Prolonged hypernutrition impairs TREM2-dependent efferocytosis to license chronic liver inflammation and NASH development.

Obesity-induced chronic liver inflammation is a hallmark of nonalcoholic steatohepatitis (NASH)-an aggressive form of nonalcoholic fatty liver disease. However, it remains unclear how such a low-grade, yet persistent, inflammation is sustained in the liver. Here, we show that the macrophage phagocytic receptor TREM2, induced by hepatocyte-derived sphingosine-1-phosphate, was required for efferocytosis of lipid-laden apoptotic hepatocytes and thereby maintained liver immune homeostasis. However, prolonged hypernutrition led to the production of proinflammatory cytokines TNF and IL-1ß in the liver to induce TREM2 shedding through ADAM17-dependent proteolytic cleavage. Loss of TREM2 resulted in aberrant accumulation of dying hepatocytes, thereby further augmenting proinflammatory cytokine production. This ultimately precipitated a vicious cycle that licensed chronic inflammation to drive simple steatosis transition to NASH. Therefore, impaired macrophage efferocytosis is a previously unrecognized key pathogenic event that enables chronic liver inflammation in obesity. Blocking TREM2 cleavage to restore efferocytosis may represent an effective strategy to treat NASH.

Accuracy of blood-based biomarkers for staging liver fibrosis in chronic liver disease: A systematic review supporting the AASLD Practice Guideline.

BACKGROUND AND AIMS: Blood-based biomarkers have been proposed as an alternative to liver biopsy for noninvasive liver disease assessment in chronic liver disease. Our aims for this systematic review were to evaluate the diagnostic utility of selected blood-based tests either alone, or in combination, for identifying significant fibrosis (F2-4), advanced fibrosis (F3-4), and cirrhosis (F4), as compared to biopsy in chronic liver disease. APPROACH AND RESULTS: We included a comprehensive search of databases including Ovid MEDLINE(R), EMBASE, Cochrane Database, and Scopus through to April 2022. Two independent reviewers selected 286 studies with 103,162 patients. The most frequently identified studies included the simple aspartate aminotransferase-to-platelet ratio index and fibrosis (FIB)-4 markers (with low-to-moderate risk of bias) in HBV and HCV, HIV-HCV/HBV coinfection, and NAFLD. Positive (LR+) and negative (LR-) likelihood ratios across direct and indirect biomarker tests for HCV and HBV for F2-4, F3-4, or F4 were 1.66-6.25 and 0.23-0.80, 1.89-5.24 and 0.12-0.64, and 1.32-7.15 and 0.15-0.86, respectively; LR+ and LR- for NAFLD F2-4, F3-4, and F4 were 2.65-3.37 and 0.37-0.39, 2.25-6.76 and 0.07-0.87, and 3.90 and 0.15, respectively. Overall, the proportional odds ratio indicated FIB-4 <1.45 was better than aspartate aminotransferase-to-platelet ratio index <0.5 for F2-4. FIB-4 >3.25 was also better than aspartate aminotransferase-to-platelet ratio index >1.5 for F3-4 and F4. There was limited data for combined tests. CONCLUSIONS: Blood-based biomarkers are associated with small-to-moderate change in pretest probability for diagnosing F2-4, F3-4, and F4 in viral hepatitis, HIV-HCV coinfection, and NAFLD, with limited comparative or combination studies for other chronic liver diseases.

Pathological response following neoadjuvant immune checkpoint inhibitors in patients with hepatocellular carcinoma: a cross-trial, patient-level analysis.

