Impaired central tolerance induces changes in the gut microbiota that exacerbate autoimmune hepatitis.

Medullary thymic epithelial cells (mTECs) induce T cell tolerance in the thymus through the elimination of self-reactive thymocytes. Commensal bacteria are also critical for shaping T cell responses in the gut and distal organs. We previously showed that mice depleted of mTECs (Traf6ΔTEC) generated autoreactive T cells and developed autoimmune hepatitis (AIH). In this report, we found that Toll-like receptor (TLR)-mediated microbial sensing on liver hematopoietic cells and the gut microbiota contributed to AIH development in Traf6ΔTEC mice. While adoptive transfer of thymic Traf6ΔTEC T cells in immune-deficient mice was sufficient for AIH development, colonization of germ-free mice with Traf6ΔTEC microbiota failed to induce AIH, suggesting that the gut microbiota contributes to but is not sufficient for AIH development. Microbiota-mediated exacerbation of AIH associated with increased numbers of hepatic Foxp3+ T cells and their increase was proportional to the degree of inflammation. The contribution of the gut microbiota to AIH development associated with an altered microbial signature whose composition was influenced by the qualitative nature of the thymic T cell compartment. These results suggest that aberrant selection of T cells in the thymus can induce changes in the gut microbiota that lead to exacerbation of organ-specific autoimmunity and AIH. Our results add to our understanding of the mechanisms of AIH development and create a platform towards developing novel therapeutic approaches for treating this disease.

Assessment of HCC response to Yttrium-90 radioembolization with gadoxetate disodium MRI: correlation with histopathology.

BACKGROUND AND AIMS: Transarterial 90Y radioembolization (TARE) is increasingly being used for hepatocellular carcinoma (HCC) treatment. However, tumor response assessment after TARE may be challenging. We aimed to assess the diagnostic performance of gadoxetate disodium MRI for predicting complete pathologic necrosis (CPN) of HCC treated with TARE, using histopathology as the reference standard. METHODS: This retrospective study included 48 patients (M/F: 36/12, mean age: 62 years) with HCC treated by TARE followed by surgery with gadoxetate disodium MRI within 90 days of surgery. Two radiologists evaluated tumor response using RECIST1.1, mRECIST, EASL, and LI-RADS-TR criteria and evaluated the percentage of necrosis on subtraction during late arterial, portal venous, and hepatobiliary phases (AP/PVP/HBP). Statistical analysis included inter-reader agreement, correlation between radiologic and pathologic percentage of necrosis, and prediction of CPN using logistic regression and ROC analyses. RESULTS: Histopathology demonstrated 71 HCCs (2.8 ± 1.7 cm, range: 0.5-7.5 cm) including 42 with CPN, 22 with partial necrosis, and 7 without necrosis. EASL and percentage of tumor necrosis on subtraction at the AP/PVP were independent predictors of CPN (p = 0.02-0.03). Percentage of necrosis, mRECIST, EASL, and LI-RADS-TR had fair to good performance for diagnosing CPN (AUCs: 0.78 - 0.83), with a significant difference between subtraction and LI-RADS-TR for reader 2, and in specificity between subtraction and other criteria for both readers (p-range: 0.01-0.04). Radiologic percentage of necrosis was significantly correlated to histopathologic degree of tumor necrosis (r = 0.66 - 0.8, p < 0.001). CONCLUSIONS: Percentage of tumor necrosis on subtraction and EASL criteria were significant independent predictors of CPN in HCC treated with TARE. Image subtraction should be considered for assessing HCC response to TARE when using MRI. KEY POINTS: • Percentage of tumor necrosis on image subtraction and EASL criteria are significant independent predictors of complete pathologic necrosis in hepatocellular carcinoma treated with90Y radioembolization. • Subtraction, mRECIST, EASL, and LI-RADS-TR have fair to good performance for diagnosing complete pathologic necrosis in hepatocellular carcinoma treated with90Y radioembolization.

The role of endoscopy in the small intestinal transplant recipient: A review.

