Non-fibrotic Liver Research Articles

Distinct Molecular Alterations in Hepatocellular Carcinoma (HCC) Arising in a Non-Fibrotic Liver Compared to a Cirrhotic Liver

Abstract Introduction/Objective HCC is known to more likely develop in a cirrhotic liver than a non-cirrhotic liver, irrespective of etiology, with TP53 and CTNNB1 being the most frequent driver mutations in this group. Whether there are distinctive molecular pathways for HCCs developing in the non-cirrhotic liver is not known. This study evaluated whether there is any difference in the mutational profile of HCCs arising in a non-fibrotic liver compared to a cirrhotic liver. Methods/Case Report Clinicopathologic and in-house next generation sequencing (NGS) data from HCC cases sequenced between 1/1/18-6/30/23 were reviewed. Cases from a targeted precision NGS panel were excluded. Results were correlated with publicly available data from The Cancer Genome Atlas (TCGA). Results (if a Case Study enter NA) 29 cases were available for review; 24 patients had background cirrhosis at the time of diagnosis, while 5 patients had no evidence of fibrosis. 21 of 24 patients (88%) patients in the cirrhosis group had a variant in TP53 and/or CTNNB1 that was considered to be disease associated or probably disease associated, whereas only 1 of 5 patients (20%) in the non-fibrotic group had a variant detected in one of these genes (Fisher’s exact: p < 0.01). BAP1 and ARID1A variants were detected in two patients in the non-fibrotic group. TP53 and CTNNB1 were generally mutually exclusive (86% of cases), however, 3 patients harbored both TP53 and CTNBB1 variants; interestingly all 3 had background cirrhosis and HCV infection. All 6 poorly differentiated tumors occurred in cirrhotic livers and were found to have TP53 variants (Figure 1). TCGA data (N = 210) similarly showed that patients with intermediate fibrosis or established cirrhosis (N = 138) were more likely than patients without background fibrosis (N = 72) to harbor TP53 or CTNNB1 variants (64% vs 42%; Chi-squared: p < 0.01; Figure 2). Aside from TP53 and CTNNB1, the most common variants seen in the TCGA non-fibrotic group were in TTN (22), ALB (10), FREM2 (9), LRP1B (9), and MUC16 (9). Conclusion Both in-house and TCGA data showed that TP53 and/or CTNNB1 variants were more likely to be seen in HCC cases arising in a cirrhotic liver compared to a non-fibrotic liver. This data suggests there may be unique and less-well characterized molecular pathways involved in the neoplastic progression of HCC in a non-fibrotic liver.

LiverMap pipeline for 3D imaging of human liver reveals volumetric spatial dysregulation of cirrhotic vasculobiliary architecture.

The liver contains an intricate microstructure that is critical for liver function. Architectural disruption of this spatial structure is pathologic. Unfortunately, 2D histopathology - the gold standard for pathological understanding of many liver diseases - can misrepresent or leave gaps in our understanding of complex 3D structural features. Here, we utilized immunostaining, tissue clearing, microscopy, and computational software to create 3D multilobular reconstructions of both non-fibrotic and cirrhotic human liver tissue. We found that spatial architecture in human cirrhotic liver samples with varying etiologies had sinusoid zonation dysregulation, reduction in glutamine synthetase-expressing pericentral hepatocytes, regression of central vein networks, disruption of hepatic arterial networks, and fragmentation of biliary networks, which together suggest a pro-portalization/decentralization phenotype in cirrhotic tissue. Further implementation of 3D pathological analyses may provide a deeper understanding of cirrhotic pathobiology and inspire novel treatments for liver disease.