BACKGROUND: Neoadjuvant use of immune checkpoint inhibitors (ICIs) before liver resection results in pathological tumour regression in patients with hepatocellular carcinoma. We aimed to describe the characteristics of pathological responses after preoperative ICI therapy for hepatocellular carcinoma and to evaluate the association between the depth of tumour regression and relapse-free survival. METHODS: In this cross-trial, patient-level analysis, we performed a pooled analysis of data from patients with hepatocellular carcinoma receiving ICI therapy before liver resection as part of a global collaborative consortium (NeoHCC) of five phase 1 and 2 clinical trials and standardised observational protocols conducted in 12 tertiary referral centres across the USA, UK, and Taiwan. Eligible patients were adults (aged ≥18 years) diagnosed with hepatocellular carcinoma by tissue core biopsy before treatment initiation, a Liver Imaging Reporting and Data System score of 5 on imaging, or both, with an Eastern Cooperative Oncology Group performance status score of 0-1, and no extrahepatic spread or previous ICI treatment. Pathological response was measured as the percentage of non-viable tumour in the resected surgical specimen, with major pathological response corresponding to at least 70% tumour regression and pathological complete response corresponding to 100% tumour regression. We correlated pathological response with radiological overall response using RECIST criteria (version 1.1) and relapse-free survival, and evaluated the threshold of tumour regression that could be optimally associated with relapse-free survival. FINDINGS: At data cutoff on Jan 31, 2024, 111 patients were included in the study, of whom data on pathological response were available for 104 (94%) patients. Patients received treatment from Oct 5, 2017, to Nov 15, 2023, mostly ICI combinations (76 [69%]), for a median of 1·4 months (IQR 0·7-2·9). 87 (78%) patients were men and 24 (22%) were women. Most patients had underlying viral chronic liver disease (73 [66%]) and Barcelona Clinic Liver Cancer stage A hepatocellular carcinoma (61 [55%]), without portal vein thrombosis (87 [78%]). We observed major pathological response in 33 (32%) patients and pathological complete response in 19 (18%) patients. Radiological overall response was associated with major pathological response, with 23 (74%) of 31 patients with radiological response showing major pathological response compared with ten (14%) of 73 patients without radiological response (p<0·0001). However, ten (30%) of 33 major pathological responses were not predicted by radiological response. After a median follow-up of 27·2 months (95% CI 22·3-32·1), median relapse-free survival for the whole cohort was 43·6 months (95% CI 28·3-not evaluable). Relapse-free survival was significantly longer in patients with major pathological response than in those who did not have a major pathological response (not reached [95% CI not evaluable-not evaluable] vs 28·3 months [12·8-43·8]; hazard ratio 0·26 [0·10-0·66]; p=0·0024) and in patients with pathological complete response than in those who did not have a pathological complete response (NR [95% CI not evaluable-not evaluable] vs 32·8 months [15·0-50·5]; 0·19 [0·05-0·78]; p=0·010). Unbiased recursive partitioning of the cohort for the risk of relapse, death, or both identified a threshold of 90% as the optimal cutoff of pathological tumour regression to predict improved relapse-free survival. INTERPRETATION: The extent of tumour regression following neoadjuvant ICI therapy could identify patients with improved relapse-free survival following liver resection. The threshold of at least 90% tumour regression should be validated for its surrogate role for relapse-free survival in phase 3 randomised controlled trials. FUNDING: None.

Serum and tissue biomarkers of plasma cell-rich rejection in liver transplant recipients.

The distinction between autoimmune and alloimmune reactions in liver transplant recipients can be challenging. Plasma cell-rich rejection (PCRR), previously known as de novo autoimmune hepatitis or plasma cell hepatitis, is an atypical and underrecognized form of allograft rejection observed post-liver transplantation, often in conjunction with features of T cell-mediated and antibody-mediated rejection. If PCRR is not recognized and treated with prompt immunosuppressive augmentation, patients can develop advanced hepatic fibrosis, necroinflammation, and allograft failure. Given the significant morbidity and mortality associated with PCRR, there exists a need to develop noninvasive biomarkers which can be used in screening, diagnosis, and treatment monitoring of PCRR. This study is a literature review of candidate serum-based and tissue-based biomarkers in adult and pediatric liver transplant PCRR. We also discuss biomarkers from plasma cell-rich processes observed in other disease states and other organ transplant recipients that might be tested in liver transplant PCRR. We conclude with proposed future directions in which biomarker implementation into clinical practice could lead to advances in personalized management of PCRR.

Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

The LATS2 tumor suppressor inhibits SREBP and suppresses hepatic cholesterol accumulation.

