Reply to: ''Network-based discovery of gene signature for vascular invasion prediction in HCC''

To the Editor:Liu and colleagues raise some issues regarding our recently pub-lished study [1] to which we would like to make the followingcomments. We acknowledge the limitations that a gene-expres-sion-based biomarker could have, and that our signature is notunique. Certainly, previous attempts to find such a signature havebeen published in the past [2]. We also know that, as in othergene expression studies, potential bias could occur. In fact,reported prognostic signatures are often not reproducible, inmost of the cases due to suboptimal study design, small samplesizes, and also because many of them have been based on retro-spectively collected tissue samples [3]. Even after taking intoaccount these sources of bias or inconsistencies, it so happensthat only a small minority of the reported signatures truly retainprognostic significance. In fact, our recent outcome analysisincluding 22 gene signatures with prognostic significance inHCC (18 from the tumor, and four from the non-tumoral adjacenttissue) showed that only two signatures retained independentprognostic value [4].In our work, we select a training cohort based upon a homo-geneous etiology to minimize the risk of molecular heterogene-ity and to identify a clean and distinct signature. Patients withHCV-related HCC were selected, since this is the most commonetiology in the Western countries. Then, we validate the signa-ture in an independent multi-etiologic cohort of patients andthe accuracy remained stable when an etiology-dependent sub-group analysis was performed [1]. The study was aimed at pro-viding a gene-set to ease the preoperative diagnosis of vascularinvasion, but was not designed for defining outcome prediction.Nonetheless, we have data indicating that the presence of a vas-cular invasion signature correlates with poor outcome, since thesignature was found to be associated with early recurrence(p = 0.057), and was enriched in patients sharing signatures ofpoor prognosis [4].Even considering that the question posed is simple (to identifya gene-signature capturing vascular invasion) the characteristicsof patients, sample collection, sampling issues, technical varia-tion, validation of results, and bioinformatics approaches are cer-tainly heterogeneous, and thus the results might vary. In mostinstances, however, the different signatures seem to be able tocapture common oncogenic mechanisms, as reflected by theircapacity to adequately allocate patients into a poor or good prog-nosis group [5]. By applying a different methodological approach(weighted gene co-expression network analysis) to our data, Liuet al. provide a 9-gene signature with similar accuracy and nooverlap with our 35-gene signature. The method applied is basedon systems biology to find clusters of highly correlated genesacross microarray samples, identify hubs of each module and cor-relate them with clinical traits [6]. This analysis is based on thehypothesis that information on signaling pathways is crucial tounderstand how genes are connected to each other and how theyinfluence cellular functions in both normal and cancer conditions.This result further underlines the need for integrating the vastamount of available data and the development of powerful bioin-formatics resources (annotation, methodologies, technical plat-forms, etc.).A more relevant question is when can our signature-alone orin combination with tumor size- be translated into clinical prac-tice. Strict rules have been proposed recently by Simon and col-leagues [7]. Following this proposal, the EASL-EORTC guidelineson management of HCC have outlined a list of requirements inorder to adopt molecular signatures in the clinical practice [8],which are as follows:1. First, the signature should be generated in the setting of ran-domized studies or in case of cohort studies, it should followthe training/validation approach.2. The signature should retain independent prognostic valuewhen tested along known clinico-pathological variables.3. The results should be confirmed by independent investigatorsin a separate set of samples.Thus, according to these rules, in order to implement our sig-nature in the decision-making process, for instance in the waitinglist of liver transplantation, it should be validated by independentinvestigators in a novel set of samples. Ideally, the signature hasto be reproduced in a device, which should give similar results.Only then, data is ready for acceptance in guidelines. It is a longpath, but the only one for translation of genomic results into ourpractice.Conflict of interestThe authors declared that they do not have anything to discloseregarding funding or conflict of interest with respect to thismanuscript.Reference

Immune Exclusion-Wnt/CTNNB1 Class Predicts Resistance to Immunotherapies in HCC

Next-generation sequencing has provided information on actionable targets and biomarkers of response in oncology. In hepatocellular carcinoma (HCC), Wnt/CTNNB1 mutations characterize the immune-excluded class (cold tumors) and might represent the biomarkers predicting resistance to immune checkpoint inhibitors. Large-scale validation of these data is needed to customize immunotherapy in advanced HCC.See related article by Harding et al.©2019 American Association for Cancer Research