Single center experience with ALPPS and timing with stage 2 in patients with fibrotic/cirrhotic liver

Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a novel procedure for major resection in patients with insufficient future liver remnant (FLR). Effective FLR augmentation is pivotal in the completion of ALPPS. Liver fibrosis/cirrhosis associated with chronic viral hepatitis impairs liver regeneration. To investigate the augmentation of FLR in associating ALPPS between patients with fibrotic/cirrhotic livers (FL) and non-fibrotic livers (NFL) and compare their short-term clinical outcomes and long-term survival. Patients were divided into two groups based on the Ishak modified staging: non-fibrotic liver group (NFL, stage 0) and fibrotic/cirrhotic liver group (FL, stage 1–5/6). Weekly liver regeneration in FLR, perioperative data, and survival outcomes were investigated. Twenty-seven patients with liver tumors underwent ALPPS (NFL, n = 7; FL, n = 20). NFL and FL patients had viral hepatitis (28.6% [n = 2] and 95% [n = 19]), absolute FLR volume increments of 134.90 ml and 161.85 ml (p = 0.825), and rates of hypertrophy were 16.46 ml/day and 13.66 ml/day (p = 0.507), respectively. In the FL group, baseline FLR volume was 360.13 ml, postoperatively it increased to a plateau (542.30 ml) in week 2 and declined (378.45 ml) in week 3. One patient (3.7%) with cirrhotic liver (stage 6) failed to proceed to ALPPS-II. The overall ALPPS-related major complication rate was 7.4%. ALPPS is feasible for fibrotic liver patients classified by Ishak modified stages ≤ 5. After ALPPS-I, 14 days for FLR augmentation seems an appropriate waiting time to reach a maximum FLR volume in these patients.

Hepatocellular Carcinoma in Non-Fibrotic Liver: A Narrative Review.

Hepatocellular carcinoma (HCC) in a non-fibrotic liver (F0) is considered to be rare, and there is a marked paucity of studies in the literature on this HCC type. A review of the literature shows some important clinical and tumor characteristics: (a) it occurs mainly in young female and elder male patients; (b) clinically, under normal hepatic function, alpha-fetoprotein level is often normal, and there are no risk factors; (c) associated with metabolic disease; (d) macroscopically, single large lesions are noted; and (e) microscopically, the lesions are well-differentiated and encapsulated. Radiological imaging results are straightforward, showing arterial hyperenhancement and later wash-out. The combined use of B-mode and contrast-enhanced (CE) ultrasound (US) is the most reliable and cost-effective diagnostic method. Few peri-and post-operative complications are noted and 5-year survival is not inferior to patients with HCC on fibrosis liver despite the lesion's large size. Most clinicians believe that HCC is unlikely to occur if patients have no symptoms and normal hepatic function. Although detailed clinical data are very limited, we expect that this review will help to improve the clinical management of HCC in non-fibrotic livers.

MRI with diffusion-weighted image in assessment of hepatic fibrosis and steatosis

Background FibroScan and diffusion MRI have been recommended for staging and noninvasive diagnosis of hepatic fibrosis and steatosis. Aim To evaluate the diagnostic efficiency of conventional MRI and diffusion-weighted imaging in diagnosis and staging of hepatic fibrosis and steatosis. Patients and methods This research was conducted on 30 patients proven to have hepatic fibrosis or steatosis based on clinical, laboratory, and/or ultrasonographic criteria. Both sexes were included. Their age ranged from 20 to 60 years. The participants were sent to the MRI Unit in the Department of Radiodiagnosis, Tanta University Hospitals, over a period of 12 months from March 2021 till March 2022. Results There was a significant difference between apparent diffusion coefficient values of F0 and F1, F3, and F4 (P=0.010, 0.041, and 0.019, respectively); however, no significant difference was observed among the other various fibrosis stages. Conclusions FibroScan is a good noninvasive route for detection of hepatic fibrosis and steatosis. It can differentiate among various phases of hepatic fibrosis and steatosis. MRI can differentiate between fibrotic and nonfibrotic liver and also steatotic from nonsteatotic liver. However, it cannot differentiate between various phases of hepatic fibrosis and steatosis, especially the intermediate stages.

Hepatic stellate cells in the injured liver: Perspectives beyond hepatic fibrosis.