The Hippo signaling pathway is a major regulator of organ size. In the liver, Hippo pathway deregulation promotes hyperplasia and hepatocellular carcinoma primarily through hyperactivation of its downstream effector, YAP. The LATS2 tumor suppressor is a core member of the Hippo pathway. A screen for LATS2-interacting proteins in liver-derived cells identified the transcription factor SREBP2, master regulator of cholesterol homeostasis. LATS2 down-regulation caused SREBP activation and accumulation of excessive cholesterol. Likewise, mice harboring liver-specific Lats2 conditional knockout (Lats2-CKO) displayed constitutive SREBP activation and overexpressed SREBP target genes and developed spontaneous fatty liver disease. Interestingly, the impact of LATS2 depletion on SREBP-mediated transcription was clearly distinct from that of YAP overexpression. When challenged with excess dietary cholesterol, Lats2-CKO mice manifested more severe liver damage than wild-type mice. Surprisingly, apoptosis, inflammation, and fibrosis were actually attenuated relative to wild-type mice, in association with impaired p53 activation. Subsequently, Lats2-CKO mice failed to recover effectively from cholesterol-induced damage upon return to a normal diet. Additionally, decreased LATS2 mRNA in association with increased SREBP target gene expression was observed in a subset of human nonalcoholic fatty liver disease cases. Together, these findings further highlight the tight links between tumor suppressors and metabolic homeostasis.

Profiling of mouse and human liver diseases identifies targets for therapeutic treatment of autoimmune hepatitis.

Autoimmune hepatitis (AIH), primary sclerosing cholangitis (PSC), and non-alcoholic steatohepatitis (NASH) are chronic liver diseases (CLDs) of distinct etiologies that represent a public health risk with limited therapeutic options. A common feature among CLDs is an aggressive T cell response resulting in destruction of liver tissue and fibrosis. Here, we assessed the presence and nature of T cell inflammation in late-stage human AIH, PSC and NASH and examined whether targeting the T cell response can improve disease pathology in a mouse model (Traf6ΔTEC) of spontaneous AIH. T cell infiltration and ensuing inflammatory pathways were present in human AIH and PSC and to a lesser extent in NASH. However, we observed qualitative differences in infiltrating T cell subsets and upregulation of inflammatory pathways among these diseases, while mouse and human AIH exhibited similar immunogenic signatures. While gene expression profiles differed among diseases, we identified 52 genes commonly upregulated across all diseases that included the JAK3 tyrosine kinase. Therapeutic targeting of chronic AIH with the JAK inhibitor tofacitinib reduced hepatic T cell infiltration, AIH histopathology and associated immune parameters in treated Traf6ΔTEC mice. Our results indicate that targeting T cell responses in established hepatic autoimmune inflammation is a feasible strategy for developing novel therapeutic approaches to treat AIH and possibly other CLDs irrespective of etiology.

Morphology of tumor and nontumor tissue in liver resection specimens for hepatocellular carcinoma following nivolumab therapy.

Nivolumab is an immune checkpoint inhibitor (ICI) approved for treatment of many cancers, including hepatocellular carcinoma (HCC). Liver injury is a known complication in patients treated with nivolumab for nonliver tumors. To date, the morphologic changes to tumor and nontumor liver have not been well-characterized in HCC patients. We identified 20 patients who underwent partial hepatectomy or liver transplantation after receiving nivolumab for HCC. Demographics, laboratory values, and imaging results were obtained from medical records. All available slides from resection specimens were evaluated for tumor necrosis, tumor-infiltrating lymphocytes (TILs), and features of liver injury. Patients in the study included 16 males and 4 females with median age of 56 years. The underlying liver disease was HBV in 10, HCV in 6, and unknown/other in 4. Twelve patients were treated with nivolumab in the neoadjuvant setting, whereas eight were treated with nivolumab, usually along with other therapies, before undergoing liver transplantation. On review of resection specimens, three patients (all from the neoadjuvant group) demonstrated marked treatment response attributable to nivolumab. TILs were present in 17/20 cases. One case that showed treatment response in the neoadjuvant group demonstrated non-necrotizing granulomas and prominent bile duct intraepithelial lymphocytes (IELs) in the nontumor liver. One case from the transplant group showed bile duct damage and prominent ductular reaction after long-term nivolumab therapy (32 doses). Our findings indicate that nivolumab is effective in a subset of patients, including in the neoadjuvant setting. Granulomas and bile duct IELs are rare findings in cases treated with nivolumab but, when seen, may indicate potential response to therapy. Bile duct damage and ductular reaction may be manifestations of long-term nivolumab therapy. Future prospective and longitudinal studies with pretreatment tumor biopsies may help identify patients apt to respond to ICI therapy and further characterize patterns of ICI-related liver injury.

Pathophysiology of SARS-CoV-2: the Mount Sinai COVID-19 autopsy experience.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection.