Liver Cancer Cell of Origin, Molecular Class, and Effects on Patient Prognosis

Primary liver cancer is the second leading cause of cancer-related death worldwide and therefore a major public health challenge. We review hypotheses of the cell of origin of liver tumorigenesis and clarify the classes of liver cancer based on molecular features and how they affect patient prognosis. Primary liver cancer comprises hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (iCCA), and other rare tumors, notably fibrolamellar carcinoma and hepatoblastoma. The molecular and clinical features of HCC versus iCCA are distinct, but these conditions have overlapping risk factors and pathways of oncogenesis. A better understanding of the cell types originating liver cancer can aid in exploring molecular mechanisms of carcinogenesis and therapeutic options. Molecular studies have identified adult hepatocytes as the cell of origin. These cells have been proposed to transform directly into HCC cells (via a sequence of genetic alterations), to dedifferentiate into hepatocyte precursor cells (which then become HCC cells that express progenitor cell markers), or to transdifferentiate into biliary-like cells (which give rise to iCCA). Alternatively, progenitor cells also give rise to HCCs and iCCAs with markers of progenitor cells. Advances in genome profiling and next-generation sequencing have led to the classification of HCCs based on molecular features and assigned them to categories such as proliferation-progenitor, proliferation-transforming growth factor β, and Wnt-catenin β1. iCCAs have been assigned to categories of proliferation and inflammation. Overall, proliferation subclasses are associated with a more aggressive phenotype and poor outcome of patients, although more specific signatures have refined our prognostic abilities. Analyses of genetic alterations have identified those that might be targeted therapeutically, such as fusions in the FGFR2 gene and mutations in genes encoding isocitrate dehydrogenases (in approximately 60% of iCCAs) or amplifications at 11q13 and 6p21 (in approximately 15% of HCCs). Further studies of these alterations are needed before they can be used as biomarkers in clinical decision making

Molecular therapies and precision medicine for hepatocellular carcinoma

The global burden of hepatocellular carcinoma (HCC) is increasing and might soon surpass an annual incidence of 1 million cases. Genomic studies have established the landscape of molecular alterations in HCC; however, the most common mutations are not actionable, and only ~25% of tumours harbour potentially targetable drivers. Despite the fact that surveillance programmes lead to early diagnosis in 40–50% of patients, at a point when potentially curative treatments are applicable, almost half of all patients with HCC ultimately receive systemic therapies. Sorafenib was the first systemic therapy approved for patients with advanced-stage HCC, after a landmark study revealed an improvement in median overall survival from 8 to 11 months. New drugs — lenvatinib in the frontline and regorafenib, cabozantinib, and ramucirumab in the second line — have also been demonstrated to improve clinical outcomes, although the median overall survival remains ~1 year; thus, therapeutic breakthroughs are still needed. Immune-checkpoint inhibitors are now being incorporated into the HCC treatment armamentarium and combinations of molecularly targeted therapies with immunotherapies are emerging as tools to boost the immune response. Research on biomarkers of a response or primary resistance to immunotherapies is also advancing. Herein, we summarize the molecular targets and therapies for the management of HCC and discuss the advancements expected in the near future, including biomarker-driven treatments and immunotherapies

Translating '–omics' results into precision medicine for hepatocellular carcinoma

A large-scale comprehensive analysis of hepatocellular carcinoma (HCC) based on the integration of six distinct data platforms has pinpointed novel oncogenic processes and prognostic subgroups. These findings confirm previously identified molecular subclasses and fuel the need for a clear strategy of precision medicine in HCC

Cancer gene discovery in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a deadly cancer, whose incidence is increasing worldwide. Albeit the main risk factors for HCC development have been clearly identified, such as hepatitis B and C virus infection and alcohol abuse, there is still preliminary understanding of the key drivers of this malignancy. Recent data suggest that genomic analysis of cirrhotic tissue - the pre-neoplastic carcinogenic field - may provide a read-out to identify at risk populations for cancer development. Given this contextual complexity, it is of utmost importance to characterize the molecular pathogenesis of this disease, and pinpoint the dominant pathways/drivers by integrative oncogenomic approaches and/or sophisticated experimental models. Identification of the dominant proliferative signals and key aberrations will allow for a more personalized therapy

QUALITY CONTROL FOR ROMANIAN MEAT PRODUCTS

Food production, trade and consumption are now globalised. This provides multiple opportunities for harmful bacteria, viruses, parasites or chemical substances to enter the food chain and contaminate food prior to consumption.  Food safety may be viewed as a cross-cutting issue and all have a role to play, food producers, manufacturers, distributors and traders are responsible for the food they produce and trade. All products must be safe as possible from pathogens and other contaminants. This study presents an objective picture opposite the meat products quality control. The study results meet european and national regulations and standards for meat products

The portrait of liver cancer is shaped by mitochondrial genetics.