Over the last two decades, our understanding of the pathological role of hepatic stellate cells (HSCs) in fibrotic liver disease has increased dramatically. AsHSCs are identified as the principal collagen-producing cells in the injured liver, several experimental and clinical studies have targeted HSCs to treat liver fibrosis. However, HSCs also play a critical role in developing nonfibrotic liver diseases such as cholestasis, portal hypertension, and hepatocellular carcinoma (HCC). Therefore, this review exclusively focuses on the role of activated HSCs beyond hepatic fibrosis. In cholestasis conditions, elevated bile salts and bile acids activate HSCs to secrete collagen and other extracellular matrix products, which cause biliary fibrosis and cholangitis. In the chronically injured liver, autocrine and paracrine signaling from liver sinusoidal endothelial cells activates HSCs to induce portal hypertension via endothelin-1 release. In the tumor microenvironment (TME), activated HSCs are the major source of cancer-associated fibroblasts (CAF). The crosstalk between activated HSC/CAF and tumor cells isassociated with tumor cell proliferation, migration, metastasis, and chemoresistance. In TME, activated HSCs convert macrophages to tumor-associated macrophages and induce the differentiation of dendritic cells (DCs) and monocytes to regulatory DCs and myeloid-derived suppressor cells, respectively. This differentiation, in turn, increases T cells proliferation and induces their apoptosis leading to reduced immune surveillance in TME. Thus, HSCs activation in chronically injured liver is a critical process involved in the progression of cholestasis, portal hypertension, and liver cancer.

Targeted truncated TGF-β receptor type II delivery to fibrotic liver by PDGFβ receptor-binding peptide modification for improving the anti-fibrotic activity against hepatic fibrosis in vitro and in vivo

Truncated transforming growth factor-β receptor type II (tTβRII) is a promising anti-fibrotic candidate because it attenuates excessive transforming growth factor-β1 (TGF-β1) and then blocks TGF-β1 activity in hepatic fibrosis. However, its use has been greatly limited due to the fact that it is expensive to chemically synthesize and it does not specifically target to the lesion site. In this study, we describe that platelet-derived growth factor β receptor (PDGFβR)-binding peptide BiPPB modified tTβRII (BiPPB-tTβRII) was prepared from the cleavage of SUMO-BiPPB-tTβRII by digestion with SUMO-specific protease. Moreover, compared to the unmodified tTβRII, the target protein BiPPB-tTβRII not only highly specific targeted activated hepatic stellate cells (HSCs) and fibrotic liver tissue, but also significantly inhibited the protein levels of fibrosis-related genes in TGF-β1-induced HSC-T6 cells and CCl4-induced liver fibrosis in mice. Furthermore, BiPPB-tTβRII markedly ameliorated liver morphology, fibrotic responses and the damage of liver function in fibrosis animal. More importantly, BiPPB-tTβRII showed a much lesser extent in binding to quiescent HSCs and non-fibrotic liver tissue. Taken together, our results suggested that the target protein BiPPB-tTβRII, with its high specific fibrotic liver-targeting potential and its improved anti-fibrotic activity in liver fibrosis, may be a potential therapeutic agent for liver fibrosis.

Integrated quantitative susceptibility and R2 * mapping for evaluation of liver fibrosis: An ex vivo feasibility study.

To develop a method for noninvasive evaluation of liver fibrosis, we investigated the differential sensitivities of quantitative susceptibility mapping (QSM) and R2 * mapping using corrections for the effects of liver iron. Liver fibrosis is characterized by excessive accumulation of collagen and other extracellular matrix proteins. While collagen increases R2 * relaxation, measures of R2 * for fibrosis are confounded by liver iron, which may be present in the liver over a wide range of concentrations. The diamagnetic collagen contribution to susceptibility values measured by QSM is much less than the contribution of highly paramagnetic iron. In 19 ex vivo liver explants with and without fibrosis, QSM (χ), R2 * and proton density fat fraction (PDFF) maps were constructed from multiecho gradient-recalled echo (mGRE) sequence acquisition at 3 T. Median parameter values were recorded and differences between the MRI parameters in nonfibrotic vs. advanced fibrotic/cirrhotic samples were evaluated using Mann-Whitney U tests and receiver operating characteristic analyses. Logistic regression with stepwise feature selection was employed to evaluate the utility of combined MRI measurements for detection of fibrosis. Median R2 * increased in fibrotic vs. nonfibrotic liver samples (P = .041), while differences in χ and PDFF were nonsignificant (P = .545 and P = .395, respectively). Logistic regression identified the combination of χ and R2 * significant for fibrosis detection (logit [prediction] = -8.45 + 0.23 R2 * - 28.8 χ). For this classifier, a highly significant difference between nonfibrotic vs. advanced fibrotic/cirrhotic samples was observed (P = .002). The model exhibited an AUC of 0.909 (P = .003) for detection of advanced fibrosis/cirrhosis, which was substantially higher compared with AUCs of the individual parameters (AUC 0.591-0.784). An integrated QSM and R2 * analysis of mGRE 3 T imaging data is promising for noninvasive diagnostic assessment of liver fibrosis.