Intestinal transplantation (ITx) is the treatment of choice for patients with intestinal failure who have developed life-threatening complications related to long-term parenteral nutrition. Patients may also undergo ITx as part of a combined liver-intestine or multivisceral transplant for a variety of indications, most commonly intestinal failure-associated liver disease or porto-mesenteric thrombosis. Endoscopy plays a critical role in the posttransplant management of these patients, most commonly in the diagnosis and management of rejection, which occurs in up to 30-40% of patients within the first-year posttransplant. With a lack of noninvasive biomarkers to identify the presence of rejection, endoscopy and biopsy remain the gold standard for its diagnosis. Endoscopic evaluation of the graft is also important in the identification of other complications post-ITx, including posttransplant lymphoproliferative disorder, graft-versus-host disease, and enteric infections. Each patient's posttransplant anatomy may be slightly different, making endoscopy sometimes technically challenging and necessitating clear and frequent communication with the surgical team in order to help identify the highest yield approach. Herein, we review the most common pathologies found endoscopically in the post-ITx patient and describe some of the unique challenges the endoscopist faces when evaluating these complex patients.

Antibody-mediated rejection of the liver allograft: An update and a clinico-pathological perspective.

Antibody-mediated rejection after liver transplantation is an under-recognised cause of allograft injury. While definitions of acute and chronic antibody-mediated rejection have increased clinical awareness, timely identification and management of antibody-mediated rejection remain difficult because of complexities in diagnosis and histopathology, lack of treatment protocols, and unclear long-term outcomes. While recent cohort studies assessing the importance of donor-specific antibodies have aided in its diagnosis, literature on the treatment of antibody-mediated rejection in liver transplantation remain limited to case reports and small series. Further increasing the awareness and timely recognition of antibody-mediated rejection post-liver transplantation is crucial in order to stimulate future research and the development of protocols for its diagnosis and treatment. This review will summarise recent advances in the clinical diagnosis and treatment of antibody-mediated rejection in liver transplantation, as well as some of the histopathologic features (on liver biopsy tissue) of acute and chronic antibody-mediated rejection.

A comparative study of cirrhosis sub-staging using the Laennec system, Beijing classification, and morphometry.

There is constant remodeling in a cirrhotic liver resulting in cirrhosis being spatially heterogeneous. The Laennec system, and, more recently the Beijing classification, have been used to sub-classify various degrees of cirrhosis. It is unknown how these two schemes compare with each other, how they are impacted by geographic variation, and how they correlate with clinical outcomes. Five needle biopsies were obtained from 20 explanted cirrhotic HCV livers at the time of transplantation. Collagen proportionate area (CPA) was measured by computerized quantitative morphometry. The Laennec system (4A-4C indicating increasing degrees of cirrhosis) and Beijing classification (P-progressive, R-regressive, I-indeterminate) were assessed and then correlated with CPA. Geographical variation using CPAs was calculated by the coefficient of variation (CoV). CPA of Laennec 4C cirrhosis was higher than 4A (p = 0.00008) or 4B (p = 0.0002). The CPA of the P pattern was greater than the R (p = 0.002) or I patterns (p = 0.037). The mean CoV of the five CPAs was 47.3 ± 4.5%, suggesting a significant degree of geographic variation. There was 100% overlap between the Beijing R pattern and Laennec 4A, and 80% overlap between the P pattern and Laennec 4C. Patients' platelet counts of P pattern were lower than R pattern (p = 0.008) or I pattern (p = 0.024), while Laennec 4C was lower than 4A (p = 0.036) and 4B patients (p = 0.7). There was no correlation between CPA, Laennec stage, or Beijing classification and MELD score, liver weights, total bilirubin, or albumin levels. The Laennec system and the Beijing classification are highly correlated with CPA in cirrhosis. This study confirms that there is a significant degree of geographic variation in terms of fibrosis content and cirrhosis morphology throughout the liver.

Individual liver plasmacytoid dendritic cells are capable of producing IFNα and multiple additional cytokines during chronic HCV infection.