Cancer heterogeneity and evolution are not fully understood. Here, we show that mitochondrial DNA of the normal liver shapes tumor progression, histology, and immune environment prior to the acquisition of oncogenic mutation. Using conplastic mice, we show that mtDNA dictates the expression of the mitochondrial unfolded protein response (UPRmt) in the normal liver. Activation of oncogenic mutations in UPRmt-positive liver increases tumor incidence and histological heterogeneity. Further, in a subset of UPRmt-positive mice, invasive liver cancers develop. RNA sequencing (RNA-seq) analysis of the normal liver reveals that, in this subset, the PAPP-A/DDR2/SNAIL axis of invasion pre-exists along with elevated collagen. Since PAPP-A promotes immune evasion, we analyzed the immune signature and found that their livers are immunosuppressed. Further, the PAPP-A signature identifies the immune exhausted subset of hepatocellular carcinoma (HCC) in humans. Our data suggest that mtDNA of normal liver shapes the entire liver cancer portrait upon acquisition of oncogenic mutations.This work was supported by an RO1 AG059635 award from the NIH to D.G.S

An Immune Gene Expression Signature Associated With Development of Human Hepatocellular Carcinoma Identifies Mice That Respond to Chemopreventive Agents

Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis.We analyzed gene expression profiles of non-tumor liver tissues from 392 patients with early-stage HCC (training set, n=167 and validation set, n=225) and liver tissue from patients with cirrhosis without HCC (n=216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of N-nitrosodiethylamine followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then given orally the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, given orally aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet.We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of non-tumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor beta signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% 1.21-4.80). Mice with chemically-induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel downregulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle.We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that associates with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and developed fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis

Mixed hepatocellular cholangiocarcinoma tumors: Cholangiolocellular carcinoma is a distinct molecular entity

Background & Aims: Mixed hepatocellular cholangiocarcinoma (HCC-CCA) is a rare and poorly understood type of primary liver cancer. We aimed to perform a comprehensive molecular characterization of this malignancy.Methods: Gene expression profiling, DNA copy number detection, and exome sequencing using formalin-fixed samples from 18 patients with mixed HCC-CCA were performed, encompassing the whole histological spectrum of the disease. Comparative genomic analysis was carried out, using independent datasets of HCC (n = 164) and intrahepatic cholangiocarcinoma (iCCA) (n = 149).Results: Integrative genomic analysis of HCC-CCAs revealed that cholangiolocellular carcinoma (CLC) represents a distinct biliaryderived entity compared with the stem-cell and classical types. CLC tumors were neural cell adhesion molecule (NCAM) positive (6/6 vs. 1/12, p < 0.001), chromosomally stable (mean chromosomal aberrations 5.7 vs. 14.1, p = 0.008), showed significant upregulation of transforming growth factor (TGF)-beta signaling and enrichment of inflammation-related and immune response signatures (p < 0.001). Stem-cell tumors were characterized by spaltlike transcription factor 4 (SALL4) positivity (6/8 vs. 0/10, p < 0.001), enrichment of progenitor-like signatures, activation of specific oncogenic pathways (i.e., MYC and insulin-like growth factor [IGF]), and signatures related to poor clinical outcome. In the classical type, there was a significant correlation in the copy number variation of the iCCA and HCC components, suggesting a clonal origin. Exome sequencing revealed an average of 63 non-synonymous mutations per tumor (2 mean driver mutations per tumor). Among those, TP53 was the most frequently mutated gene (6/21, 29%) in HCC-CCAs.Conclusions: Mixed HCC-CCA represents a heterogeneous group of tumors, with the stem-cell type characterized by features of poor prognosis, and the classical type with common lineage for HCC and iCCA components. CLC stands alone as a distinct biliary-derived entity associated with chromosomal stability and active TGF-b signaling.Lay summary: Molecular analysis of mixed hepatocellular cholangiocarcinoma (HCC-CCA) showed that cholangiolocellular carcinoma (CLC) is distinct and biliary in origin. It has none of the traits of hepatocellular carcinoma (HCC). However, within mixed HCC-CCA, stem-cell type tumors shared an aggressive nature and poor outcome, whereas the classic type showed a common cell lineage for both the HCC and the intrahepatic CCA component. The pathological classification of mixed HCC-CCA should be redefined because of the new molecular data provided. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved