Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome.

The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype.

A gut microbial metabolite of dietary polyphenols reverses obesity-driven hepatic steatosis.

The molecular mechanisms by which dietary fruits and vegetables confer cardiometabolic benefits remain poorly understood. Historically, these beneficial properties have been attributed to the antioxidant activity of flavonoids. Here, we reveal that the host metabolic benefits associated with flavonoid consumption hinge, in part, on gut microbial metabolism. Specifically, we show that a single gut microbial flavonoid catabolite, 4-hydroxyphenylacetic acid (4-HPAA), is sufficient to reduce diet-induced cardiometabolic disease (CMD) burden in mice. The addition of flavonoids to a high fat diet heightened the levels of 4-HPAA within the portal plasma and attenuated obesity, and continuous delivery of 4-HPAA was sufficient to reverse hepatic steatosis. The antisteatotic effect was shown to be associated with the activation of AMP-activated protein kinase α (AMPKα). In a large survey of healthy human gut metagenomes, just over one percent contained homologs of all four characterized bacterial genes required to catabolize flavonols into 4-HPAA. Our results demonstrate the gut microbial contribution to the metabolic benefits associated with flavonoid consumption and underscore the rarity of this process in human gut microbial communities.

Clinicopathologic Features of Gastrointestinal Tract Langerhans Cell Histiocytosis.

Gastrointestinal (GI) tract involvement by Langerhans cell histiocytosis (LCH) is rare and its clinicopathologic characteristics have only been described in case reports and small series. We reviewed hematoxylin and eosin and CD1a, S100, and Langerin immunohistochemical-stained slides from 47 patients with well-documented demographic and clinical findings. Our cases included 8 children and 39 adults, with a mean follow-up of 63 months. All pediatric patients had concurrent multisystem LCH, presented with GI symptoms, and showed nonpolypoid lesions. Seven (88%) showed multifocal GI disease, including 5 with multiple GI organ involvement. All sampled lesions from children exhibited infiltrative growth. More than half had died of the disease or manifested persistent LCH at last follow-up. Twenty-five of 39 (64%) adults had LCH involving only the GI tract (single system), with the remaining 14 (36%) exhibiting multisystem disease. Adult single-system GI LCH was typically encountered incidentally on screening/surveillance endoscopy (72%). Most exhibited isolated colorectal involvement (88%) as a solitary polyp (92%), with a well-demarcated/noninfiltrative growth pattern (70%), and excellent prognosis (100%). In comparison, adult patients with multisystem LCH more frequently presented with GI symptoms (92%, P < .001), noncolorectal GI site involvement (50%, P = .02), multifocal GI lesions (43%, P = .005), nonpolypoid lesions (71%, P < .001), infiltrative histologic growth pattern (78%, P = .04), and persistent disease (57%, P < .001). Adult patients with multisystem LCH appear to exhibit similar clinicopathologic features to those of pediatric patients. These results demonstrated that adults with single-system LCH involving the GI tract have an excellent prognosis, whereas multisystem LCH occurring at any age carries an unfavorable prognosis. High-risk features of GI LCH include pediatric age, GI symptomatology, noncolorectal GI involvement, multifocal GI disease, nonpolypoid lesions, and infiltrative growth pattern.

Validation of Metallothionein Immunohistochemistry as a Highly Sensitive Screening Test for Wilson Disease.

Wilson's disease (WD) is a rare autosomal recessive condition with protean clinical manifestations that results from biallelic ATP7B mutations. However, non-destructive tissue tests to be applied clinically to tissue specimens are not widely available to effectively assess patients for possible WD. Previously, we showed that metallothionein (MTH) immunohistochemistry has a high sensitivity and specificity for WD diagnosis and, thus, represents a potentially powerful diagnostic tool that can be used in routine histologic sections. We sought to validate this finding in a large cohort of bona fide WD patients and to correlate metallothionein expression with other histologic features. We identified 91 cases of WD, which included 28 needle biopsies and 64 explants from 14 centers worldwide. Histologic features were evaluated, and a histopathological pattern was assigned to each case. All cases were evaluated with Masson trichrome and MTH immunohistochemistry (clone UC1MT, Abcam) using previously published technique. Liver tissues from chronic cholestatic diseases (n= 42) were used as controls. The median age of the cohort was 28.5 years. Out of 91 total cases, 83 were positive for MTH immunostain. In the controls, all 42 cases were negative for MTH immunostain. The sensitivity and specificity of MTH immunostain for WD were 91.20% and 100%, respectively. MTH immunohistochemistry is a highly sensitive and specific cost-effective screening tool for WD. It can be used for patients across age groups, varied histologic patterns, and fibrosis stages. This marker could prove to be a valuable tool in the evaluation of patients with possible WD.