Plasmacytoid dendritic cells (pDCs) are "natural" interferon α (IFNα)-producing cells. Despite their importance to antiviral defense, autoimmunity, and ischemic liver graft injury, because DC subsets are rare and heterogeneous, basic questions about liver pDC function and capacity to make cytokines remain unanswered. Previous investigations failed to consistently detect IFNα mRNA in HCV-infected livers, suggesting that pDCs may be incapable of producing IFNα. We used a combination of molecular, biochemical, cytometric, and high-dimensional techniques to analyze DC frequencies/functions in liver and peripheral blood mononuclear cells (PBMCs) of hepatitis C virus (HCV)-infected patients, to examine correlations between DC function and gene expression of matched whole liver tissue and liver mononuclear cells (LMCs), and to determine if pDCs can produce multiple cytokines. T cells often produce multiple cytokines/chemokines but until recently technical limitations have precluded tests of polyfunctionality in individual pDCs. Mass cytometry (CyTOF) revealed that liver pDCs are the only LMC that produces detectable amounts of IFNα in response TLR-7/8 stimulation. Liver pDCs secreted large quantities of IFNα (~2 million molecules of IFNα/cell/hour) and produced more IFNα than PBMCs after stimulation, p = 0.0001. LMCs secreted >14-fold more IFNα than IFNλ in 4 hours. Liver pDC frequency positively correlated with whole liver expression of "IFNα-response" pathway (R2 = 0.58, p = 0.007) and "monocyte surface" signature (R2 = 0.54, p = 0.01). Mass cytometry revealed that IFNα-producing pDCs were highly polyfunctional; >90% also made 2-4 additional cytokines/chemokines of our test set of 10. Liver BDCA1 DCs, but not BDCA3 DCs, were similarly polyfunctional. pDCs from a healthy liver were also polyfunctional. Our data show that liver pDCs retain the ability to make abundant IFNα during chronic HCV infection and produce many other immune modulators. Polyfunctional liver pDCs are likely to be key drivers of inflammation and immune activation during chronic HCV infection.

Consensus report from the 9th International Forum for Liver Magnetic Resonance Imaging: applications of gadoxetic acid-enhanced imaging.

OBJECTIVES: The 9th International Forum for Liver Magnetic Resonance Imaging (MRI) was held in Singapore in September 2019, bringing together radiologists and allied specialists to discuss the latest developments in and formulate consensus statements for liver MRI, including the applications of gadoxetic acid-enhanced imaging. METHODS: As at previous Liver Forums, the meeting was held over 2 days. Presentations by the faculty on days 1 and 2 and breakout group discussions on day 1 were followed by delegate voting on consensus statements presented on day 2. Presentations and discussions centered on two main meeting themes relating to the use of gadoxetic acid-enhanced MRI in primary liver cancer and metastatic liver disease. RESULTS AND CONCLUSIONS: Gadoxetic acid-enhanced MRI offers the ability to monitor response to systemic therapy and to assist in pre-surgical/pre-interventional planning in liver metastases. In hepatocellular carcinoma, gadoxetic acid-enhanced MRI provides precise staging information for accurate treatment decision-making and follow-up post therapy. Gadoxetic acid-enhanced MRI also has potential, currently investigational, indications for the functional assessment of the liver and the biliary system. Additional voting sessions at the Liver Forum debated the role of multidisciplinary care in the management of patients with liver disease, evidence to support the use of abbreviated imaging protocols, and the importance of standardizing nomenclature in international guidelines in order to increase the sharing of scientific data and improve the communication between centers. KEY POINTS: • Gadoxetic acid-enhanced MRI is the preferred imaging method for pre-surgical or pre-interventional planning for liver metastases after systemic therapy. • Gadoxetic acid-enhanced MRI provides accurate staging of HCC before and after treatment with locoregional/biologic therapies. • Abbreviated protocols for gadoxetic acid-enhanced MRI offer potential time and cost savings, but more evidence is necessary. The use of gadoxetic acid-enhanced MRI for the assessment of liver and biliary function is under active investigation.

Hemophagocytic lymphohistiocytosis occurring after liver transplantation: A case series and review of the literature.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease characterized by excessive inflammation and tissue destruction due to a dysregulated immune response. Its secondary form is most commonly triggered by viral infection or malignancy. There have previously been 11 cases of acquired HLH described following liver transplantation in adult transplant recipients, most occurring within the first year following transplantation. Herein, we describe two cases of HLH in liver transplant recipients that both occurred remotely following transplantation. In the first case, HLH was thought to be triggered by the development of a post-transplant lymphoproliferative disorder in a patient who was initially diagnosed with recurrent autoimmune hepatitis. In the second, it was thought to be triggered by a newly acquired human herpesvirus-8 infection. In both cases, the syndrome was not recognized until treatment for the initial putative diagnoses was unsuccessful. Despite treatment, both patients unfortunately died from multiorgan failure. HLH in the post-liver transplant setting is likely under-recognized and has a high mortality; early diagnosis and intervention may lead to improved outcomes.