Advanced MRI of Liver Fibrosis and Treatment Response in a Rat Model of Nonalcoholic Steatohepatitis.

Background Liver biopsy is the reference standard to diagnose nonalcoholic steatohepatitis (NASH) but is invasive with potential complications. Purpose To evaluate molecular MRI with type 1 collagen-specific probe EP-3533 and allysine-targeted fibrogenesis probe Gd-Hyd, MR elastography, and native T1 to characterize fibrosis and to assess treatment response in a rat model of NASH. Materials and Methods MRI was performed prospectively (June-November 2018) in six groups of male Wistar rats (a) age- and (b) weight-matched animals received standard chow (n = 12 per group); (c) received choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) for 6 weeks or (d) 9 weeks (n = 8 per group); (e) were fed 6 weeks of CDAHFD and switched to standard chow for 3 weeks (n = 12); (f) were fed CDAHFD for 9 weeks with daily treatment of elafibranor beginning at week 6 (n = 14). Differences in imaging measurements and tissue analyses among groups were tested with one-way analysis of variance. The ability of each imaging measurement to stage fibrosis was quantified by using area under the receiver operating characteristic curve (AUC) with quantitative digital pathology (collagen proportionate area [CPA]) as reference standard. Optimal cutoff values for distinguishing advanced fibrosis were used to assess treatment response. Results AUC for distinguishing fibrotic (CPA >4.8%) from nonfibrotic (CPA ≤4.8%) livers was 0.95 (95% confidence interval [CI]: 0.91, 1.00) for EP-3533, followed by native T1, Gd-Hyd, and MR elastography with AUCs of 0.90 (95% CI: 0.83, 0.98), 0.84 (95% CI: 0.74, 0.95), and 0.65 (95% CI: 0.51, 0.79), respectively. AUCs for discriminating advanced fibrosis (CPA >10.3%) were 0.86 (95% CI: 0.76, 0.97), 0.96 (95% CI: 0.90, 1.01), 0.84 (95% CI: 0.70, 0.98), and 0.74 (95% CI: 0.63, 0.86) for EP-3533, Gd-Hyd, MR elastography, and native T1, respectively. Gd-Hyd MRI had the highest accuracy (24 of 26, 92%; 95% CI: 75%, 99%) in identifying responders and nonresponders in the treated groups compared with MR elastography (23 of 26, 88%; 95% CI: 70%, 98%), EP-3533 (20 of 26, 77%; 95% CI: 56%, 91%), and native T1 (14 of 26, 54%; 95% CI: 33%, 73%). Conclusion Collagen-targeted molecular MRI most accurately detected early onset of fibrosis, whereas the fibrogenesis probe Gd-Hyd proved most accurate for detecting treatment response. © RSNA, 2020 Online supplemental material is available for this article.

Diffusion MRI Derived per Area Vessel Density as a Surrogate Biomarker for Detecting Viral Hepatitis B-Induced Liver Fibrosis: A Proof-of-Concept Study.

