogeneity in a subset of patients. Different hypotheses have been proposed to explain tumour evolution, including clonal selection or neutral and punctuated acquisition of genetic alterations. In parallel, data indicate a fundamental contribution of nonmalignant cells of the tumour microenvironment to cancer clonal evolution. Delineating these dynamics is crucial to improve the treatment of patients with HCC, and particularly to help understand how HCC evolution drives resistance to systemic therapies. A number of new minimally invasive techniques, such as liquid biopsies, could help track cancer evolution in HCC. These tools might improve our understanding of how systemic therapies affect tumour clonal composition and could facilitate implementation of real-time molecular monitoring of patients with HCC.

Restoration of a healthy intestinal microbiota normalizes portal hypertension in a rat model of nonalcoholic steatohepatitis.

Portal hypertension (PH) drives most of the clinical complications in chronic liver diseases. However, its progression in nonalcoholic steatohepatitis (NASH) and its association with the intestinal microbiota (IM) have been scarcely studied. Our aim was to investigate the role of the IM in the mechanisms leading to PH in early NASH. The experimental design was divided in two stages. In stage 1, Sprague-Dawley rats were fed for 8 weeks a high-fat, high-glucose/fructose diet (HFGFD) or a control diet/water (CD). Representative rats were selected as IM donors for stage 2. In stage 2, additional HFGFD and CD rats underwent intestinal decontamination, followed by IM transplantation with feces from opposite-diet donors (heterologous transplant) or autologous fecal transplant (as controls), generating four groups: CD-autotransplanted, CD-transplanted, HFGFD-autotransplanted, HFGFD-transplanted. After IM transplantation, the original diet was maintained for 12-14 days until death. HFGFD rats developed obesity, insulin resistance, NASH without fibrosis but with PH, intrahepatic endothelial dysfunction, and IM dysbiosis. In HFGFD rats, transplantation with feces from CD donors caused a significant reduction of PH to levels comparable to CD without significant changes in NASH histology. The reduction in PH was due to a 31% decrease of intrahepatic vascular resistance compared to the HFGFD-autotransplanted group (P < 0.05). This effect occurs through restoration of the sensitivity to insulin of the hepatic protein kinase B-dependent endothelial nitric oxide synthase signaling pathway. CONCLUSION: The IM exerts a direct influence in the development of PH in rats with diet-induced NASH and dysbiosis; PH, insulin resistance, and endothelial dysfunction revert when a healthy IM is restored. (Hepatology 2018;67:1485-1498).

A Nitric Oxide-Donating Statin Decreases Portal Pressure with a Better Toxicity Profile than Conventional Statins in Cirrhotic Rats.

Statins present many beneficial effects in chronic liver disease, but concerns about safety exist. We evaluated the hepatic effects of a nitric oxide-releasing atorvastatin (NCX 6560) compared to conventional statins. Simvastatin, atorvastatin and NCX 6560 were evaluated in four-week bile duct-ligated rats (BDL) simulating decompensated cirrhosis and in thirteen-week carbon tetrachloride (CCl4) intoxicated rats, a model of early cirrhosis. In the BDL model, simvastatin treated rats showed high mortality and the remaining animals presented muscular and hepatic toxicity. At equivalent doses, NCX 6560 eliminated hepatic toxicity and reduced muscular toxicity (60-74%) caused by atorvastatin in the more advanced BDL model; toxicity was minimal in the CCl4 model. Atorvastatin and NCX 6560 similarly reduced portal pressure without changing systemic hemodynamics in both models. Atorvastatin and NCX 6560 caused a mild decrease in liver fibrosis and inflammation and a significant increase in intrahepatic cyclic guanosine monophosphate. NCX 6560 induced a higher intrahepatic vasoprotective profile (activated endothelial nitric oxide synthase and decreased platelet/endothelial cell adhesion molecule-1), especially in the CCl4 model, suggesting a higher benefit in early cirrhosis. In conclusion, NCX 6560 improves the liver profile and portal hypertension of cirrhotic rats similarly to conventional statins, but with a much better safety profile.

Cerebellar neurodegeneration in a new rat model of episodic hepatic encephalopathy.

Hepatic encephalopathy has traditionally been considered a reversible disorder. However, recent studies suggested that repeated episodes of hepatic encephalopathy cause persistent impairment leading to neuronal loss. The aims of our study were the development of a new animal model that reproduces the course of episodic hepatic encephalopathy and the identification of neurodegeneration evidences. Rats with portacaval anastomosis underwent simulated episodes of hepatic encephalopathy, triggered by the regular administration of ammonium acetate, and/or lipopolysaccharide. The neurological status was assessed and neuronal loss stereologically quantified in motor areas. During the simulated episodes, ammonia induced reversible motor impairment in portacaval anastomosis rats. In cerebellum, stereology showed a reduction in Purkinje cell population in portacaval anastomosis and PCA+NH3 groups and morphological changes. An increase in astrocyte size in PCA+NH3 group and activated microglia in groups treated with ammonium acetate and/or lipopolysaccharide was observed. A modulation of neurodegeneration-related genes and the presence of apoptosis in Bergmann glia were observed. This new animal model reproduces the clinical course of episodic hepatic encephalopathy when ammonia is the precipitant factor and demonstrates the existence of neuronal loss in cerebellum. The persistence of over-activated microglia and reactive astrocytes could participate in the apoptosis of Bergmann glia and therefore Purkinje cell degeneration.

Gene expression profiling of brain cortex microvessels may support brain vasodilation in acute liver failure rat models.

Development of brain edema in acute liver failure can increase intracranial pressure, which is a severe complication of the disease. However, brain edema is neither entirely cytotoxic nor vasogenic and the specific action of the brain microvasculature is still unknown. We aimed to analyze gene expression of brain cortex microvessels in two rat models of acute liver failure. In order to identify global gene expression changes we performed a broad transcriptomic approach in isolated brain cortex microvessels from portacaval shunted rats after hepatic artery ligation (HAL), hepatectomy (HEP), or sham by array hybridization and confirmed changes in selected genes by RT-PCR. We found 157 and 270 up-regulated genes and 143 and 149 down-regulated genes in HAL and HEP rats respectively. Western blot and immunohistochemical assays were performed in cortex and ELISA assays to quantify prostaglandin E metabolites were performed in blood of the sagittal superior sinus. We Identified clusters of differentially expressed genes involving inflammatory response, transporters-channels, and homeostasis. Up-regulated genes at the transcriptional level were associated with vasodilation (prostaglandin-E synthetase, prostaglandin-E receptor, adrenomedullin, bradykinin receptor, adenosine transporter), oxidative stress (hemoxygenase, superoxide dismutase), energy metabolism (lactate transporter) and inflammation (haptoglobin). The only down-regulated tight junction protein was occludin but slightly. Prostaglandins levels were increased in cerebral blood with progression of liver failure. In conclusion, in acute liver failure, up-regulation of several genes at the level of microvessels might suggest an involvement of energy metabolism accompanied by cerebral vasodilation in the cerebral edema at early stages.