Cirrhotic Samples Research Articles

Differential diagnosis of small hepatocellular nodules incirrhosis: surrogate histological criteria of TERT promotermutations.

The differential diagnosis of small hepatocellular nodules in cirrhosis between dysplastic nodules and hepatocellular carcinoma (HCC) remains challenging on biopsy. As TERT promoter (pTERT) mutations may indicate the nodules already engaged in the malignant process, the aim of this study was to identify histological criteria associated with pTERT mutations by detecting these mutations by ddPCR in small formalin-fixed paraffin-embedded (FFPE) hepatocellular nodules arising in cirrhosis. We built a bicentric cohort data set of 339 hepatocellular nodules < 2 cm from cirrhotic samples, divided into a test cohort of 299 resected samples and a validation cohort of 40 biopsies. Pathological review, based on the evaluation of 14 histological criteria, classified all nodules. pTERT mutations were identified by ddPCR in FFPE samples. Among the 339 nodules, ddPCR revealed pTERT mutations in 105 cases (31%), including 90 and 15 cases in the test and validation cohorts, respectively. On multivariate analysis, three histological criteria were associated with pTERT mutations in the test cohort: increased cell density (P = 0.003), stromal invasion (P = 0.036) and plate-thickening anomalies (P < 0.001). With the combination of at least two of these major criteria, the AUC for predicting pTERT mutations was 0.84 in the test cohort (sensitivity: 86%, specificity: 83%) and 0.81 in the validation cohort (sensitivity: 87%, specificity: 76%). We identified three histological criteria as surrogate markers of pTERT mutations that may be used in routine biopsy to more clearly classify small hepatocellular nodules arising in cirrhosis.

Unraveling the activation process and core driver genes of HSCs during cirrhosis by single-cell transcriptome.

Worldwide, cirrhosis is a common cause of death, manifesting itself as fibrosis of the liver tissue. When the liver is damaged, the liver produces fibrotic, proliferative myofibroblasts, which are formed by the differentiation of activated hepatic stellate cells. There are no effective antifibrotic treatment options. To deeply explore the activation process of hepatic stellate cells (HSCs) and to discover better therapeutic target genes, single-cell RNA sequencing data on 13 non-cirrhotic liver tissue samples and 10 cirrhotic liver tissue samples were analyzed. We identified activated HSCs from the mesenchymal cell population with high expression of ACTA2. By pseudo-time analysis, we found that the key genes for the differentiation of HSCs into myofibroblasts were C3, CCDC80, COL1A1, COL3A1, DCN, FBLN1, IGFBP3, MXRA5, SERPINE1, and MYH11. Then, we found that the main regulators of HSCs from inactive to activated state were NTF3, NTRK3, NTRK2, JAG1, NOTCH3, ESAM, and CD46 by cell-cell communication analysis. In addition, we found that the top2 hub genes of activated HSCs were CRIP1 and ACTA2. The experimental results show that the top2 hub genes were significantly overexpressed in cirrhotic samples. Our work dissected key intercellular regulators and core driver genes during hepatic stellate cell activation during cirrhosis through single-cell transcriptome data analysis, providing a research strategy to discover rational therapeutic targets for cirrhosis and some important information for gene targeting therapy.

Use of ATR-FTIR spectroscopy to differentiate between cirrhotic/non-cirrhotic HCV patients

BackgroundConventional techniques to diagnose (HCV) and assess non-cirrhotic/cirrhotic status of the patient for appropriate treatment regime are expensive and invasive. Present available diagnostic tests are expensive as they include multiple screening steps. Therefore, there is a need of cost-effective, less time consuming and minimally invasive alternative diagnostic approaches can be used for effective screening. We propose that (ATR-FTIR) in conjunction with (PCA-LDA),(PCA-QDA) and (SVM) multivariate algorithms can be used as a sensitive tool for detection of HCV infection and to assess non-cirrhotic/cirrhotic status of patients. MethodsWe used 105 sera samples, of which, 55 were from healthy and 50 were from HCV positive individuals. These 50 HCV positive patients were further classified into cirrhotic and non-cirrhotic categories using serum markers and imaging techniques. These samples were freeze dried prior to spectral acquisition then multivariate data classification algorithms were employed to classify these sample types. ResultsPCA-LDA and SVM model computed the diagnostic accuracy of 100% for detection of HCV infection. To further classify the non-cirrhotic/cirrhotic status of a patient, diagnostic accuracy of 90.91% for PCA-QDA and 100% for SVM was observed. Internal and external validation for SVM based classifications observed 100% sensitivity and specificity. The confusion matrix generated by PCA-LDA model computed the validation and calibration accuracy showed 100% sensitivity and specificity, by using 2 PCs for HCV infected and healthy individuals. However, when the PCA QDA analysis was done to classify the non-cirrhotic sera samples from cirrhotic sera samples the diagnostic accuracy achieved was 90.91% based on 7 PC's. SVM was also employed for classification and developed model showed the best results with 100% sensitivity and specificity when external validation was applied. ConclusionsThis study provides an initial insight that ATR-FTIR spectroscopy in conjugation with multivariate data classification tools holds a potentialnot onlytoeffectively diagnosis HCV infection but also to assess non-cirrhotic/cirrhotic status of patients.

Glisson’s capsule matrix structure and function is altered in patients with cirrhosis irrespective of aetiology

Background & AimsGlisson’s capsule is the interstitial connective tissue that surrounds the liver. As part of its normal physiology, it withstands significant daily changes in liver size. The pathophysiology of the capsule in disease is not well understood. The aim of this study was to characterise the changes in capsule matrix, cellular composition, and mechanical properties that occur in liver disease and to determine whether these correlate with disease severity or aetiology. MethodsSamples from ten control patients, and six with steatosis, seven with moderate fibrosis, and 37 with cirrhosis were collected from autopsies, intraoperative biopsies, and liver explants. Matrix proteins and cell markers were assessed by staining and second harmonic generation imaging. Mechanical tensile testing was performed on a test frame. ResultsCapsule thickness was significantly increased in cirrhotic samples compared with normal controls irrespective of disease aetiology (70.12 ± 14.16 μm and 231.58 ± 21.82 μm, respectively), whereas steatosis and moderate fibrosis had no effect on thickness (90.91 ± 11.40 μm). Changes in cirrhosis included an increase in cell number (fibroblasts, vascular cells, infiltrating immune cells, and biliary epithelial cells). Key matrix components (collagens 1 and 3, hyaluronan, versican, and elastin) were all deposited in the lower capsule, although only the relative amounts per area of hyaluronan and versican were increased. Organisational features, including crimping and alignment of collagen fibres, were also altered in cirrhosis. Unexpectedly, capsules from cirrhotic livers had decreased resistance to loading compared with controls. ConclusionsThe liver capsule, similar to the parenchyma, is an active site of disease, demonstrating changes in matrix and cell composition as well as mechanical properties. Impact and implicationsWe assessed the changes in composition and response to stretching of the liver outer sheath, the capsule, in human liver disease. We found an increase in key structural components and numbers of cells as well as a change in matrix organisation of the capsule during the later stages of disease. This allows the diseased capsule to stretch more under any given force, suggesting that it is less stiff than healthy tissue.

Monitoring One-carbon metabolism by mass spectrometry for early diagnosis of cirrhosis and HCC

Introduction: Liver cancer represents one of the most frequent causes of death by cancer, ranked as the sixth most prevalent and the second in lethality. Hepatocellular carcinoma (HCC) is the predominant type of liver cancer and presents an increasing incidence during the last years. Late diagnosis is one of the reasons explaining the low survival rate of HCC patients (5 years survival after diagnosis below 20%). Remarkably, 80% of HCC cases develop in cirrhotic tissue (1). Main risk factors for HCC are well known and include hepatitis B and C viral infections or abusive alcohol consumption. However, the underlying molecular mechanisms remain unknown and its research will lead to the characterization of new biomolecular markers for the early diagnosis, prognosis and therapy of HCC. Metabolic remodeling is a common feature among several hepatic disorders, from steatosis to HCC (2). Tumoral hepatocytes modify their metabolism to satisfy cancer’s proliferative requirements. One-carbon metabolism (OCM) plays a fundamental role maintaining the differentiation and quiescent state of hepatocytes, and is recognized as the link between intermediate metabolism and epigenetic regulation. Owing to these reasons, it might be a potential source of biomarkers for early diagnosis and prognosis of HCC. Accordingly, it has been demonstrated that some OCM enzymes are differentially expressed in murine HCC models (3). Materials and methods: We have developed a robust targeted mass spectrometry-based method, using SRM mode (Selected Reaction Monitoring), for the systematic quantification of 13 enzymes that participate in OCM. For this purpose, purified synthetic heavy standard peptides, as well as OCM recombinant proteins have been used. Sixty-four human liver samples, including 28 control samples, 21 tumoral samples and15 cirrhotic samples have been used. Results and conclusions: We have demonstrated that there is a profound remodeling of the OCM cycle in HCC versus control samples, while cirrhotic samples tend to show intermediate expression levels between both physiological situations. Machine learning- based analysis of our results suggests that monitoring a panel of functionally related proteins might be useful for future clinical developments and improve the management of HCC patients. However, further experiments with larger cohorts are required to confirm these findings. References 1) Ryerson, A. B., et al (2016). Annual Report to the Nation on the Status of Cancer, 1975-2012, featuring the increasing incidence of liver cancer. Cancer, 122(9), 1312-1337. 2) Vander Heiden, et al (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science, 324(5930), 1029-1033 3) Mora, M. I., Corrales, F. J., et al (2017). Prioritizing popular proteins in liver cancer: remodelling one-carbon metabolism. Journal of proteome research, 16(12), 4506-4514.

Identification of miRNA-target gene regulatory networks in liver fibrosis based on bioinformatics analysis.

BackgroundLiver cirrhosis is one of the leading causes of death worldwide. MicroRNAs (miRNAs) can regulate liver fibrosis, but the underlying mechanisms are not fully understood, and the interactions between miRNAs and mRNAs are not clearly elucidated.MethodsmiRNA and mRNA expression arrays of cirrhotic samples and control samples were acquired from the Gene Expression Omnibus database. miRNA-mRNA integrated analysis, functional enrichment analysis and protein-protein interaction (PPI) network construction were performed to identify differentially expressed miRNAs (DEMs) and mRNAs (DEGs), miRNA-mRNA interaction networks, enriched pathways and hub genes. Finally, the results were validated with in vitro cell models.ResultsBy bioinformatics analysis, we identified 13 DEMs between cirrhotic samples and control samples. Among these DEMs, six upregulated (hsa-miR-146b-5p, hsa-miR-150-5p, hsa-miR-224-3p, hsa-miR-3135b, hsa-miR-3195, and hsa-miR-4725-3p) and seven downregulated (hsa-miR-1234-3p, hsa-miR-30b-3p, hsa-miR-3162-3p, hsa-miR-548aj-3p, hsa-miR-548x-3p, hsa-miR-548z, and hsa-miR-890) miRNAs were further validated in activated LX2 cells. miRNA-mRNA interaction networks revealed a total of 361 miRNA-mRNA pairs between 13 miRNAs and 245 corresponding target genes. Moreover, PPI network analysis revealed the top 20 hub genes, including COL1A1, FBN1 and TIMP3, which were involved in extracellular matrix (ECM) organization; CCL5, CXCL9, CXCL12, LCK and CD24, which participated in the immune response; and CDH1, PECAM1, SELL and CAV1, which regulated cell adhesion. Functional enrichment analysis of all DEGs as well as hub genes showed similar results, as ECM-associated pathways, cell surface interaction and adhesion, and immune response were significantly enriched in both analyses.ConclusionsWe identified 13 differentially expressed miRNAs as potential biomarkers of liver cirrhosis. Moreover, we identified 361 regulatory pairs of miRNA-mRNA and 20 hub genes in liver cirrhosis, most of which were involved in collagen and ECM components, immune response, and cell adhesion. These results would provide novel mechanistic insights into the pathogenesis of liver cirrhosis and identify candidate targets for its treatment.

Abstract 2544: TGF-β receptors 1 and 2 are functional biomarkers that stratify risk of hepatocellular cancer (HCC). Artificial intelligence based validation at three centers

Abstract Background: Hepatocellular carcinoma (HCC) is the fastest rising cancer in the USA and is the fourth leading cause of cancer deaths globally. A critical unmet need to reduce high mortality associated with advanced HCC is the ability to identify high-risk individuals for cost-effective, targeted screening, early detection, and prevention strategies. Human genomics and animal models have revealed etiological patterns and multiple genes and signaling pathways such as TGF-β, WNT, VEGF to be associated with the initiation and progression of HCC. We used integrated functional approaches (combining bioinformatics analysis, in vivo mouse models, and in vitro biological and biochemical methods) and identified potential biomarkers for HCC. We examined and validated 10 functional biomarkers for HCC risk prediction. We further harnessed machine learning tools and Artificial Intelligence (AI)-based technology to validate manual analyses of Immunohistochemical (IHC) labeled slides across different centers. Methods: &amp;gt;280 liver tissue samples were collected from patients with HCC and cirrhosis. Immunohistochemistry was performed against 10 functional biomarkers (TGFBR1, TGFBR2, SPTBN1, HMGA2, RSPO3, ITIH4, EPCAM, PLK1, SIRT6, FANCD2). Artificial intelligence-based deep learning technology by AIFORIA was used for the unbiased, intensive, and rapid validation of IHC. Results: The most promising results were for TGFBR1 and TGFBR2, on &amp;gt;80 HCC and cirrhotic samples, IHC labeling revealed: • Both TGFBR1 (p&amp;lt;0.001) and TGFBR2 (p&amp;lt;0.02) showed considerably reduced expression levels in tumor tissue within HCC samples, compared to non-tumorous adjacent tissue and cirrhotic samples. • The results were validated with another set of 70 patient samples at two other centers. Results obtained at George Washington University (GW), University of Maryland (UMD), and the University of Hawaii (UH) for TGFBR2 were consistent. For TGFBR1, GW and UMD showed reproducible results while results at UH lacked statistical significance. • AI-based analysis for TGFBR2 (p&amp;lt;0.002) validated the results from manual analysis ensuring the consistency of results across different sites. • Of the other biomarkers, PLK1 also displayed potentially substantial alterations with increased labeling in tumor tissue as compared to non-tumor tissue. Conclusions: TGFBR2 is a promising functional predictive HCC tissue biomarker. • Tissue biomarkers are important as they provide spatial and contextual information. • Computerized analysis is a powerful tool to capture microscopic patterns in visual data that are not normally assessed in manual analysis such as the pattern of immune cells in cirrhosis and HCC. • AI adds consistency and confidence to results obtained at different sites. Citation Format: Sobia Zaidi, Kirty Shetty, Herbert Yu, Linda Wong, Shuyun Rao, Wilma Jogunoori, Richard Amdur, Shulin Li, Patricia latham, Bao-Ngoc Nguyen, Lopa Mishra. TGF-β receptors 1 and 2 are functional biomarkers that stratify risk of hepatocellular cancer (HCC). Artificial intelligence based validation at three centers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2544.

Comprehensive Analysis of Competing Endogenous RNA Network Focusing on Long Noncoding RNA Involved in Cirrhotic Hepatocellular Carcinoma.

The role of long noncoding RNAs- (lncRNAs-) associated competing endogenous RNA (ceRNA) in the field of hepatocellular carcinoma (HCC) biology is well established, but the involvement of lncRNAs competing interactions in the progression of liver cirrhosis to HCC is still unclear. We aimed to explore the differential expression profiles of lncRNAs, microRNAs (miRNA), and messenger RNAs (mRNAs) to construct a functional ceRNA network in cirrhotic HCC. The lncRNA, miRNA, and mRNA expression datasets were obtained from Gene Expression Omnibus and The Cancer Genome Atlas. Based on miRanda and TargetScan, the HCC-specific ceRNA network was constructed to illustrate the coexpression regulatory relationship of lncRNAs, miRNAs, and mRNAs. The potential prognostic indicators in the network were confirmed by survival analysis and validated by qRT-PCR. A total of 74 lncRNAs, 36 intersection miRNAs, and 949 mRNAs were differentially expressed in cirrhotic HCC samples compared with cirrhosis samples. We constructed a ceRNA network, including 47 lncRNAs, 35 miRNAs, and 168 mRNAs. Survival analysis demonstrated that 2 lncRNAs (EGOT and SERHL), 4 miRNAs, and 40 mRNAs were significantly associated with the overall survival of HCC patients. Two novel regulatory pathways, EGOT-miR-32-5p-XYLT2 axis and SERHL-miR-1269a/miR-193b-3p-BCL2L1/SYK/ARNT/CHST3/LPCAT1 axis, were built up and contribute to the underlying mechanism of HCC pathogenesis. The higher-expressed SERHL was associated with a higher risk of all-cause death. The expressions of SERHL-miR-1269a-BCL2L1 were significantly different using qRT-PCR in vitro studies. lncRNAs EGOT and SERHL might serve as effective prognostic biomarkers and potential therapeutic targets in cirrhotic HCC treatment.

High-resolution three-dimensional visualization of hepatic sinusoids in cirrhotic rats via serial histological sections.

As a specialized intraparenchymal vascular conduit, hepatic sinusoids play a key role in liver microcirculation. This study aimed to explore the three-dimensional (3D) morphological changes of cirrhotic sinusoids by serial histological sections. Cirrhosis was induced by tail vein injection of albumin in Wistar rats with a positive antibody. A total of 356 serial histological sections were prepared from liver tissue blocks of normal and cirrhotic rats. The optical microscope images were registered and reconstructed, and 3D reconstructions of the fine structures of fibrous tissues and sinusoids were subsequently visualized. The fibrosis area of the cirrhotic sample was 6-16 times that of the normal sample (P<0.001). Cirrhosis led to obvious changes in the distribution and morphology of sinusoids, which were mainly manifested as dilation, increased quantity and disordered distribution. Compared with normal liver, cirrhotic liver has a significantly increased volume ratio, number and volume of sinusoids (1.63-, 0.53-, and 1.75-fold, respectively, P<0.001). Furthermore, the samples were further divided into three zones according to the oxygen supply, and there were significant differences in the morphology of the sinusoids in the normal and cirrhotic samples (P<0.05). In particular, morphological parameters of the cirrhotic sinusoids near the portal area were obviously greater than those in the normal liver (P<0.05). 3D morphological structures of hepatic sinusoids were reconstructed, and the adaptive microstructure changes of cirrhotic sinusoids were accurately measured, which has an important implications for the study of hepatic microcirculation and pathological changes of cirrhosis.

IGF2 mRNA Binding Protein 2 Transgenic Mice Are More Prone to Develop a Ductular Reaction and to Progress Toward Cirrhosis.

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2–12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival.

Identification of a novel four-lncRNA signature as a prognostic indicator in cirrhotic hepatocellular carcinoma.

BackgroundMany studies have shown that long noncoding RNAs (lncRNA) are closely associated with the occurrence and development of various tumors and have the potential to be prognostic markers. Moreover, cirrhosis is an important prognostic risk factors in patients with liver cancer. Some studies have reported that lncRNA-related prognostic models have been used to predict overall survival (OS) and recurrence-free survival (RFS) in patients with hepatocellular carcinoma (HCC). However, no one has constructed a prognostic lncRNA model only in patients with cirrhotic HCC. Thus, it is necessary to screen novel potential lncRNA markers for improve the prognosis of cirrhotic HCC patients.MethodsThe probe expression profile dataset (GSE14520–GPL3921) from the Gene Expression Omnibus (GEO), which included 204 cirrhotic HCC samples, was reannotated and the lncRNA and mRNA expression dataset was obtained. The patients were randomly assigned to either the training set (n = 103) and testing set (n = 100). Univariate cox regression and the least absolute shrinkage and selection operator (LASSO) model were applied to screen lncRNAs linked to the OS of cirrhotic HCC in the training set. The lncRNAs having significant correlation with OS were then selected and the multivariate Cox regression model was implemented to construct the prognostic score model. Whether or not this model was related to RFS in the training set was simultaneously determined. The testing set was used to validate the lncRNA risk score model. A risk score based on the lncRNA signature was used for stratified analysis of different clinical features to test their prognostic performance. The prognostic lncRNA-related protein genes were identified by the co-expression matrix of lncRNA-mRNA, and the function of these lncRNAs was predicted through the enrichment of these co-expression genes.ResultsThe signature consisted of four lncRNAs:AC093797.1,POLR2J4,AL121748.1 and AL162231.4. The risk model was closely correlated with the OS of cirrhotic HCC in the training cohort, with a hazard ratio (HR) of 3.650 (95% CI [1.761–7.566]) and log-rank P value of 0.0002. Moreover, this model also showed favorable prognostic significance for RFS in the training set (HR: 2.392, 95% CI [1.374–4.164], log-rank P = 0.0015). The predictive performance of the four-lncRNA model for OS and RFS was verified in the testing set. Furthermore, the results of stratified analysis revealed that the four-lncRNA model was an independent factor in the prediction of OS and RFS of patients with clinical characteristics such as TNM (Tumor, Node, Metastasis system) stages I–II, Barcelona Clinic Liver Cancer (BCLC) stages 0–A, and solitary tumors in both the training set and testing set. The results of functional prediction showed that four lncRNAs may be potentially involve in multiple metabolic processes, such as amino acid, lipid, and glucose metabolism in cirrhotic HCC.

PIN79 ACCESS TO HCV CURE UNDER MEDICAID PROGRAMS IN THE UNITED STATES

Restrictive policies on access to new, curative hepatitis C virus (HCV) treatments represent a substantial barrier to care. In this study, we evaluated the access rates to HCV treatment for Medicaid patients in 27 States in 2016-2017. Data were collected using Trio Health’s disease management program and are specific to Medicaid-insured patients prescribed anti-HCV therapy in 2016-2017. Access was defined as dispensed direct-acting antivirals (DAA). Access rates were only calculated for sample sizes of >30 patients. To determine dispense, patients were followed for 150+ days from prescription date. Cirrhotic status was assigned based on physician-reported fibrosis scores. Of the total HCV Medicaid subjects in the cohort, 3802/5493 (69%) had access to treatment. In the 27 States evaluated, 9 States had 2-year access rates <50%, 10 had rates of 50%-70%, and 8 had rates >70%. In 11 States with sufficient cirrhotic (F4) and non-cirrhotic samples (F0-3), access rates were higher in each State for patients with cirrhosis. The greatest disparities between F0-3 and F4 were observed for Idaho (56% access F0-3 vs. 84% access F4) and California (60% access F0-3 vs. 83% access F4). Smallest differences between F0-3 and F4 were observed for Washington (93% access F0-3 vs. 95% access F4) and Connecticut (96% access F0-3 vs. 100% access F4). Year over year changes were assessed for 18 States: access rates increased in 15 States, decreased in 2 States, and remained flat for 1 State. Despite a decrease in the price of HCV medications, access to HCV treatment remains a barrier to reducing HCV-associated morbidity and mortality. Prioritization of care appears to remain, with many States displaying higher access rates for patients with cirrhosis compared to non-cirrhotic patients.

Differentially methylated loci in NAFLD cirrhosis are associated with key signaling pathways

Altered DNA methylation events contribute to the pathogenesis and progression of metabolic disorders, including nonalcoholic fatty liver disease (NAFLD). Investigations of global DNA methylation patterns in liver biopsies representing severe NAFLD fibrosis have been limited. We used the HumanMethylation 450K BeadChip to analyze genome-wide methylation in patients with biopsy-proven grade 3/4 NAFLD fibrosis/cirrhosis (N = 14) and age- and sex-matched controls with normal histology (N = 15). We identified 208 CpG islands (CGIs), including 99 hypomethylated and 109 hypermethylated CGIs, showing statistically significant evidence (adjusted P value < 0.05) for differential methylation between cirrhotic and normal samples. Comparison of β values for each CGI to the read count of its corresponding gene obtained from RNA-sequencing analysis revealed negative correlation (adjusted P value < 0.05) for 34 transcripts. These findings provide supporting evidence for a role for CpG methylation in the pathogenesis of NAFLD-related cirrhosis, including confirmation of previously reported differentially methylated CGIs, and contribute new insight into the molecular mechanisms underlying the initiation and progression of liver fibrosis and cirrhosis.

Systems analysis of key genes and pathways in the progression of hepatocellular carcinoma

The carcinogenesis of hepatocellular carcinoma (HCC) is a complex process, starting from a chronically altered hepatic microenvironment due to liver cirrhosis and ultimately progressing to HCC. However, the sequential molecular alterations driving the malignant transformation in liver cirrhosis are not clearly defined.In this study, we obtained gene expression profiles of HCC, including 268 tumor tissues, 243 adjacent tumor tissues, and 40 cirrhotic tissues (GSE25097) from Gene Expression Omnibus (GEO), to comprehensively define changes in the transcriptome of HCC during the sequential evolution of liver cirrhosis into HCC.We showed that changes in the molecular profiles of cirrhotic and adjacent tumor samples were small and quite uniform, whereas there was a striking increase in the heterogeneity of tumors in HCC tissues at the mRNA level. A massive deregulation of key oncogenic molecules and pathways was observed from cirrhosis to HCC tumors. In addition, we focused on FOXO1 and DCN, 2 critical tumor suppressor genes that play an important role in liver cirrhosis and HCC development. FOXO1 and DCN expression levels were significantly reduced in tumor tissues compared with adjacent tumor tissues in HCC. Kaplan-Meier analysis revealed that FOXO1 and DCN expression was positively correlated with overall survival, defining FOXO1 and DCN as adverse prognostic biomarkers for HCC.This system-level research provided new insights into the molecular mechanisms of HCC carcinogenesis. FOXO1 and DCN may be applied as potential targets for HCC treatment in the future.