Liver vessel density can be evaluated by an imaging biomarker diffusion-derived vessel density (DDVD): DDVD/area(b0b2) = Sb0/ROIarea0 - Sb2/ROIarea2, where Sb0 and Sb2 refer to the liver signal when b is 0 or 2 (s/mm2); ROIarea0 and ROIarea2 refer to the region of interest (ROI) on b = 0 or 2 images; and Sb2 may be replaced by Sb15 (b = 15). This concept was validated in this study. Liver diffusion images were acquired at 1.5 T. For a scan-rescan repeatability study of six subjects, b values of 0 and 2 were used. The validation study was composed of 26 healthy volunteers and 19 consecutive suspected chronic viral hepatitis B patients, and diffusion images with b values of 0, 2, 15, 20, 30, 45, 50, 60, 80, 100, 200, 300, 600, and 800 were acquired. Four patients did not have liver fibrosis, and the rest were four stage 1, three stage 2, four stage 3, and one stage 4 patients, respectively. The intraclass correlation coefficient for repeatability was 0.994 for DDVD/area(Sb0Sb2) and 0.978 for DDVD/area(Sb0Sb15). In the validation study, DDVD/area(Sb0Sb2) and area(Sb0Sb15) were 14.80 ± 3.06 and 26.58 ± 3.97 for healthy volunteers, 10.51 ± 1.51 and 20.15 ± 2.21 for stage 1-2 fibrosis patients, and 9.42 ± 0.87 and 19.42 ± 1.89 for stage 3-4 fibrosis patients. For 16 patients where IVIM analysis was performed, a combination of DDVD/area, PF, and Dfast achieved the best differentiation for nonfibrotic livers and fibrotic livers. DDVD/area were weakly correlated with PF or Dfast. Both DDVD/area(Sb0Sb2) and area(Sb0Sb15) are useful imaging biomarkers to separate fibrotic and nonfibrotic livers, with fibrotic livers having lower measurements.

CPEB4 Increases Expression of PFKFB3 to Induce Glycolysis and Activate Mouse and Human Hepatic Stellate Cells, Promoting Liver Fibrosis

We investigated mechanisms of hepatic stellate cell (HSC) activation, which contributes toliver fibrogenesis. We aimed to determine whether activated HSCs increase glycolysis, which is regulated by6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and whether this pathway might serve as a therapeutic target. We performed studies with primary mouse HSCs, human LX2 HSCs, human cirrhotic liver tissues, rats and mice with liver fibrosis (due to bile duct ligation [BDL] or administration of carbon tetrachloride), and CPEB4-knockout mice. Glycolysis was inhibited in cells and mice by administration of a small molecule antagonist of PFKFB3 (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one [3PO]). Cells were transfected with small interfering RNAs that knock down PFKFB3 or CPEB4. Up-regulation of PFKFB3 protein and increased glycolysis were early and sustained events during HSC activation and accompanied by increased expression of markers of fibrogenesis; incubation of HSCs with 3PO or knockdown of PFKFB3 reduced their activation and proliferation. Mice with liver fibrosis after BDL had increased hepatic PFKFB3; injection of 3PO immediately after the surgery prevented HSC activation and reduced the severity of liver fibrosis compared with mice given vehicle. Levels of PFKFB3 protein were increased in fibrotic liver tissues from patients compared with non-fibrotic liver. Up-regulation of PFKFB3 in activated HSCs did not occur via increased transcription, but instead via binding of CPEB4 to cytoplasmic polyadenylation elements within the 3'-untranslated regions of PFKFB3 messenger RNA. Knockdown of CPEB4 in LX2 HSCs prevented PFKFB3 overexpression and cell activation. Livers from CPEB4-knockout had decreased PFKFB3 and fibrosis after BDL or administration of carbon tetrachloride compared with wild-type mice. Fibrotic liver tissues from patients and rodents (mice and rats) have increased levels of PFKFB3 and glycolysis, which are essential for activation of HSCs. Increased expression of PFKFB3 is mediated by binding of CPEB4 to its untranslated messenger RNA. Inhibition or knockdown of CPEB4 or PFKFB3 prevents HSC activation and fibrogenesis in livers of mice.

Hepatocarcinoma–intestine–pancreas/pancreatitis-associated protein (HIP/PAP) confers protection against hepatic fibrosis through downregulation of transforming growth factor β receptor II

