Purpose: Morphologic intratumor heterogeneity is well known to exist in hepatocellular carcinoma (HCC), but very few systematic analyses of this phenomenon have been performed. The aim of this study was to comprehensively characterize morphologic intratumor heterogeneity in HCC. Also, taken into account were well-known immunohistochemical markers and molecular changes in liver cells that are considered in proposed classifications of liver cell neoplasms or discussed as molecular therapeutic targets.Experimental Design: In HCC of 23 patients without medical pretreatment, a total of 120 tumor areas were defined. Analyzed were cell and tissue morphology, expression of the liver cell markers cytokeratin (CK)7, CD44, a-fetoprotein (AFP), epithelial cell adhesion molecule (EpCAM), and glutamine synthetase (GS) along with mutations of TP53 and CTNNB1, assayed by both Sanger and next-generation sequencing.Results: Overall, intratumor heterogeneity was detectable in the majority of HCC cases (20 of 23, 87%). Heterogeneity solely on the level of morphology was found in 6 of 23 cases (26%), morphologic heterogeneity combined with immunohistochemical heterogeneity in 9 of 23 cases (39%), and heterogeneity with respect to morphologic, immunohistochemical, and mutational status of TP53 and CTNNB1 in 5 of 23 cases (22%).Conclusions: Our findings demonstrate that intratumor heterogeneity represents a challenge for the establishment of a robust HCC classification and may contribute to treatment failure and drug resistance in many cases of HCC.

SummaryConcomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Murine liver tumors often fail to recapitulate the complexity of human hepatocellular carcinoma (HCC), which might explain the difficulty to translate preclinical mouse studies into clinical science. The aim of this study was to evaluate a subtyping approach for murine liver cancer models with regard to etiology-defined categories of human HCC, comparing genomic changes, histomorphology, and IHC profiles. Sequencing and analysis of gene copy-number changes [by comparative genomic hybridization (CGH)] in comparison with etiology-dependent subsets of HCC patients of The Cancer Genome Atlas (TCGA) database were conducted using specimens (75 tumors) of five different HCC mouse models: diethylnitrosamine (DEN) treated wild-type C57BL/6 mice, c-Myc and AlbLTab transgenic mice as well as TAK1 LPC-KO and Mcl-1 Dhep mice. Digital microscopy was used for the assessment of morphology and IHC of liver cell markers (A6-CK7/ 19, glutamine synthetase) in mouse and n ¼ 61 human liver tumors. Tumor CGH profiles of DEN-treated mice and c-Myc transgenic mice matched alcohol-induced HCC, including morphologic findings (abundant inclusion bodies, fatty change) in the DEN model. Tumors from AlbLTab transgenic mice and TAK1 LPC-KO models revealed the highest overlap with NASH-HCC CGH profiles. Concordant morphology (steatosis, lymphocyte infiltration, intratumor heterogeneity) was found in AlbLTab murine livers. CGH profiles from the Mcl-1 Dhep model displayed similarities with hepatitis-induced HCC and characteristic human-like phenotypes (fatty change, intertumor and intratumor heterogeneity). Implications: Our findings demonstrate that stratifying preclinical mouse models along etiology-oriented genotypes and human-like phenotypes is feasible. This closer resemblance of preclinical models is expected to better recapitulate HCC subgroups and thus increase their informative value.

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Genome-wide CRISPR phenotypic screens are clarifying many fundamental biological phenomena. While pooled screens can be used to study selectable features, arrayed CRISPR libraries extend the screening territory to cell-nonautonomous, biochemical and morphological phenotypes. Using a novel high-fidelity liquid-phase plasmid cloning technology, we generated two human genome-wide arrayed libraries termed T.spiezzo (gene ablation, 19,936 plasmids) and T.gonfio (gene activation and epigenetic silencing, 22,442 plasmids). Each plasmid encodes four non-overlapping single-guide RNAs (sgRNAs), each driven by a unique housekeeping promoter, as well as lentiviral and transposable vector sequences. The sgRNAs were designed to tolerate most DNA polymorphisms identified in 10,000 human genomes, thereby maximizing their versatility. Sequencing confirmed that ~90% of each plasmid population contained ≥3 intact sgRNAs. Deletion, activation and epigenetic silencing experiments showed efficacy of 75-99%, up to 10,000x and 76-92%, respectively; lentiviral titers were ~10^7/ml. As a proof of concept, we investigated the effect of individual activation of each human transcription factor (n=1,634) on the expression of the cellular prion protein PrPC. We identified 24 upregulators and 12 downregulators of PrPC expression. Hence, the T.spiezzo and T.gonfio libraries represent a powerful resource for the individual perturbation of human protein-coding genes.

Background and Aims: ALPPS (associating liver partition and portal vein ligation for staged hepatectomy), a novel 2-staged hepatectomy, dramatically accelerates liver regeneration and thus enables extensive liver tumor resection. The signaling networks underlying the ALPPS-induced accelerated regeneration process are largely unknown.Methods: We performed transcriptome profiling (TP) of liver tissue obtained from a mouse model of ALPPS, standard hepatectomy (68% model), and additional control surgeries (sham, PVL and Tx). We also performed TP using human liver biopsies (n = 5) taken from the occluded lobe and the future liver remnant (FLR) during the first step of ALPPS surgery (4–5 h apart). We used Oncofinder computational tools, which covers 378 ISPs, for unsupervised, unbiased quantification of ISP activity.Results: Gene expression cluster analysis revealed an ALPPS specific signature: the IGF1R Signaling Pathway (Cell survival), the ILK Pathway (Induced cell proliferation), and the IL-10 Pathway (Stability determination) were significantly enriched, whereas the activity of the Interferon Pathway (Transcription) was reduced (p < 0.05). Further, the PAK- and ILK-associated ISPs were activated at an earlier time point, reflecting significant acceleration of liver regeneration (p < 0.001). These pathways, which were also recovered in human liver biopsies, control cell growth and proliferation, inflammatory response, and hypoxia-related processes.Conclusions: ALPPS is not a straightforward addition of portal vein ligation (PVL) plus transection—it is more. The early stages of normal and accelerated liver regeneration are clearly discernible by a significantly increased and earlier activation of a small number of signaling pathways. Compounds mimicking these responses may help to improve the ALPPS method and further reduce the hospitalization time of the patient.

Background Brown adipose tissue (BAT) is a specialized form of adipose tissue, able to increase energy expenditure by heat generation in response to various stimuli. Recently, its pathological activation has been implicated in the pathogenesis of cancer cachexia. To establish a causal relationship, we retrospectively investigated the longitudinal changes in BAT and cancer in a large FDG-PET/CT cohort. Methods We retrospectively analyzed 13 461 FDG-PET/CT examinations of n = 8 409 patients at our institution from the winter months of 2007-2015. We graded the activation strength of BAT based on the anatomical location of the most caudally activated BAT depot into three tiers, and the stage of the cancer into five general grades. We validated the cancer grading by an interreader analysis and correlation with histopathological stage. Ambient temperature data (seven-day average before the examination) was obtained from a meteorological station close to the hospital. Changes of BAT, cancer, body mass index (BMI) and temperature between the different examinations were examined with Spearman's test and a mixed linear model for correlation, and with a causal inference algorithm for causality. Results We found n = 283 patients with at least two examinations and active BAT in at least one of them. There was no significant interaction between the changes in BAT activation, cancer burden or BMI. Temperature changes exhibited a strong negative correlation with BAT activity (% =-0.57, p<0.00001). These results were confirmed with the mixed linear model. Causal

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