Long Noncoding RNA HOTTIP Promotes Mouse Hepatic Stellate Cell Activation via Downregulating miR-148a

Background/Aims: HOTTIP is a critical modulator in human diseases including liver cancer, but its role and molecular biological mechanisms in liver fibrosis are still unclear. Methods: The expression profile of HOTTIP during the progression of liver fibrosis was detected in human liver samples and in CCl₄-treated mice using qRT-PCR. The expressing sh-HOTTIP adenoviral vector was used to reduce HOTTIP levels in vivo. Dual-Luciferase Reporter Assay was performed to validate the interaction between miR-148a and HOTTIP, TGFBR1, or TGFBR2. Results: HOTTIP expressions in fibrotic liver samples and cirrhotic liver samples were significantly upregulated compared with healthy liver controls, and cirrhotic samples exhibited the highest levels of HOTTIP. Moreover, HOTTIP expressions were substantially induced in the liver tissues and hepatic stellate cells (HSC) of CCl₄-treated mice. Ad-shHOTTIP delivery could alleviate CCl₄- induced liver fibrosis in mice. Down-regulation of HOTTIP inhibited the viability and activation of HSCs in vitro, and HOTTIP negatively regulated miR-148a expression in HSCs. miR-148a had a negative effect on HSC activation by targeting TGFBR1 and TGFBR2. Conclusion: HOTTIP is involved in the progression of liver fibrosis by promoting HSC activation. The high level of HOTTIP downregulates miR-148a, thus to increase the level of TGFBR1 and TGFBR2 and contribute to liver fibrosis.

Bile acids promote diethylnitrosamine-induced hepatocellular carcinoma via increased inflammatory signaling.

Hepatocellular carcinoma (HCC) is the most common hepatic malignancy and the third leading cause of cancer related deaths. Previous studies have implicated bile acids in pathogenesis of HCC, but the mechanisms are not known. We investigated the mechanisms of HCC tumor promotion by bile acids the diethylnitrosamine (DEN)-initiation-cholic acid (CA)-induced tumor promotion protocol in mice. The data show that 0.2% CA treatment resulted in threefold increase in number and size of DEN-induced liver tumors. All tumors observed in DEN-treated mice were well-differentiated HCCs. The HCCs observed in DEN-treated CA-fed mice exhibited extensive CD3-, CD20-, and CD45-positive inflammatory cell aggregates. Microarray-based global gene expression studies combined with Ingenuity Pathway Analysis revealed significant activation of NF-κB and Nanog in the DEN-treated 0.2% CA-fed livers. Further studies showed significantly higher TNF-α and IL-1β mRNA, a marked increase in total and phosphorylated-p65 and phosphorylated IκBα (degradation form) in livers of DEN-treated 0.2% CA-fed mice. Treatment of primary mouse hepatocytes with various bile acids showed significant induction of stemness genes including Nanog, KLF4, Sox2, and Oct4. Quantification of total and 20 specific bile acids in liver, and serum revealed a tumor-associated bile acid signature. Finally, quantification of total serum bile acids in normal, cirrhotic, and HCC human samples revealed increased bile acids in serum of cirrhotic and HCC patients. Taken together, these data indicate that bile acids are mechanistically involved pathogenesis of HCC and may promote HCC formation via activation of inflammatory signaling.

Insights into translational utility of rodent models of hepatic fibrosis

Activation of hepatic stellate cells (HSCs) has been established as the dominant process in the progression of hepatic fibrosis. A number of phenotypic alterations occur during activation of HSCs, including proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis and retinoid loss. These phenotypic changes could serve as potential targets for anti‐fibrotic therapy. In the past, several rodent models have been used to delineate the mechanism of fibrogenesis. However, the translatability of these animal models for clinical development remains largely questionable. The goal of this study is to compare the number of proliferating HSCs in commonly used mouse models of hepatic fibrosis [carbon tetrachloride (CCl4), bile duct ligation (BDL) and metabolic disease (ob/ob high fat diet)] and human cirrhotic liver samples. Dual immunostaining for Ki67 (proliferative marker) and a marker for activated HSCs (aHSCs) on formalin fixed paraffin embedded liver sections were performed. Whole slide quantitative image analysis on dually immunostained human cirrhotic liver samples (n=5) showed only 1.9% (range 1.3–3%) of aHSCs cells were positive for Ki67. In contrast, BDL, ob/ob high fat diet, and CCl4 mouse models showed approximately 4%, 5%, and 17% of aHSCs were positive for Ki67, respectively. In summary, cognizance of the discrepancy in the number of proliferating HSCs between human cirrhotic samples and mouse models of fibrosis will assist in careful design of preclinical studies.