EpCam is required for maintaining the integrity of the biliary epithelium.

BACKGROUND & AIMS: Tufting enteropathy (TE) is a rare congenital disorder often caused by mutations in the gene encoding epithelial cell adhesion molecule (EpCam). The disease leads to diarrhoea, intestinal failure and dependence on total parenteral nutrition (TPN). These patients often have liver impairments, but the pathology and mechanism of the damage are not well understood. We evaluated liver biopsies from TE patients to understand the pathophysiology. METHODS: We identified three patients with TE who underwent liver biopsy. Two normal controls and 45 patients on TPN secondary to short gut syndrome were selected for comparison (five were age- and TPN duration matched to the TE patients). RESULTS: We found that all TE patients showed a complete loss of EpCam expression in enterocytes and biliary epithelial cells, while the normal and TPN groups show basolateral expression. Histologically TE patients showed ductopenia, which was not seen in control groups. E-cadherin and ß-catenin are normally located along the lateral membrane of biliary epithelial cells. However, they were relocated to the apical membrane in TE patients, indicating a defect in the apical-basal polarity of cholangiocytes. We examined hepatic reparative cells and found near absence of hepatic progenitor cells and intermediate hepatobiliary cells with mild reactive ductular cells in TE patients. CONCLUSION: Our findings show that TE is associated with disrupted polarity of cholangiocyte and ductopenia. We demonstrate for the first time a role of EpCam in the maintenance of integrity of biliary epithelium. We also provided evidence for a disrupted development of hepatic reparative cells.

Initial assessment and ongoing monitoring of lysosomal acid lipase deficiency in children and adults: Consensus recommendations from an international collaborative working group.

BACKGROUND: Lysosomal acid lipase (LAL) deficiency is an ultra-rare, progressive, autosomal recessive disorder. Functional mutations in LIPA, the gene that encodes LAL, result in accumulation of cholesteryl esters and triglycerides in hepatocytes and in the macrophages of the intestines, vascular endothelial system, and numerous other organs. LAL deficiency has a broad clinical spectrum; children and adults can present with dyslipidemia, liver enzyme elevations, hepatosplenomegaly, hepatic steatosis, liver fibrosis and/or cirrhosis, and vascular disease, which may lead to significant morbidity and premature mortality in some patients. Given the systemic involvement and the wide range of healthcare specialists who manage patients with LAL deficiency, there is a need for guidelines to assess and monitor disease involvement. OBJECTIVES: To provide a set of recommendations for the initial assessment and ongoing monitoring of patients with LAL deficiency to help physicians in various disciplines effectively manage the disease based on the observed presentation and progression in each case. METHODS: A group of internationally recognized healthcare specialists with expertise in clinical genetics, pathology, hepatology, gastroenterology, cardiology, and lipidology convened to develop an evidence-based consensus of best practices for the initial assessment and ongoing monitoring of children and adults with LAL deficiency, regardless of treatment status; infants with LAL deficiency have been excluded from these guidelines because they require specialized care. RESULTS: The authors present guidance for the assessment and monitoring of patients with LAL deficiency based on age and disease manifestations that include the hepatic, cardiovascular, and gastrointestinal systems. A schedule for ongoing monitoring of disease progression is provided. In addition, the need to establish an interdisciplinary and integrated care team to optimize the approach to managing this systemic disease is highlighted. CONCLUSIONS: There is currently no published guidance on the assessment and monitoring of patients with LAL deficiency. These consensus recommendations for the initial assessment and ongoing monitoring of children and adults with LAL deficiency are intended to help improve the management of these patients.

The clinical characteristics, pre- and post-liver transplantation outcomes in patients having autoimmune overlap syndromes.