Hepatocarcinoma–intestine–pancreas/pancreatitis-associated protein (HIP/PAP) has antimicrobial, antioxidant, anti-inflammatory, mitogenic, and antiapoptotic effects and thus exerts important functions in the maintenance of integrity and homeostasis of several organs, such as the gastrointestinal tract, pancreas, and liver. Although the potent hepatoprotective effect of HIP/PAP has been validated, its impact on liver fibrosis has not been reported. In this study, we evaluated the role of HIP/PAP on hepatic fibrosis and explored the possible underlying mechanisms. We found that the expression of HIP/PAP and its mouse counterpart, Reg3B, was markedly upregulated in fibrotic human or mouse livers. Intraperitoneal (i.p.) interleukin (IL)-10, IL-6, and TNF-α but not TGF-β1 significantly induced hepatic overexpression of Reg3B in mice. In both CCl4 and BDL liver fibrosis models, adenovirus-mediated ectopic expression of HIP/PAP markedly alleviated liver injury, inflammation, collagen deposition, hepatic stellate cell activation, and the overexpression of profibrotic cytokines, including transforming growth factor β1 (TGF-β1), platelet-derived growth factor (PDGF)-A, B, connective tissue growth factor (CTGF), and plasminogen activator inhibitor-1 (PAI-1), in mice. In vitro experiments demonstrated that, in addition to suppressing hepatic stellate cell proliferation and accelerating hepatocyte proliferation, HIP/PAP mitigated TGF-β1-induced hepatic stellate cell activation, hepatocyte epithelial-mesenchymal transition (EMT) and upregulated expression of profibrotic cytokines in both hepatic stellate cells and hepatocytes. Moreover, HIP/PAP attenuated the overexpression of TGF-β receptor II (TGF-βRII) in fibrotic mouse livers and decreased the basal expression of TGF-βRII in nonfibrotic mouse livers as well as in cultured hepatocytes and hepatic stellate cells, which is at least partly attributable to the TGF-β1-antagonizing function of HIP/PAP. This study indicates that increased expression of hepatic HIP/PAP serves as a countermeasure against liver injury and fibrosis. Exogenous supplementation of HIP/PAP might be a promising therapeutic agent for hepatic fibrosis as well as liver injury.

Elevated Autotaxin and LPA Levels During Chronic Viral Hepatitis and Hepatocellular Carcinoma Associate with Systemic Immune Activation.

Circulating autotaxin (ATX) is elevated in persons with liver disease, particularly in the setting of chronic hepatitis C virus (HCV) and HCV/HIV infection. It is thought that plasma ATX levels are, in part, attributable to impaired liver clearance that is secondary to fibrotic liver disease. In a discovery data set, we identified plasma ATX to be associated with parameters of systemic immune activation during chronic HCV and HCV/HIV infection. We and others have observed a partial normalization of ATX levels within months of starting interferon-free direct-acting antiviral (DAA) HCV therapy, consistent with a non-fibrotic liver disease contribution to elevated ATX levels, or HCV-mediated hepatocyte activation. Relationships between ATX, lysophosphatidic acid (LPA) and parameters of systemic immune activation will be discussed in the context of HCV infection, age, immune health, liver health, and hepatocellular carcinoma (HCC).

Normal and fibrotic liver parenchyma respond differently to irreversible electroporation

BackgroundThe safety and efficacy of irreversible electroporation (IRE) in treating hepatic, biliary, and pancreatic malignancies are active areas of clinical investigation. In addition, recent studies have shown that IRE may enable regenerative surgery and in vivo tissue engineering. To use IRE effectively in these clinical applications, it is important to understand how different tissue microenvironments impact the response to IRE. In this study, we characterize the electrical and histological properties of non-fibrotic and fibrotic liver parenchyma before and after IRE treatment. MethodsElectrical resistivity and histology of fibrotic liver from C57BL/6 mice fed a 0.1% 3,5-diethylcarbonyl-1,4-dihydrocollidine (DDC) diet were compared to those of non-fibrotic liver from matched control mice before and after IRE treatment. ResultsAt baseline, the electrical resistivity of fibrotic liver was lower than that of non-fibrotic liver. Post-IRE, resistivity of non-fibrotic liver declined and then recovered back to baseline with time, correlating with hepatocyte repopulation of the ablated parenchyma without deposition of fibrotic scar. In contrast, resistivity of fibrotic liver remained depressed after IRE treatment, correlating with persistent inflammation. ConclusionNon-fibrotic and fibrotic liver respond to IRE differently. The underlying tissue microenvironment is an important modifying factor to consider when designing IRE protocols for tissue ablation.