Immunohistochemical angiogenic biomarkers in hepatocellular carcinoma and cirrhosis: correlation with pathological features

OBJECTIVE:To investigate immunohistochemical markers of angiogenesis and their association with pathological prognostic features in hepatocellular carcinoma and cirrhotic liver.METHODS:Vascular endothelial growth factor, CD105, and cyclooxygenase-2 were immunohistochemically detected in 52 hepatocellular carcinoma tissue samples and 48 cirrhotic liver tissue samples. Semiquantitative measurements of vascular endothelial growth factor and cyclooxygenase-2 were evaluated considering the degree and intensity of immunostaining based on a 7-point final scoring scale. CD105 microvascular density (MVD-CD105) was measured using automated analysis. Morphological aspects evaluated in the hepatocellular carcinoma samples included size (≤2 and >2 cm), differentiation grade, and microvascular invasion.RESULTS:The mean vascular endothelial growth factor immunoreactivity score was slightly higher in the hepatocellular carcinoma samples (4.83±1.35) than the cirrhotic liver (4.38±1.28) samples. There was a significant and direct correlation between these mean scores (rs=0.645, p=0.0001). Cyclooxygenase-2 was expressed in all the cirrhotic liver samples but was only found in 78% of the hepatocellular carcinoma samples. The mean cyclooxygenase-2 score was higher in the cirrhotic liver samples (4.85±1.38) than the hepatocellular carcinoma samples (2.58±1.68), but there was no correlation between the scores (rs=0.177, p=0.23). The mean CD105 percentage in the hepatocellular carcinoma samples (11.2%) was lower than that in the cirrhotic samples (16.9%). There was an inverse relationship in MVD-CD105 expression between the hepatocellular carcinoma and cirrhotic samples (rs=-0.78, p=0.67). There were no significant associations between vascular endothelial growth factor expression and morphological characteristics. Cyclooxygenase-2 and CD105 were associated with hepatocellular carcinoma differentiation grade (p=0.003 and p=0.05, respectively).CONCLUSION:Vascular endothelial growth factor, cyclooxygenase-2, and MVD-CD105 were highly expressed in cirrhotic liver compared to hepatocellular carcinoma and might be involved in liver carcinogenesis. Additionally, cyclooxygenase-2 and CD105 might be involved in hepatocellular carcinoma differentiation grade.

Bile Acids Promote Diethylnitrosamine‐induced Hepatocellular Carcinoma via Increased Inflammatory Signaling.

Hepatocellular carcinoma (HCC) is the most common hepatic malignancy and the third leading cause of cancer related deaths. Previous studies have implicated bile acids in pathogenesis of HCC but the mechanisms are not known. We investigated the mechanisms of bile acid‐mediated promotion of HCC using a diethylnitrosamine (DEN)‐mediated initiation‐cholic acid (CA)‐induced tumor promotion protocol in mice. The data show that 0.2% CA treatment resulted in 3‐fold increase in number and size of DEN‐induced liver tumors. All tumors observed in DEN‐treated mice were well‐differentiated HCCs. Microarray based global gene expression studies combined with Ingenuity Pathway Analysis revealed NF‐κB pathway as the main signaling pathways activated in the DEN‐treated 0.2% CA‐fed livers. Further studies showed significantly higher TNFα, IL‐1β, MCP‐1 mRNA, a significant increase in total and phosphorylated‐p65 and phosphorylated IκBa (degradation form) and higher EGR1 mRNA and protein in livers of DEN‐treated 0.2% CA‐fed mice. Interestingly, total bile acid quantification revealed increase in hepatic and serum bile acids in both DEN‐treated and DEN‐treated 0.2% CA‐fed mice, which was higher in the latter group. Finally, quantification of total serum bile acids in normal, cirrhotic and HCC human samples revealed increased bile acids in serum of cirrhotic and HCC patients. Taken together, these data indicate that bile acids are mechanistically involved pathogenesis of HCC and may promote HCC formation via activation of inflammatory signaling.

Molecular Determinants of Prognosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma is one of the leading causes of death by cancer worldwide. Prognosis of hepatocellular carcinoma is determined by characteristics of the tumor and the surrounding cirrhotic liver. Several molecular signatures reflecting tumor biology and derived from tumor analyses predict early tumor recurrence and survival. In contrast, molecular signatures from cirrhotic non-tumor samples are enriched in immunity/inflammation related genes and could predict late tumor recurrence. Moreover, combination of clinical, pathological, and molecular features may refine prognosis prediction in these patients. Finally, molecular signatures from both tumor and non-tumor tissues will be helpful in the future to guide treatments in different clinical settings.