Non-tumor Liver Research Articles

TP53 R249S mutation in hepatic organoids captures the predisposing cancer risk.

Major genomic drivers of hepatocellular carcinoma (HCC) are nowadays well recognized, although models to establish their roles in human HCC initiation remain scarce. Here, we used human liver organoids in experimental systems to mimic the early stages of human liver carcinogenesis from the genetic lesions of TP53 loss and L3 loop R249S mutation. In addition, chromatin immunoprecipitation sequencing (ChIP-seq) of HCC cell lines shed important functional insights into the initiation of HCC consequential to the loss of tumor-suppressive function from TP53 deficiency and gain-of-function activities from mutant p53. Human liver organoids were generated from surgical nontumor liver tissues. CRISPR knockout of TP53 in liver organoids consistently demonstrated tumor-like morphological changes, increased in stemness and unrestricted in vitro propagation. To recapitulate TP53 status in human HCC, we overexpressed mutant R249S in TP53 knockout organoids. A spontaneous increase in tumorigenic potentials and bona fide HCC histology in xenotransplantations were observed. ChIP-seq analysis of HCC cell lines underscored gain-of-function properties from L3 loop p53 mutants in chromatin remodeling and overcoming extrinsic stress. More importantly, direct transcriptional activation of PSMF1 by mutant R249S could increase organoid resistance to endoplasmic reticulum stress, which was readily abrogated by PSMF1 knockdown in rescue experiments. In a patient cohort of primary HCC tumors and genome-edited liver organoids, quantitative polymerase chain reaction corroborated ChIP-seq findings and verified preferential genes modulated by L3 mutants, especially those enriched by R249S. We showed differential tumorigenic effects from TP53 loss and L3 mutations, which together confer normal hepatocytes with early clonal advantages and prosurvival functions.

Analysis of viral integration reveals new insights of oncogenic mechanism in HBV-infected intrahepatic cholangiocarcinoma and combined hepatocellular-cholangiocarcinoma

BackgroundIntegration of HBV DNA into the human genome could progressively contribute to hepatocarcinogenesis. Both intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC) are known to be associated with HBV infection. However, the integration of HBV and mechanism of HBV-induced carcinogenesis in ICC and CHC remains unclear.Methods41 patients with ICC and 20 patients with CHC were recruited in the study. We conducted HIVID analysis on these 61 samples to identify HBV integration sites in both the tumor tissues and adjacent non-tumor liver tissues. To further explore the effect of HBV integration on gene alteration, we selected paired tumors and adjacent non-tumor liver tissues from 3 ICC and 4 CHC patients for RNA-seq and WGS.ResultsWe detected 493 HBV integration sites in ICC patients, of which 417 were from tumor samples and 76 were from non-tumor samples. And 246 HBV integration sites were detected in CHC patients, of which 156 were located in the genome of tumor samples and 90 were in non-tumor samples. Recurrent HBV integration events were detected in ICC including TERT, ZMAT4, MET, ANKFN1, PLXNB2, and in CHC like TERT, ALKBH5. Together with our established data of HBV-infected hepatocellular carcinoma, we found that HBV preferentially integrates into the specific regions which may affect the gene expression and regulation in cells and involved in carcinogenesis. We further performed genomic and transcriptomic sequencing of three ICC and four CHC patients, and found that HBV fragments could integrate near some important oncogene like TERT, causing large-scale genome variations on nearby genomic sequences, and at the same time changing the expression level of the oncogenes.ConclusionComparative analysis demonstrates numerous newly discovered mutational events in ICC and CHC resulting from HBV insertions in the host genome. Our study provides an in-depth biological and clinical insights into HBV-induced ICC and CHC.

Distinctive Metabolism-Associated Gene Clusters That Are Also Prognostic in Intrahepatic Cholangiocarcinoma and Hepatocellular Carcinoma.

Objective To offer new prognostic evaluations by exploring potentially distinctive genetic features of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Methods There were 12 samples for gene expression profiling processes in this study. These included three HCC lesion samples and their matched adjacent nontumor liver tissues obtained from patients with HCC, as well as three ICC samples and their controls collected similarly. In addition to the expression matrix generated on our own, profiles of other cohorts from The Cancer Genome Atlas (TCGA) program and the Gene Expression Omnibus (GEO) were also employed in later bioinformatical analyses. Differential analyses, functional analyses, protein interaction network analyses, and gene set variation analyses were used to identify key genes. To establish the prognostic models, univariate/multivariate Cox analyses and subsequent stepwise regression were applied, with the Akaike information criterion evaluating the goodness of fitness. Results The top three pathways enriched in HCC were all metabolism-related; they were fatty acid degradation, retinol metabolism, and arachidonic acid metabolism. In ICC, on the other hand, additional pathways related to fat digestion and absorption and cholesterol metabolism were identified. Consistent characteristics of such a metabolic landscape were observed across different cohorts. A prognostic risk score model for calculating HCC risk was constructed, consisting of ADH4, ADH6, CYP2C9, CYP4F2, and RDH16. This signature predicts the 3-year survival with an AUC area of 0.708 (95%CI = 0.644 to 0.772). For calculating the risk of ICC, a prognostic risk score model was built upon the expression levels of CYP26A1, NAT2, and UGT2B10. This signature predicts the 3-year survival with an AUC area of 0.806 (95% CI = 0.664 to 0.947). Conclusion HCC and ICC share commonly abrupted pathways associated with the metabolism of fatty acids, retinol, arachidonic acids, and drugs, indicating similarities in their pathogenesis as primary liver cancers. On the flip side, these two types of cancer possess distinctive promising biomarkers for predicting overall survival or potential targeted therapies.

CDC20 May Serve as a Potential Biomarker-Based Risk Score System in Predicting the Prognosis of Patients with Hepatocellular Carcinoma.

Background The specificity and sensitivity of hepatocellular carcinoma (HCC) diagnostic markers are limited, hindering the early diagnosis and treatment of HCC patients. Therefore, improving prognostic biomarkers for patients with HCC is urgently needed. Methods HCC-related datasets were downloaded from the public databases. Differentially expressed genes (DEGs) between HCC and adjacent nontumor liver tissues were then identified. Moreover, the intersection of DEGs in four datasets (GSE138178, GSE77509, GSE84006, and TCGA) was used in the functional enrichment, and module genes were obtained by a coexpression network. Cox and Kaplan-Meier analyses were used to identify overall survival- (OS-) related genes from module genes. Area under the curve (AUC) > 0.9 of OS-related genes was then carried out in order to perform the protein-protein interaction network. The feature genes were identified by least absolute shrinkage and selection operator (LASSO). Furthermore, the hub gene was identified through the univariate Cox model, after which the correlation analysis between the hub gene and pathways was explored. Finally, infiltration in immune cell types in HCC was analyzed. Results A total of 2,227 upregulated genes and 1,501 downregulated DEGs were obtained in all four datasets, which were mainly found to be involved in the cell cycle and retinol metabolism. Accordingly, 998 OS-related genes were screened to construct the LASSO model. Finally, 8 feature genes (BUB1, CCNB1, CCNB2, CCNA2, AURKB, CDC20, OIP5, and TTK) were obtained. CDC20 was shown to serve as a poor prognostic gene in HCC and was mainly involved in the cell cycle. Moreover, a positive correlation was noted between the high degree of infiltration with Th2 and CDC20. Conclusion High expression of CDC20 predicted poor survival, as potential target in the treatment for HCC.

Dietary Fat Composition Affects Hepatic Angiogenesis and Lymphangiogenesis in Hepatitis C Virus Core Gene Transgenic Mice

Introduction: Previous research has demonstrated that an isocaloric diet rich in trans-fatty acid (TFA), saturated fatty acid (SFA), and cholesterol (Chol) promoted steatosis-derived hepatic tumorigenesis in hepatitis C virus core gene transgenic (HCVcpTg) mice in different manners. Growth factor signaling and ensuing angiogenesis/lymphangiogenesis are key factors in hepatic tumorigenesis that have become recent therapeutic targets for hepatocellular carcinoma. However, the influence of dietary fat composition on these factors remains unclear. This study investigated whether the type of dietary fat would have a specific impact on hepatic angiogenesis/lymphangiogenesis in HCVcpTg mice. Methods: Male HCVcpTg mice were treated with a control diet, an isocaloric diet containing 1.5% cholesterol (Chol diet), or a diet replacing soybean oil with hydrogenated coconut oil (SFA diet) for a period of 15 months or with shortening (TFA diet) for 5 months. The degree of angiogenesis/lymphangiogenesis and the expression of growth factors, including fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF), were evaluated in non-tumorous liver tissues using quantitative mRNA measurement, immunoblot analysis, and immunohistochemistry. Results: Long-term feeding of SFA and TFA diets to HCVcpTg mice increased the expressions of vascular endothelial cell indicators, such as CD31 and TEK receptor tyrosine kinase, in addition to lymphatic vessel endothelial hyaluronan receptor 1, indicating that angiogenesis/lymphangiogenesis were upregulated only by these fatty acid-enriched diets. This promoting effect correlated with elevated VEGF-C and FGF receptor 2 and 3 levels in the liver. c-Jun N-terminal kinase (JNK) and hypoxia-inducible factor (HIF) 1α, both key regulators of VEGF-C expression, were enhanced in the SFA- and TFA-rich diet groups as well. The Chol diet significantly increased the expressions of such growth factors as FGF2 and PDGF subunit B, without any detectable impact on angiogenesis/lymphangiogenesis. Conclusion: This study revealed that diets rich in SFA and TFA, but not Chol, might stimulate hepatic angiogenesis/lymphangiogenesis mainly through the JNK-HIF1α-VEGF-C axis. Our observations indicate the importance of dietary fat species for preventing hepatic tumorigenesis.

Accuracy and Safety of Scout Dose Resin Yttrium-90 Microspheres for Radioembolization Therapy Treatment Planning: A Prospective Single-Arm Clinical Trial

PurposeTo compare the accuracy and safety of 0.56 GBq resin yttrium-90 (90Y) (scout90Y) microspheres with those of technetium-99m macroaggregated albumin (MAA) in predicting the therapeutic 90Y (Rx90Y) dose for patients with hepatocellular carcinoma (HCC). Materials and MethodsThis prospective single-arm clinical trial (Clinicaltrials.gov: NCT04172714) recruited patients with HCC. Patients underwent same-day mapping with MAA and scout90Y. Rx90Y activity was administered 3 days after mapping. Using paired t test and Pearson correlation, the tumor-to-normal ratio (TNR), lung shunt fraction (LSF), predicted mean tumor dose (TD), and nontumoral liver dose (NTLD) by MAA and scout90Y were compared with those by Rx90Y. Bland-Altman plots compared the level of agreement between the TNR and LSF of scout90Y and MAA with that of Rx90Y. The safety of scout90Y was evaluated by examining the discrepancy in extrahepatic activity between MAA and scout90Y. ResultsThirty patients were treated using 19 segmental and 14 nonsegmental (ie, 2 contiguous segments or nonsegmental) therapies. MAA had weak LSF, moderate TNR, and moderate TD linear correlation with Rx90Y. Scout90Y had a moderate LSF, strong TNR, strong TD, and very strong NTLD in correlation with those of Rx90Y. Furthermore, the TNR and LSF of scout90Y had a stronger agreement with those of Rx90Y than with those of MAA. In the nonsegmental subgroup, MAA had no significant correlation with the TD and NTLD of Rx90Y, whereas scout90Y had a very strong correlation with both of these factors. In the segmental subgroup, both MAA and scout90Y had a strong linear correlation with the TD and NTLD of Rx90Y. ConclusionsCompared with MAA, scout90Y is a more accurate surrogate for Rx90Y biodistribution for nonsegmental therapies.

Hepatic decompensation after transarterial radioembolization: A retrospective analysis of risk factors and outcome in patients with hepatocellular carcinoma.

Transarterial radioembolization (TARE) is a well‐established therapy for intermediate and advanced tumor stages of hepatocellular carcinoma (HCC). Treatment‐associated toxicities are rare. Previous studies have outlined that the prognosis after TARE is determined primarily by tumor stage and liver function. The subset of patients benefiting from TARE remains to be defined. Sixty‐one patients with HCC treated with TARE between 2015 and 2020 were retrospectively included in the study. Hepatic decompensation was defined as an increase of bilirubin or newly developed ascites that was not explained by tumor progression within 3 months after TARE. Predictive factors of hepatic decompensation and prognostic factors were assessed. Hepatic decompensation was observed in 27.9% (n = 17) of TARE‐treated patients during follow‐up. Albumin‐bilirubin (ALBI) score at baseline and radiation dose on nontumor liver proved to be independent risk factors for the development of hepatic decompensation in multivariable regression models (ALBI score: odds ratio [OR] 6.425 [1.735;23.797], p < 0.005; radiation dose: OR 1.072 [1.016;1.131], p < 0.011). The occurrence of hepatic decompensation markedly impaired the prognosis of the patients. Survival was significantly worsened. Hepatic decompensation has shown to be an independent negative prognostic factor for death, adjusted for Barcelona Clinic Liver Cancer stage, age and ALBI grade (hazard ratio 5.694 [2.713;11.952], p < 0.001). Conclusion: Hepatic decompensation after TARE for HCC treatment is a highly relevant complication with major effects on the prognosis of patients. Main risk factors are the pretreatment ALBI score and radiation dose. There is an urgent need to define safe cutoff values and exclusion criteria for TARE to limit complications and improve patient outcomes.

Abstract PO011: Proteomic profiling of childhood liver cancer: identification of novel diagnostic and prognostic biomarkers

Abstract Childhood liver cancers hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are rare diseases but with a rising incidence. HEMNOS (Hepatocellular malignant neoplasm, not otherwise specified) is a recent entity with histopathological features of HB and HCC. Current chemotherapy treatments are effective to shrink tumor before surgery, nonetheless can cause severe lifelong adverse effects and are not effective for patients with aggressive and metastatic HB or HCC (~20% die due to the disease). The HB and HCC diagnosis is key to assign therapeutic regimens in the ongoing Pediatric Hepatic International Tumor Trial (PHITT); however, differential diagnosis for some patients is challenging due to the lack of specific biomarkers. Nowadays, there is an urgent need to identify diagnostic and prognostic biomarkers to improve the clinical management of childhood liver cancer. Herein, we aimed to uncover the proteomic profiles of different types of childhood liver cancer and identify new biomarkers for improving tumor diagnosis and early detection of the most aggressive cases. For this, 99 frozen tissue samples from 71 patients (mean age: 54.5 months, 60.6% boys, 40.9% metastasis, 23.7% deaths) including 70 primary tumors (57 HB, 8 HCC and 5 HEM-NOS), 22 non-tumors and 7 metastases were analyzed by label free mass spectrometry analysis. A total of 5,417 proteins were sequenced in the different samples. Through supervised analysis, we identified a total of 1302 differential expressed proteins in tumors as compared to non-tumor livers (FC +2 and FDR &amp;lt;0.01). A total of 246 proteins of them (19%) were commonly differently expressed in all tumors types; having HEMNOS a mixture of HB and HCC protein profiling. A panel of potential diagnostic biomarkers including 179, 120 and 81 upregulated proteins (FC &amp;gt;2) was defined for HB, HCC and HEM-NOS, respectively. Hierarchical unsupervised clustering and principal component analysis showed two main cluster of tumors: one including HB and HEM-NOS and another one with a mixture of HB, HEM-NOS and HCC. Interestingly, this second cluster was associated with clinical and molecular features of aggressive tumors such as multifocality (p = 0.029), Epigenetic Epi-CB subclass (p = 0.006), high-risk Molecular Risk Stratification (p = 0.009) and poor survival (log rank test=0.012). A total of 2082 proteins were found to be differently expressed between the two prognostic clusters (FC +2 and FDR &amp;lt; 0.01); the GEO enrichment analysis revealed that these proteins were significantly enriched in post-transcriptional gene silencing and RNA splicing mechanisms (FDR &amp;lt;10−7). In addition, a 25-protein signature associated to liver cancer prognosis was defined (FC&amp;gt;6 and FDR &amp;lt; 10−14). In conclusion, we identified a list of potential diagnostic and prognostic protein biomarkers that after a validation in large patient cohort, could be used to improve the clinical management of childhood liver cancer. Citation Format: Álvaro Del Río-Álvarez, Juan Carrillo-Reixach, Laura Royo, Montse Domingo-Sàbat, Mikel Azkargorta, Roland Kapler, Stefano Cairo, Christian Vokuhl, Ronald de Krijger, Rita Alaggio, Marta Garrido, Gabriela Guillen, Constantino Sábado, Laura Guerra, Francisco Hernandez, Maria Elena Mateos, Manuel López-Satamaría, Barbara Torres, Maria Pilar Abad, Bajčiová Viera, Piotr Czauderna, Marie Annick Buendia, Felix Elortza, Keith Wheatley, Bruce Morland, Carolina Armengol. Proteomic profiling of childhood liver cancer: identification of novel diagnostic and prognostic biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO011.

Abstract PO010: Molecular characterization of pediatric hepatocellular carcinoma: genomic, methylomic and transcriptomic analysis

Abstract Background: The main pediatric liver tumors are hepatoblastoma (HB) and, to a lesser extent, pediatric hepatocellular carcinoma (HCC). HB appears at an early age (&amp;lt;3 years) while HCC is diagnosed mainly in older children (&amp;gt;8 years) or adolescents and it is usually associated to underlying metabolic diseases. HEM-NOS (Hepatocellular malignant neoplasm, not otherwise specified) is a recent entity with histopathological features of HB and HCC. Due to the rarity of the disease (&amp;lt;1 case per million children), the biology of HCC and their similarity with HB has been poorly explored. Aims: To molecularly characterize pediatric HCC at the genomic, transcriptomic and epigenomic level and to perform a comparative study with HB and HEM-NOS. Methodology: We performed a genomic (Cytoscan, ThermoFisher), epigenomic (EPIC/850k, Illumina) and transcriptomic (HTA 2.0 array, ThermoFisher) studies on 8 HCC and compared them with already published omics data of 31 HB, 2 HEM-NOS and 19 control non-tumor liver (NT) samples (Carrillo-Reixach et al, J Hepatol, 2020). The results obtained were validated in an independent series of 19 HCC, including 10 fibrolamellar HCC (FL-HCC), 56 HB, 7 HEM-NOS and 5 NTs using QUAlu and Nanostring techniques for assessing DNA methylation and gene expression, respectively. Results: We identified CTNNB1 gene mutations in 73%, 50% and 14% of HB, HEM-NOS and HCC, respectively. Contrarily, TERT mutations were only found in HEM-NOS and HCC tumors. FL-HCC had the DNAJB1:PRKACA fusion transcript and in one case without this fusion, a deletion in the PRKCA gene was identified. The genome of HCC and HEM-NOS had higher number of chromosomal losses than HB. Moreover, HBs and HEM-NOS showed a higher DNA hypomethylation than HCC whereas HCC was characterized by CpG island hypermethylation. Two imprinting regions were differently expressed in 14q32 (DLK1/DIO3) and 15q11.2 (SNORD115/116) in HB and HCC, respectively. Finally, we identified the SPINK1 gene (Serine peptidase inhibitor, kazal type 1) as an overexpressed gene in HCC that was strongly associated with prognosis of patients with liver cancer in both training (Log-Rank=0.008) and validation sets (Log-rank=0.028). Using XENA database, we found also that different SPINK1 expression was also associated with adult HCC prognosis (n=357, Log-rank=0.017). Conclusions: The characterization of HCC and its comparison with HB allowed us to identify potential diagnostic and prognostic markers that could help to improve the clinical management of childhood liver cancer. Citation Format: Juan Carrillo-Reixach, Álvaro del Río-Álvarez, Laura Royo, Montserrat Domingo-Sàbat, Rita Alaggio, Ronald de Krijger, Christian Vokuhl, Marta Garrido, Laura Guerra, Constantino Sábado, Francisco Hernández, Manuel López-Santamaría, Viera Bajčiová, Rudolf Maibach, Margaret Childs, Piotr Czauderna, Keith Wheatley, Roland Kappler, Stefano Cairo, Bruce Morland, Margarita Sala, Maria Rosa Sarrias, Carolina Armengol. Molecular characterization of pediatric hepatocellular carcinoma: genomic, methylomic and transcriptomic analysis [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO010.

Leukocyte cell-derived chemotaxin 2 regulates epithelial-mesenchymal transition and cancer stemness in hepatocellular carcinoma

Leukocyte cell-derived chemotaxin 2 (LECT2) acts as a tumor suppressor in hepatocellular carcinoma (HCC). However, the antineoplastic mechanism of LECT2, especially its influence on hepatic cancer stem cells (CSCs), remains largely unknown. In The Cancer Genome Atlas cohort, LECT2 mRNA expression was shown to be associated with stage, grade, recurrence, and overall survival in human HCC patients, and LECT2 expression was downregulated in hepatoma tissues compared with the adjacent nontumoral liver. Here, we show by immunofluorescence and immunoblot analyses that LECT2 was expressed at lower levels in tumors and in poorly differentiated HCC cell lines. Using functional assays, we also found LECT2 was capable of suppressing oncogenic behaviors such as cell proliferation, anchorage-independent growth, migration, invasiveness, and epithelial-mesenchymal transition in hepatoma cells. Moreover, we show exogenous LECT2 treatment inhibited CSC functions such as tumor sphere formation and drug efflux. Simultaneously, hepatic CSC marker expression was also downregulated, including expression of CD133 and CD44. This was supported by infection with adenovirus encoding LECT2 (Ad-LECT2) in HCC cells. Furthermore, in animal experiments, Ad-LECT2 gene therapy showed potent efficacy in treating HCC. We demonstrate LECT2 overexpression significantly promoted cell apoptosis and reduced neovascularization/CSC expansion in rat hepatoma tissues. Mechanistically, we showed using immunoblot and immunofluorescence analyses that LECT2 inhibited β-catenin signaling via the suppression of the hepatocyte growth factor/c-MET axis to diminish CSC properties in HCC cells. In summary, we reveal novel functions of LECT2 in the suppression of hepatic CSCs, suggesting a potential alternative strategy for HCC therapy.

Value of Non-tumoral Liver Volume in the Prognosis of Large Hepatocellular Carcinoma Patients After R0 Resection.

Hepatectomy is an effective treatment for selected patients with large hepatocellular carcinoma (HCC). This study aimed to develop a nomogram incorporating non-tumoral liver volume (non-TLV) and liver function markers to predict the patients' overall survival (OS) and disease-free survival (DFS). Data of 198 consecutive large HCC patients who underwent hepatectomy at the Zhongshan Hospital Xiamen University were collected. Another 68 patients from the Mengchao Hepatobiliary Surgery Hospital served as an external validation cohort. The nomograms were developed based on the independent prognostic factors screened by multivariate Cox regression analyses. Concordance index (C-index), calibration curves, and time-dependent receiver operating characteristic (ROC) curves were used to measure the discrimination and predictive accuracy of the models. High HBV DNA level, low non-TLV/ICG, vascular invasion, and a poorly differentiated tumor were confirmed as independent risk factors for both OS and DFS. The model established in this study predicted 5-year post-operative survival and DFS in good agreement with the actual observation confirmed by the calibration curves. The C-indexes of the nomograms in predicting OS and DFS were 0.812 and 0.823 in the training cohort, 0.821 and 0.846 in the internal validation cohort, and 0.724 and 0.755 in the external validation cohort. The areas under the ROC curves (AUCs) of nomograms for predicted OS and DFS at 1, 3, and 5 year were 0.85, 0.86, 0.83 and 0.76, 0.76, 0.63, respectively. Nomograms with non-TLV/ICG predicted the prognosis of single large HCC patients accurately and effectively.

Ectodysplasin-A mRNA in exosomes released from activated hepatic stellate cells stimulates macrophage response

IntroductionThe interaction between activated hepatic stellate cells (aHSCs) and macrophages is central to liver fibrosis development. The cargo contained within aHSC exosomes (aHSC-EXOs) and how aHSC-EXOs affect macrophage function is poorly understood. MethodsRNA from aHSC-EXOs was separated into small (<200-basepairs) and large (≥200-basepairs) RNA species, transfected into macrophages, and macrophage IL-6 and TNFα mRNA expression and protein secretion measured. Next generation sequencing was performed on EXOs from rat quiescent and aHSCs and human aHSCs. aHSCs were transfected with siRNA against ectodysplasin-A (EDA), EXOs collected, and their effect on macrophage function analyzed. Human cirrhotic liver was analyzed for EDA mRNA expression and compared to non-tumor liver (NTL). ResultsTransfection with large RNA from aHSC-EXOs stimulated macrophage IL-6 and TNFα mRNA expression and protein secretion. EDA mRNA was highly expressed in aHSCs and transfection of aHSCs with EDA-siRNA decreased aHSC-EXO EDA mRNA and blunted the effect of aHSC-EXOs on macrophage function (IL-6/TNFα expression and macrophage migration). Human cirrhotic liver exhibited high EDA mRNA compared to NTL. ConclusionsHSC activation leads to altered EXO mRNA/miRNA profiles with aHSC-EXOs mRNAs exerting a dominant role in altering macrophage function. Ectodysplasin-A mRNA is an important component in aHSC-EXOs in regulating macrophage function.

Integrated analysis using ToppMiR uncovers altered miRNA- mRNA regulatory networks in pediatric hepatocellular carcinoma-A pilot study.

Pediatric hepatocellular carcinoma (HCC) is a group of liver cancers whose mechanisms behind their pathogenesis and progression are poorly understood. We aimed to identify alterations in the expression of miRNAs and their putative target mRNAs in not only tumor tissues of patients with pediatric HCC but also in corresponding non-tumorous background livers by using liver tissues without underlying liver disease as a control. We performed a small-scale miRNA and mRNA profiling of pediatric HCC (consisting of fibrolamellar carcinoma [FLC] and non-FLC HCC) and paired liver tissues to identify miRNAs whose expression levels differed significantly from control livers without underlying liver disease. ToppMiR was used to prioritize both miRNAs and their putative target mRNAs in a gene-annotation network, and the mRNA profile was used to refine the prioritization. Our analysis generated prioritized lists of miRNAs and mRNAs from the following three sets of analyses: (a) pediatric HCC versus control; (b) FLC versus control; and (c) corresponding non-tumorous background liver tissues from the same patients with pediatric HCC versus control. No liver disease liver tissues were used as the control group for all analyses. Many miRNAs whose expressions were deregulated in pediatric HCC were consistent with their roles in adult HCC and/or other non-hepatic cancers. Our gene ontology analysis of target mRNAs revealed enrichment of biological processes related to the sustenance and propagation of cancer and significant downregulation of metabolic processes. Our pilot study indicates that alterations in miRNA-mRNA networks were detected in not only tumor tissues but also corresponding non-tumorous liver tissues from patients with pediatric HCC, suggesting multi-faceted roles of miRNAs in disease progression. Our results may lead to novel hypotheses for future large-scale studies.

Identification of Ferroptosis-related molecular model and immune subtypes of hepatocellular carcinoma for individual therapy.

Excessive iron accumulation and lipid peroxidation are primary characteristics of ferroptosis in hepatocellular carcinoma (HCC). Ferroptosis inducer combined with immunotherapy has become a new hope for HCC patients. Therefore, the construction and validation of subtype-specific sensitivity to ferroptosis inducer will be helpful for hierarchical management and precise individual therapy. RNA-seq transcriptome and clinical data of HCC patients were extracted from International Cancer Genome Consortium (ICGC) dataset and The Cancer Genome Atlas (TCGA) dataset. Consistency matrix and data clustering of the both cohorts were constructed by 'ConsensusClusterPlus' package. Single-sample gene set enrichment analysis (ssGSEA) analysis was performed to evaluate immune infiltration. Cox analysis was utilized to construct a ferroptosis phenotype-related prognostic model (FRPM) in HCC. The predictive efficiency of the constructed FRPM was evaluated through Kaplan Meier (K-M) survival analyses and Receiver Operating Characteristic (ROC) curves. The expression levels of candidate genes were detected and validated by Real-Time PCR between liver cancer tissues and adjacent non-tumor liver tissues. 45 differentially expressed ferroptosis-related genes (FRGs) were identified between HCC tissues and non-tumor liver tissues. Furthermore, four ferroptosis-associated clusters (FACs) of HCC were established via consensus clustering. Subsequently, we established a FRPM, consisting of four prognostic genes (SLC7A11, SLC1A5, GCLM and SAT1), to evaluate the survival of HCC patients, based on which, patients were divided into high-risk group and low-risk group. The high-risk group exhibited worse survival compared to low-risk group (p< 0.0001 both in TCGA and ICGC cohorts). Patients belong to different FACs or different risk scores showed distinct clinical characteristics. Moreover, in the validation experiment, the transcriptional expression levels of the four prognostic genes were consistent with the results drew from datasets. We revealed a novel FRGs signature, which may provide the molecular characteristic data for effectively prognostic evaluation and potential personalized therapy for HCC patients.

Human liver organoids for disease modeling of fibrolamellar carcinoma.

SummaryFibrolamellar carcinoma (FLC) is a rare, often lethal, liver cancer affecting adolescents and young adults, for which there are no approved therapeutics. The development of therapeutics is hampered by a lack of in vitro models. Organoids have shown utility as a model system for studying many diseases. In this study, tumor tissue and the adjacent non-tumor liver were obtained at the time of surgery. The tissue was dissociated and grown as organoids. We developed 21 patient-derived organoid lines: 12 from metastases, three from the liver tumor and six from adjacent non-tumor liver. These patient-derived FLC organoids recapitulate the histologic morphology, immunohistochemistry, and transcriptome of the patient tumor. Patient-derived FLC organoids were used in a preliminary high-throughput drug screen to show proof of concept for the identification of therapeutics. This model system has the potential to improve our understanding of this rare cancer and holds significant promise for drug testing and development.

Differences in tumor-to-normal organ SUV ratios measured with 68 Ga-DOTATATE PET compared with 177 Lu-DOTATATE SPECT in patients with neuroendocrine tumors.

Our goal is to quantitatively compare radiotracer biodistributions within tumors and major normal organs on pretherapy 68 Ga-DOTATATE PET to post-therapy 177 Lu-DOTATATE single-photon emission computed tomography (SPECT) in patients receiving peptide receptor radionuclide therapy (PRRT). PET/CT at ~ 60 min postinjection of Ga-68 DOTATATE and research 177 Lu-SPECT/CT imaging ~ at 4 h (SPECT1) and ~ 24 h (SPECT2) post-cycle#1 were available. Manual contours of lesions on baseline CT or MRI were applied to co-registered SPECT/CT and PET/CT followed by deep learning-based CT auto-segmentation of organs. Tumor-to-normal organ ratios (TNR) were calculated from standardized uptake values (SUV) mean and SUV peak for tumor, and SUV mean for non-tumoral liver (nliver), spleen and kidney. There were 90 lesons in 24 patients with progressive metastatic neuroendocrine tumor. The correlation between PET and SPECT SUV TNRs were poor/moderate: PET versus SPECT1 R 2 = 0.19, 0.21, 0.29; PET versus SPECT2 R 2 = 0.06, 0.16, 0.33 for TNR nliver ,TNR spleen ,TNR kidney , respectively. Across all patients, the average value of the TNR measured on PET was significantly lower than on SPECT at both time points ( P < 0.001). Using SUV mean for tumor, average TNR values and 95% confidence intervals (CI) were PET: TNR nliver = 3.5 [CI: 3.0-3.9], TNR spleen = 1.3 [CI, 1.2-1.5], TNR kidney = 1.7 [CI: 1.6-1.9]; SPECT1: TNR nliver = 10 [CI: 8.2-11.7], TNR spleen = 2.9 [CI: 2.5-3.4], TNR kidney = 2.8 [CI: 2.3-3.3]; SPECT2: TNR nliver = 16.9 [CI: 14-19.9], TNR spleen = 3.6 [CI: 3-4.2], TNR kidney = 3.6 [CI: 3.0-4.2]. Comparison of PET and SPECT results in a sphere phantom study demonstrated that these differences are not attributed to imaging modality. Differences in TNR exist for the theranostic pair, with significantly higher SUV TNR on 177 Lu SPECT compared with 68 Ga PET. We postulate this phenomenon is due to temporal differences in DOTATATE uptake and internalization in tumor as compared to normal organs.

Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5′) pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics.

Toxicity comparison of yttrium-90 resin and glass microspheres radioembolization.

To investigate the clinical, hematological and biochemical toxicity differences between glass and resin yttrium-90 (90Y)-microspheres radioembolization treatment of primary and metastatic liver disease. Between May 2014 and November 2016 all consecutive glass and resin 90Y microspheres radioembolization treatments were retrospectively analyzed. Biochemical, hematological and clinical data were collected at treatment day, two weeks, one month and three months follow-up. Post-treatment 90Y PET/CTs were assessed for the absorbed doses in non-tumorous liver volume (DNTLV) and tumor volume (DTV). Biochemical, hematological and clinical toxicity were compared between glass and resin using chi square tests and repeated ANOVA measures. Biochemical and clinical toxicity was correlated with DNTLV,total by means of Pearson correlation and independent T-tests. A total of 85 patients were included (n=44 glass, n=41 resin). Clinical toxicity the day after treatment (i.e. abdominal pain (p=0.000), nausea (p=0.000) and vomiting (p=0.003)) was more prevalent for resin. Biochemical and hematological toxicities were similar for both microspheres. The DNTLV,total was significantly higher in patients with REILD grade ≥3 in the resin group (43.5 versus 33.3 Gy (p=0.050)). A similar non-significant trend was seen in the glass group: 95.0 versus 69.0 Gy (p=0.144). The clinical, hematological and biochemical toxicity of radioembolization treatment with glass and resin is comparable, however, post-embolization syndrome related complaints are more common for resin.

Genetic variants of NTCP gene and hepatitis B vaccine failure in Taiwanese children of hepatitis B e antigen positive mothers.

Hepatitis B virus (HBV) vaccine failure remains a hurdle to the global elimination of HBV infections in the vaccination era. We aimed to elucidate the relationships between HBV entry receptor sodium taurocholate co-transporting polypeptide (NTCP) and vaccine failure in children born to highly infectious mothers. The genetic variants rs7154439, rs4646285, rs4646287, and rs2296651 were genotyped in 170 children with chronic HBV infections and 138 control children of mothers positive for hepatitis B e antigen (HBeAg). All children received hepatitis B immunoglobulin and complete HBV vaccination. Total RNAs from 82 adult non-tumor liver tissues were quantified for NTCP, type I interferons and interferon-induced transmembrane protein 3 (IFITM3) levels. A higher rate of the GA/AA genotype (28.3% vs. 15.3%, p = 0.006) of the genetic variant rs4646287 in intron 1 of the NTCP gene was detected in control children compared to the carrier children. The rs4646287 G > A genotype was associated with younger ages at which spontaneous HBeAg seroconversion occurred (10.8 ± 8.4 vs. 14.6 ± 8.7years, p = 0.003) in chronic HBV-infected children. Unique correlation patterns of NTCP and innate immunity-related genes (type I interferons and IFITM3) were found in HBV-infected liver tissues with the rs4646287 G > A genotype. The rs4646287 G > A genotype of the NTCP gene may be associated with lower risk for HBV vaccine failure in children born to highly infectious mothers. The protective effect of rs4646287 G > A was also present in carrier children, evidenced by earlier spontaneous HBeAg seroconversion.

Abstract No. 202 Correlation of non-tumoral liver dose with treatment related adverse events in HCC patients treated with glass-based Y-90 radioembolization

To evaluate the relationship between non-tumor liver (NTL) dose and clinical or biochemical adverse event (AE) in patients with hepatocellular carcinoma (HCC) treated with glass-based Yttrium-90 radioembolization (Y-90-RE) in lobar (RL) or segmental (RS) fashion