Mannose Phosphate Isomerase and Mannose Regulate Hepatic Stellate Cell Activation and Fibrosis in Zebrafish and Humans.

The growing burden of liver fibrosis and lack of effective antifibrotic therapies highlight the need for identification of pathways and complementary model systems of hepatic fibrosis. A rare, monogenic disorder in which children with mutations in mannose phosphate isomerase (MPI) develop liver fibrosis led us to explore the function of MPI and mannose metabolism in liver development and adult liver diseases. Herein, analyses of transcriptomic data from three human liver cohorts demonstrate that MPI gene expression is down-regulated proportionate to fibrosis in chronic liver diseases, including nonalcoholic fatty liver disease and hepatitis B virus. Depletion of MPI in zebrafish liver in vivo and in human hepatic stellate cell (HSC) lines in culture activates fibrotic responses, indicating that loss of MPI promotes HSC activation. We further demonstrate that mannose supplementation can attenuate HSC activation, leading to reduced fibrogenic activation in zebrafish, culture-activated HSCs, and in ethanol-activated HSCs. Conclusion: These data indicate the prospect that modulation of mannose metabolism pathways could reduce HSC activation and improve hepatic fibrosis.

Aberrant von Willebrand factor expression of sinusoidal endothelial cells and quiescence of hepatic stellate cells in nodular regenerative hyperplasia and obliterative portal venopathy.

AIMS: Nodular regenerative hyperplasia (NRH) and obliterative portal venopathy (OPV), entities that comprise idiopathic non-cirrhotic portal hypertension (INCPH), are under-recognised diseases of uncertain aetiology and the diagnosis can be easily missed on liver biopsy. The expression of CD34 and von Willebrand factor (vWF) in liver sinusoidal endothelial cells (LSEC) and alpha-smooth muscle actin (ASMA) in hepatic stellate cells (HSCs) is unknown in NRH and OPV. We sought to investigate the pathogenesis and potential immunomarkers that might aid in making the diagnosis of NRH and OPV. METHODS AND RESULTS: Immunohistochemical (IHC) staining for CD34, vWF and ASMA was performed in clinically and histologically well-characterised NRH (n = 15) and OPV (n = 47) liver specimens. Among the 47 OPV cases, 37 (78.7%) had concurrent features of NRH. CD34 positive staining was mainly confined to small vessels in the portal tracts and LSECs in periportal areas, a finding similar to that in non-NRH/OPV livers. However, expression of vWF in LSECs was positive in the compressed sinusoids of NRH and in a patchy or geographic pattern, particularly prominent in the perivenular areas and dilated sinusoids of OPV cases. HSCs were negative for ASMA in all NRH and OPV cases. CONCLUSION: Our findings indicate that NRH may be a subtle but common concurrent morphological feature in OPV. The aberrant expression of vWF in LSECs suggests that endothelial injury may play a role in the pathogenesis, which may thus aid in the recognition and diagnosis of NRH and OPV, particularly when confronted with otherwise apparent normal liver histology on needle biopsy.

Hepatocellular carcinoma detection in liver cirrhosis: diagnostic performance of contrast-enhanced CT vs. MRI with extracellular contrast vs. gadoxetic acid.