BACKGROUND: There are little data on the pre- and post-liver transplantation (LT) outcomes of patients having autoimmune hepatitis-primary biliary cholangitis (AIH-PBC), AIH-primary sclerosing cholangitis (AIH-PSC), and AIH-small-duct PSC (AIH-SDPSC). The aim of this study was to analyze pre- and post-LT outcomes and survival of patients having different overlap syndromes (OS) undergoing LT. METHODS: Patients with compatible clinical and pathologic features of AIH-PBC (n = 86), AIH-PSC (n = 22), and AIH-SDPSC (n = 9) were included in the study. Demographic, laboratory, clinical, and survival data were analyzed. Multivariable analyses were performed to determine factors predicting transplant-free survival. RESULTS: AIH-primary sclerosing cholangitis patients were less treatment-responsive and were more likely to undergo LT than other OS. No survival difference was noted among the 3 groups. Liver decompensation was independently associated with higher mortality (HR 21.78; 95% CI 2.50-190.01). Thirteen patients with OS underwent LT. One-year survival post-LT was 91.7%. Overall recurrence rate for OS post-LT was 8%. CONCLUSIONS: AIH-primary sclerosing cholangitis patients were more likely to require LT compared with patients having AIH-PBC. Transplant-free survival was similar among the three AIH-overlap syndromes. Allograft recurrence of OS occurred in about 10% of cases. Patients with OS appear to have good short- and medium-term post-LT outcomes in terms of graft function and overall survival.

Hypo-vascular hepatocellular carcinoma and liver transplantation: Morphological characteristics and implications on outcomes.

BACKGROUND: The clinical importance of hypovascular liver lesions in cirrhotic patients awaiting liver transplantation (LT) has not been fully investigated. The objective of this study was to characterize the clinicopathologic features and management of these tumors and to assess their impact on post-LT outcomes. METHODS: We performed a retrospective review of cirrhotic patients with lesions suspicious for hypovascular hepatocellular carcinoma (HCC) who underwent LT at a single institution from 2011- 2017. RESULTS: We identified 22 pre-LT patients with radiologic diagnosis of a lesion(s) suspicious for hypovascular HCC. There were 28 hypovascular lesions within the 22 patient cohort; 9 lesions (32%) converted to hypervascular HCC before LT and 19 lesions remained hypovascular at LT. 88% of hypovascular lesions were HCC on explant pathology. Compared to patients with hyper-vascular HCC lesions, hypovascular HCC lesions underwent less preoperative tumor ablation (58% vs 89%; P < .01). Hypovascular HCC were more likely to be well-differentiated (67% vs 11%; P < .01), but there were no differences in the microvascular invasion, tumor recurrence, or survival post-LT. CONCLUSIONS: Hypovascular HCC has similar clinical outcomes and needs for transplantation as hypervascular HCC. The high prevalence of HCC within suspicious hypovascular lesions supports a similar monitoring and locoregional therapy strategy as for hypervascular HCC.

Lysosomal acid lipase deficiency allograft recurrence and liver failure- clinical outcomes of 18 liver transplantation patients.

Lysosomal acid lipase deficiency (LAL-D) results in progressive microvesicular hepatosteatosis, fibrosis, cirrhosis, dyslipidemia, and vascular disease. Interventions available prior to enzyme replacement therapy development, including lipid lowering medications, splenectomy, hematopoietic stem cell and liver transplantation were unsuccessful at preventing multi-systemic disease progression, and were associated with significant morbidity and mortality. We report two sisters, diagnosed in infancy, who succumbed to LAL-D with accelerated disease progression following splenectomy and liver transplantation. The index patient died one year after hematopoietic stem cell transplant and liver transplantation. Her younger sister survived five years post liver-transplantation, complicated by intermittent, acute rejection. Typical LAL-D hepatopathology, including progressive, microvesicular steatosis, foamy macrophage aggregates, vacuolated Kupffer cells, advanced fibrosis and micronodular cirrhosis recurred in the liver allograft. She died before a second liver transplant could occur for decompensated liver failure. Neither patient received sebelipase alfa enzyme replacement therapy, human, recombinant, lysosomal acid lipase enzyme, FDA approved in 2015. Here are reviewed 18 LAL-D post-liver transplantation cases described in the literature. Multi-systemic LAL-D progression occurred in 11 patients (61%) and death in six (33%). These reports demonstrate that liver transplantation may be necessary for LAL-D-associated liver failure, but is not sufficient to prevent disease progression, or liver disease recurrence, since the pathophysiology is predominantly mediated by deficient enzyme activity in bone marrow-derived monocyte-macrophages. Enzyme replacement therapy addresses systemic disease and hepatopathology, potentially improving liver-transplantation outcomes. This is the first systematic review of liver transplantation for LAL-D, and the first account of liver allograft LAL-D-associated hepatopathology recurrence.

The impact of donor liver allograft fibrosis on patients undergoing liver transplantation.