Risk Factors of Hepatocellular Carcinomas Developed on Non-fibrotic Liver: Case-control Study (NoFlic Study)

Risk Factors of Hepatocellular Carcinomas Developed on Non-fibrotic Liver: Case-control Study (NoFlic Study)

PNPLA3 and TM6SF2 variants as risk factors of hepatocellular carcinoma across various etiologies and severity of underlying liver diseases.

Few single nucleotide polymorphisms (SNPs) have been reproducibly associated with hepatocellular carcinoma (HCC). Our aim was to test the association between nine SNPs and HCC occurrence. SNPs in genes linked to HCC (DEPDC5, GRIK1, KIF1B, STAT4, MICA, DLC1, DDX18) or to liver damage (PNPLA3-rs738409, TM6SF2-rs58542926) in GWAS were genotyped in discovery cohorts including 1,020 HCC, 2,021 controls with chronic liver disease and 2,484 healthy individuals and replication was performed in prospective cohorts of cirrhotic patients with alcoholic liver disease (ALD, n = 249) and hepatitis C (n = 268). In the discovery cohort, PNPLA3 and TM6SF2 SNPs were associated with HCC (OR = 1.67 [CI95%:1.16-2.40], p = 0.005; OR = 1.45 [CI95%:1.08-1.94], p = 0.01) after adjustment for fibrosis, age, gender and etiology. In contrast, STAT4-rs7574865 was associated with HCC only in HBV infected patients (p = 0.03) and the other tested SNP were not linked with HCC risk. PNPLA3 and TM6SF2 variants were independently associated with HCC in patients with ALD (OR = 3.91 [CI95%:2.52-6.06], p = 1.14E-09; OR = 1.79 [CI95%:1.25-2.56], p = 0.001) but not with other etiologies. PNPLA3 SNP was also significantly associated with HCC developed on a nonfibrotic liver (OR = 2.19 [CI95%:1.22-3.92], p = 0.007). The association of PNPLA3 and TM6SF2 with HCC risk was confirmed in the prospective cohort with ALD. A genetic score including PNPLA3 and TM6SF2 minor alleles showed a progressive significant increased risk of HCC in ALD patients. In conclusion, PNPLA3-rs738409 and TM6SF2-rs58542926 are inherited risk variants of HCC development in patients with ALD in a dose dependent manner. The link between PNPLA3 and HCC on nonfibrotic liver suggests a direct role in liver carcinogenesis.

Hepatic Parenchymal Injury in Crigler-Najjar Type I.

Crigler-Najjar syndrome type I (CNI) arises from biallelic variants of UGT1A1 that abrogate uridine diphosphate glucuronosyltransferase (UGT1A1) activity resulting in unconjugated hyperbilirubinemia. Historically, liver parenchyma in CNI was considered structurally and histologically normal. Recent review of CNI liver explants revealed fibrosis. Our aim was to investigate the association between hepatic histology and disease phenotype in CNI. We extracted data from the medical record at the time of liver transplant from 22 patients with CNI at the Children's Hospital of Pittsburgh, and reviewed explant histology. Continuous data were normally distributed, are presented as mean (±1 SD), and analyzed using two-tailed Student t-test. Categorical data were analyzed using the Chi-square test. Both alanine transaminase (ALT; mean 87.4 IU/L) and aspartate transaminase (AST; mean 54.6 IU/L) were elevated. Nine (41%) of 22 explants had significant fibrosis. Pericentral (n = 5), periportal (n = 2), and mixed (n = 2) patterns of fibrosis occurred. A significant difference in mean age of subjects with fibrotic versus non-fibrotic livers (16.1 years vs 10.5 years; P = 0.02) was seen. There were no indices of synthetic liver dysfunction or portal hypertension. Neither a history of gallstone disease nor excess weight appeared to contribute to the development of fibrosis. For the first time, we report a 41% prevalence of clinically silent, yet histologically significant fibrosis among subjects with Crigler-Najjar type 1. Risk for fibrosis appears to accrue with time, indicating that earlier intervention may be prudent whenever considering alternative treatments such as hepatocyte transplant, auxiliary liver transplant, or viral gene therapy.

Arkadia protein expression is reduced in the liver during the progression of hepatic fibrosis.