OBJECTIVES: To evaluate the diagnostic performance of contrast-enhanced CT vs. MRI with extracellular contrast agents (EC-MRI) vs. MRI with gadoxetic acid (EOB-MRI) for HCC detection in patients with liver cirrhosis using liver explant as the reference. The additional value of hepatobiliary phase (HBP) post Gadoxetic acid was also assessed. METHODS: Two-hundred seventy-seven consecutive patients who underwent liver transplantation over a 9 year period and imaging within 90 days of were retrospectively included. Imaging consisted in CT (n = 100), EC-MRI (n = 77) and EOB-MRI (n = 100), the latter subdivided into dynamic EOB-MRI and full EOB-MRI (dynamic+HBP). Three radiologists retrospectively categorized lesions ≥ 1 cm using the LI-RADSv2017 algorithm. Dynamic EOB-MRI was re-evaluated with the addition of HBP. Results were correlated with explant pathology. RESULTS: Pathology demonstrated 265 HCCs (mean size 2.1 ± 1.4 cm) in 177 patients. Per-patient sensitivities were 86.3% for CT, 89.5% for EC-MRI, 92.8% for dynamic EOB-MRI and 95.2% for full EOB-MRI (pooled reader data), with a significant difference between CT and dynamic/full EOB-MRI (p = 0.032/0.002), and between EC-MRI and full EOB-MRI (p = 0.047). Per-lesion sensitivities for CT, EC-MRI, dynamic EOB-MRI and full EOB-MRI were 59.5%,78.5%,69.7% and 76.8%, respectively, with a significant difference between MRI groups and CT (p-range:0.001-0.04), and no difference between EC-MRI and dynamic EOB-MRI (p = 0.949). For HCCs 1-1.9 cm, sensitivities were 34.4%, 64.6%, 57.3% and 67.3%, respectively, with all MRI groups significantly superior to CT (p ≤ 0.01) and full EOB-MRI superior to dynamic EOB-MRI (p = 0.002). CONCLUSIONS: EOB-MRI outperforms CT and EC-MRI for per-patient HCC detection sensitivity, and is equivalent to EC-MRI for per-lesion sensitivity. MRI methods outperform CT for detection of HCCs 1-1.9 cm. KEY POINTS: • MRI is superior to CT for HCC detection in patients with liver cirrhosis. • EOB-MRI outperforms CT and MRI using extracellular contrast agents (EC-MRI) for per-patient HCC detection sensitivity, and is equivalent to EC-MRI for per-lesion sensitivity. • The addition of hepatobiliary phase images improves HCC detection when using gadoxetic acid.

Idiopathic noncirrhotic portal hypertension.

Cirrhosis is the most common cause of portal hypertension but there are many causes of noncirrhotic portal hypertension. Many of these etiologies may be diagnosed by liver biopsy. Idiopathic noncirrhotic portal hypertension is being increasingly diagnosed and has varied histopathological findings as well as overlapping definitions. Many of these histological changes can be subtle, thus making it a challenging diagnosis for the pathologist to make. This review summarizes the clinical aspects of idiopathic noncirrhotic portal hypertension and outlines the different definitions and histological features of the entity. In addition, pearls and pitfalls for the pathologist in making this diagnosis are included.

A simple diet- and chemical-induced murine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancer.

BACKGROUND AND AIMS: Although the majority of patients with non-alcoholic fatty liver disease (NAFLD) have only steatosis without progression, a sizeable fraction develop non-alcoholic steatohepatitis (NASH), which can lead to cirrhosis and hepatocellular carcinoma (HCC). Many established diet-induced mouse models for NASH require 24-52 weeks, which makes testing for drug response costly and time consuming. METHODS: We have sought to establish a murine NASH model with rapid progression of extensive fibrosis and HCC by using a western diet (WD), which is high-fat, high-fructose and high-cholesterol, combined with low weekly dose of intraperitoneal carbon tetrachloride (CCl4), which serves as an accelerator. RESULTS: C57BL/6J mice were fed a normal chow diet ±â€¯CCl4 or WD ±â€¯CCl4 for 12 and 24 weeks. Addition of CCl4 exacerbated histological features of NASH, fibrosis, and tumor development induced by WD, which resulted in stage 3 fibrosis at 12 weeks and HCC development at 24 weeks. Furthermore, whole liver transcriptomic analysis indicated that dysregulated molecular pathways in WD/CCl4 mice and immunologic features were similar to those of human NASH. CONCLUSIONS: Our mouse NASH model exhibits rapid progression of advanced fibrosis and HCC, and mimics histological, immunological and transcriptomic features of human NASH, suggesting that it will be a useful experimental tool for preclinical drug testing. LAY SUMMARY: A carefully characterized model has been developed in mice that recapitulates the progressive stages of human fatty liver disease, from simple steatosis, to inflammation, fibrosis and cancer. The functional pathways of gene expression and immune abnormalities in this model closely resemble human disease. The ease and reproducibility of this model make it ideal to study disease pathogenesis and test new treatments.

Development of a novel histologic diagnostic algorithm for hepatic graft-versus-host disease.

The histopathologic diagnosis of hepatic graft-versus-host disease post bone marrow and stem cell transplantation can be challenging, but timely and unambiguous diagnosis is essential for appropriate patient management. To address this diagnostic dilemma, we identified histologic features specific for hepatic graft-versus-host disease and developed a diagnostic algorithm. Two hepatopathologists blindly evaluated 40 liver biopsies from patients with clinically and biologically confirmed graft-versus-host disease, as well as 44 controls, for percent bile duct loss, bile duct damage, intraepithelial lymphocytes, ductular reaction, acidophilic bodies/10 high power fields (HPF), cholestasis, portal and lobular inflammation, and endotheliitis. Compared with controls, graft-versus-host disease cases had significantly more bile duct loss (P<0.0001), bile duct damage (P=0.0002), cholestasis (P<0.0001), and acidophilic bodies/10 HPF (P=0.0006), as well as significantly less ductular reaction (P<0.0001). Significance was maintained with a drug-induced liver injury-only control group. No histologic differences were noted in acute versus chronic graft-versus-host disease, nor cholestatic versus hepatitic types. An algorithm to predict likelihood of graft-versus-host disease was developed, with a three-tiered scoring system: 1-2 not, 3-4 probable, and 5-8 unequivocal graft-versus-host disease. This algorithm had a sensitivity of 93%, specificity of 93%, and accuracy of 92%. We identified histologic features with specificity for hepatic graft-versus-host disease and developed a simple algorithm for pathologists to predict its likelihood, distinguishing this critical diagnosis promptly from mimickers having vastly different treatments and prognoses.

Hepatitis C virus double-stranded RNA is the predominant form in human liver and in interferon-treated cells.

Hepatitis C virus (HCV) is unique among RNA viruses in its ability to establish chronic infection in the majority of exposed adults. HCV persists in the liver despite interferon (IFN)-stimulated gene (ISG) induction; robust induction actually predicts treatment failure and viral persistence. It is unclear which forms of HCV RNA are associated with ISG induction and IFN resistance during natural infections. To thoroughly delineate HCV RNA populations, we developed conditions that fully separate the strands of long double-stranded RNA (dsRNA) and allow the released RNAs to be quantified in reverse transcription/polymerase chain reaction assays. These methods revealed that dsRNA, a pathogen-associated molecular pattern (PAMP), comprised 52% (standard deviation, 28%) of the HCV RNA in the livers of patients with chronic infection. HCV dsRNA was proportionally higher in patients with the unfavorable IL28B TT (rs12979860) genotype. Higher ratios of HCV double-stranded to single-stranded RNA (ssRNA) correlated positively with ISG induction. In Huh-7.5 cells, IFN treatment increased the total amount of HCV dsRNA through a process that required de novo viral RNA synthesis and shifted the ratio of viral dsRNA/ssRNA in favor of dsRNA. This shift was blocked by ribavirin (RBV), an antiviral drug that reduces relapse in HCV patients. Northern blotting established that HCV dsRNA contained genome-length minus strands. CONCLUSION: HCV dsRNA is the predominant form in the HCV-infected liver and has features of both a PAMP and a genomic reservoir. Interferon treatment increased rather than decreased HCV dsRNA. This unexpected finding suggests that HCV produces dsRNA in response to IFN, potentially to antagonize antiviral defenses. (Hepatology 2017;66:357-370).

Histological assessment of the liver explant in transplanted hepatitis C virus patients achieving sustained virological response with direct-acting antiviral agents.

AIMS: The use of direct-acting anti-viral agents (DAAs) has resulted in extremely high sustained virological response (SVR) rates in patients being treated while on liver transplantation (LT) waiting lists. The aim of this study was to evaluate the histological findings of hepatitis C virus (HCV) patients who achieved SVR after receiving DAA treatment [SVR(+)] prior to LT, and compare them with HCV patients who had not achieved SVR [SVR(-)]. METHODS AND RESULTS: Fifty-eight adult HCV patients who underwent LT at our institution from 2014 to 2016 were included in the study. Two pathologists, blinded to SVR status, simultaneously evaluated the histological sections. Assessment included the Histology Activity Index (HAI/modified Knodell score), fibrosis stage (Ishak score), and Laennec cirrhosis stage. The study group comprised 25 SVR(+) patients (56% male; mean age, 63.8 years), and the control group comprised 33 SVR(-) patients (69% male; mean age, 61.7 years). There was no significant difference in HAI between the groups (P = 0.414). Patients who achieved SVR also did not show less portal inflammation (P = 0.787), interface hepatitis (P = 0.999), confluent necrosis (P = 0.627) or spotty necrosis (P = 0.093) than the control group. There was a trend towards a higher degree of inflammation in patients who achieved SVR in <24 weeks (P = 0.07). The degree of focal lytic necrosis/apoptosis and portal inflammation was more prominent in SVR(+) patients with shorter SVR-LT intervals. CONCLUSIONS: Our study is the first to report persistent inflammation in HCV patients who received DAAs prior to LT. This supports the notion that inflammation is immunologically driven and that inflammation persists despite the absence of virus.