BACKGROUND: The utilization of extended criteria liver allografts (ECD) shortens time to transplantation. OBJECTIVE: To characterize the effect of liver allograft fibrosis on graft and patient survival after liver transplantation (LT), with particular attention to fibrosis progression. METHODS: Retrospective database search of donor and recipient liver allograft histology of liver transplants performed between 2007 and 2011. Donor and patient characteristics were analyzed. RESULTS: One hundred and one patients underwent LT with donor liver allografts with early-stage fibrosis (stage 1 fibrosis and stage 2 fibrosis). The level of liver fibrosis did not progress in 40% of the patients tested, and there was a regression of fibrosis in 30%. At a median follow-up of 71 months, of 101 patients transplanted with fibrotic livers, 63 patients (63%) were alive with functioning initial grafts, six patients (6%) were retransplanted, and 35 patients expired. The graft survival rates were 82% and 69% at 1 and 5 years, respectively. Graft survival differences were not found to be statistically significant between the degrees of liver allograft fibrosis: 5-year graft survival (73% for stage 1 fibrosis and 62% for stage 2 fibrosis, P = .24). The entire fibrosis group was further compared with a control group of 208 consecutive primary liver transplant patients with allografts having no fibrosis. The 5-year graft survival was not significantly different between the groups (69% for the fibrosis group vs 75% for the nonfibrosis group, P = .19). Survival was also not statistically different between the groups (5-year survival of 73% for the fibrosis group vs 79% for the nonfibrosis group, P = .2). In patients with HCV, graft survival differences were not found to be statistically significant with the use of early-stage fibrotic livers: 5-year graft survival of 60% for fibrosis group vs 70% for the nonfibrosis group, P = .22). CONCLUSION: This study demonstrates that allografts with early-stage fibrosis achieve acceptable long-term survival after liver transplantation. Given these preliminary results, the use of organs with early-stage fibrosis warrants further studies at a larger scale to validate these results.

Trunk mutational events present minimal intra- and inter-tumoral heterogeneity in hepatocellular carcinoma.

BACKGROUND & AIMS: According to the clonal model of tumor evolution, trunk alterations arise at early stages and are ubiquitous. Through the characterization of early stages of hepatocarcinogenesis, we aimed to identify trunk alterations in hepatocellular carcinoma (HCC) and study their intra- and inter-tumor distribution in advanced lesions. METHODS: A total of 151 samples representing the multistep process of hepatocarcinogenesis were analyzed by targeted-sequencing and a single nucleotide polymorphism array. Genes altered in early lesions (31 dysplastic nodules [DNs] and 38 small HCCs [sHCC]) were defined as trunk. Their distribution was explored in: a) different regions of large tumors (43 regions, 21 tumors), and b) different nodules of the same patient (39 tumors, 17 patients). Multinodular lesions were classified as intrahepatic metastases (IMs) or synchronous tumors based on chromosomal aberrations. RESULTS: TERT promoter mutations (10.5%) and broad copy-number aberrations in chromosomes 1 and 8 (3-7%) were identified as trunk gatekeepers in DNs and were maintained in sHCCs. Trunk drivers identified in sHCCs included TP53 (23%) and CTNNB1 (11%) mutations, and focal amplifications or deletions in known drivers (6%). Overall, TERT, TP53 and CTNNB1 mutations were the most frequent trunk events and at least one was present in 51% of sHCCs. Around 90% of mutations in these genes were ubiquitous among different regions of large tumors. In multinodular HCCs, 35% of patients harbored IMs; 85% of mutations in TERT, TP53 and/or CTNNB1 were retained in primary and metastatic tumors. CONCLUSIONS: Trunk events in early stages (TERT, TP53, CTNNB1 mutations) were ubiquitous across different regions of the same tumor and between primary and metastatic nodules in >85% of cases. This concept supports the knowledge that single biopsies would suffice to capture trunk mutations in HCC. LAY SUMMARY: Trunk alterations arise at early stages of cancer and are shared among all malignant cells of the tumor. In order to identify trunk alterations in HCC, we characterized early stages of hepatocarcinogenesis represented by dysplastic nodules and small lesions. Mutations in TERT, TP53 and CTNNB1 genes were the most frequent. Analyses in more advanced lesions showed that mutations in these same genes were shared between different regions of the same tumor and between primary and metastatic tumors, suggesting their trunk role in this disease.