Arkadia is able to degrade key signaling molecules in the transforming growth factor (TGF)-β1 signaling pathway; however, the expression of Arkadia in the liver during development and progression of TGF-β1/Smad signaling-regulated hepatic fibrosis remains to be elucidated. The present study aimed to examine Arkadia expression in the livers of two rat models of hepatic fibrosis induced by bile duct ligation and carbon tetrachloride intoxication, and in human liver samples from patients with hepatic fibrosis. Expression was analyzed by quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. The results indicated that Arkadia was predominantly expressed in the cytoplasm of cholangiocytes and hepatocytes. The protein expression levels of Arkadia were significantly decreased in fibrotic livers, whereas the mRNA expression levels of Arkadia were significantly increased in fibrotic livers compared with in nonfibrotic livers. In conclusion, these data indicated that Arkadia may regulate the pathogenesis and progression of hepatic fibrosis.

MiR-185 Inhibits Fibrogenic Activation of Hepatic Stellate Cells and Prevents Liver Fibrosis.

Recent studies have shown the effect of microRNAs on HSC activation and transformation, which is essential for the pathogenesis of liver fibrosis. In our study, we explored the role of miR-185 in liver fibrosis. Plasma miR-185 was detected in hepatitis B virus-related liver fibrosis patients (S2/3, n = 10) by Illumina HiSeq sequencing, and healthy volunteers were selected (n = 8) as the control group. We found that the plasma miR-185 level in fibrosis patients was significantly downregulated. CCl4-induced fibrosis tissues in mouse livers and TGF-β1-activated HSCs also presented downregulated miR-185 concomitant with an increased expression of RHEB and RICTOR. To explore the correlations, LX-2 cells were transiently transfected with miR-185 mimics. The expression levels of α-SMA, collagen I, and collagen III were decreased as well as RHEB and RICTOR. Inhibition of endogenous miR-185 increased fibrogenic activity. Furthermore, dual-luciferase reporter assays indicated that miR-185 inhibited the expression of RHEB and RICTOR by directly targeting their 3′ UTRs. Moreover, silencing RHEB and RICTOR suppressed α-SMA and collagen expression levels. In conclusion, miR-185 prevents liver fibrogenesis by inhibiting HSC activation via inhibition of RHEB and RICTOR. These results provide new insights into the mechanisms behind the anti-fibrotic effect of miR-185.

Diagnostic Value of Glypican3, Heat Shock Protein 70 and Glutamine Synthetase in Hepatocellular Carcinoma Arising in Cirrhotic and Non-Cirrhotic Livers

Histopathological distinction of various nodular lesions in liver with sufficient sensitivity and specificity is a challenge even in an expert set up. The panel of immunohistochemical markers composed of glutamine synthetase (GS), Glypican3 (GPC3) and heat shock protein 70 (HSP70) was recommended by the International Consensus Group for Hepatocellular Neoplasia group for the differentiation of high grade dysplastic nodule and early hepatocellular carcinoma (HCC). The panel has been extensively validated in the western population. This study aims to test this panel on Indian population on resected, explanted and autopsy cirrhotic and non-cirrhotic liver specimens of HCC. This study was conducted on 39 such liver specimens (12 cirrhotic, 12 pre-cirrhotic and 11 non-cirrhotic, non-fibrotic livers), including 35 cases of HCC over a period of 12 years. Immunohistochemistry was performed with antibodies against GS, GPC3 and HSP70 on the sections containing both malignant and dysplastic nodules. The diagnostic yield depended upon the nature of background liver pathology and was found to be high for only those HCCs arising in cirrhotic background, when positivity of any two markers was taken to be in favor of HCC (sensitivity-58.33%; specificity-100%). GS had a sensitivity and Negative predictive value of 100% for HCCs arising in cirrhotic livers. Strong positivity for GS is a highly sensitive marker for HCC in a cirrhotic background regardless of the differentiation of the tumor in Indian population. This may be due to preferential activation of Wnt pathway in Indian patients with cirrhosis. The sensitivity of the panel was too low for detecting HCCs arising in non-cirrhotic livers, even in the pre-cirrhotic chronically inflamed livers, even though the specificity was high. GPC3 and HSP70 appear to be useful as individual markers for HCCs arising in non-cirrhotic livers.