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

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

A pilot systematic genomic comparison of recurrence risks of hepatitis B virus-associated hepatocellular carcinoma with low- and high-degree liver fibrosis.

BACKGROUND: Chronic hepatitis B virus (HBV) infection leads to liver fibrosis, which is a major risk factor in hepatocellular carcinoma (HCC) and an independent risk factor of recurrence after HCC tumor resection. The HBV genome can be inserted into the human genome, and chronic inflammation may trigger somatic mutations. However, how HBV integration and other genomic changes contribute to the risk of tumor recurrence with regards to the different degree of liver fibrosis is not clearly understood. METHODS: We sequenced mRNAs of 21 pairs of tumor and distant non-neoplastic liver tissues of HBV-HCC patients and performed comprehensive genomic analyses of our RNAseq data and public available HBV-HCC sequencing data. RESULTS: We developed a robust pipeline for sensitively identifying HBV integration sites based on sequencing data. Simulations showed that our method outperformed existing methods. Applying it to our data, 374 and 106 HBV host genes were identified in non-neoplastic liver and tumor tissues, respectively. When applying it to other RNA sequencing datasets, consistently more HBV integrations were identified in non-neoplastic liver than in tumor tissues. HBV host genes identified in non-neoplastic liver samples significantly overlapped with known tumor suppressor genes. More significant enrichment of tumor suppressor genes was observed among HBV host genes identified from patients with tumor recurrence, indicating the potential risk of tumor recurrence driven by HBV integration in non-neoplastic liver tissues. We also compared SNPs of each sample with SNPs in a cancer census database and inferred samples' pathogenic SNP loads. Pathogenic SNP loads in non-neoplastic liver tissues were consistently higher than those in normal liver tissues. Additionally, HBV host genes identified in non-neoplastic liver tissues significantly overlapped with pathogenic somatic mutations, suggesting that HBV integration and somatic mutations targeting the same set of genes are important to tumorigenesis. HBV integrations and pathogenic mutations showed distinct patterns between low and high liver fibrosis patients with regards to tumor recurrence. CONCLUSIONS: The results suggest that HBV integrations and pathogenic SNPs in non-neoplastic tissues are important for tumorigenesis and different recurrence risk models are needed for patients with low and high degrees of liver fibrosis.

Imaging-based surrogate markers of transcriptome subclasses and signatures in hepatocellular carcinoma: preliminary results.

OBJECTIVES: In this preliminary study, we examined whether imaging-based phenotypes are associated with reported predictive gene signatures in hepatocellular carcinoma (HCC). METHODS: Thirty-eight patients (M/F 30/8, mean age 61 years) who underwent pre-operative CT or MR imaging before surgery as well as transcriptome profiling were included in this IRB-approved single-centre retrospective study. Eleven qualitative and four quantitative imaging traits (size, enhancement ratios, wash-out ratio, tumour-to-liver contrast ratios) were assessed by three observers and were correlated with 13 previously reported HCC gene signatures using logistic regression analysis. RESULTS: Thirty-nine HCC tumours (mean size 5.7 ± 3.2 cm) were assessed. Significant positive associations were observed between certain imaging traits and gene signatures of aggressive HCC phenotype (G3-Boyault, Proliferation-Chiang profiles, CK19-Villanueva, S1/S2-Hoshida) with odds ratios ranging from 4.44-12.73 (P <0.045). Infiltrative pattern at imaging was significantly associated with signatures of microvascular invasion and aggressive phenotype. Significant but weak associations were also observed between each enhancement ratio and tumour-to-liver contrast ratios and certain gene expression profiles. CONCLUSIONS: This preliminary study demonstrates a correlation between phenotypic imaging traits with gene signatures of aggressive HCC, which warrants further prospective validation to establish imaging-based surrogate markers of molecular phenotypes in HCC. KEY POINTS: • There are associations between imaging and gene signatures of aggressive hepatocellular carcinoma. • Infiltrative type is associated with gene signatures of microvascular invasion and aggressiveness. • Infiltrative type may be a surrogate marker of microvascular invasion gene signature.