5S rRNA pseudogene transcripts are associated with interferon production and inflammatory responses in alcohol-associated hepatitis.

BACKGROUND AND AIMS: Interferon (IFN) signaling is critical to the pathogenesis of alcohol-associated hepatitis (AH), yet the mechanisms for activation of this system are elusive. We hypothesize that host-derived 5S rRNA pseudogene (RNA5SP) transcripts regulate IFN production and modify immunity in AH. APPROACH AND RESULTS: Mining of transcriptomic datasets revealed that in patients with severe alcohol-associated hepatitis (sAH), hepatic expression of genes regulated by IFNs was perturbed and gene sets involved in IFN production were enriched. RNA5SP transcripts were also increased and correlated with expression of type I IFNs. Interestingly, inflammatory mediators upregulated in sAH, but not in other liver diseases, were positively correlated with certain RNA5SP transcripts. Real-time quantitative PCR demonstrated that RNA5SP transcripts were upregulated in peripheral blood mononuclear cells (PBMCs) from patients with sAH. In sAH livers, increased 5S rRNA and reduced nuclear MAF1 (MAF1 homolog, negative regulator of RNA polymerase III) protein suggested a higher activity of RNA polymerase III (Pol III); inhibition of Pol III reduced RNA5SP expression in monocytic THP-1 cells. Expression of several RNA5SP transcript-interacting proteins was downregulated in sAH, potentially unmasking transcripts to immunosensors. Indeed, siRNA knockdown of interacting proteins potentiated the immunostimulatory activity of RNA5SP transcripts. Molecular interaction and cell viability assays demonstrated that RNA5SP transcripts adopted Z-conformation and contributed to ZBP1-mediated caspase-independent cell death. CONCLUSIONS: Increased expression and binding availability of RNA5SP transcripts was associated with hepatic IFN production and inflammation in sAH. These data identify RNA5SP transcripts as a potential target to mitigate inflammation and hepatocellular injury in AH.

Macrophage-derived MLKL in alcohol-associated liver disease: Regulation of phagocytosis.

BACKGROUND AND AIMS: Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3)-/- mice are completely protected from ethanol-induced liver injury, Mlkl-/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. APPROACH AND RESULTS: Bone marrow transplants between Mlkl-/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl-/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→ Mlkl-/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1-mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl-/- mice phagocytosed fewer bioparticles than KCs of WT mice. CONCLUSION: Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.

Biomarker Discovery in Liver Disease Using Untargeted Metabolomics in Plasma and Saliva.

Chronic liver diseases, including non-alcoholic fatty liver disease (NAFLD), cirrhosis, and hepatocellular carcinoma (HCC), continue to be a global health burden with a rise in incidence and mortality, necessitating a need for the discovery of novel biomarkers for HCC detection. This study aimed to identify novel non-invasive biomarkers for these different liver disease states. We performed untargeted metabolomics in plasma (Healthy = 9, NAFLD = 14, Cirrhosis = 10, HCC = 34) and saliva samples (Healthy = 9, NAFLD = 14, Cirrhosis = 10, HCC = 22) to test for significant metabolite associations with each disease state. Additionally, we identified enriched biochemical pathways and analyzed correlations of metabolites between, and within, the two biofluids. We identified two salivary metabolites and 28 plasma metabolites significantly associated with at least one liver disease state. No metabolites were significantly correlated between biofluids, but we did identify numerous metabolites correlated within saliva and plasma, respectively. Pathway analysis revealed significant pathways enriched within plasma metabolites for several disease states. Our work provides a detailed analysis of the altered metabolome at various stages of liver disease while providing some context to altered pathways and relationships between metabolites.

Neoadjuvant therapy leads to objective response in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is the second most common hepatic malignancy and has a poor prognosis. Surgical resection is the standard of care for patients with resectable disease, representing 30-40% of cases. Increasingly, neoadjuvant systemic therapy is being utilized in patients due to high-risk anatomic or biologic considerations. However, data on the clinical effect of this approach are limited. We performed a cohort study to evaluate the effect of neoadjuvant therapy in patients with oncologically high-risk iCCA. METHODS: iCCA patients (n = 181) between the years 2014-2020 were reviewed for clinical, histopathologic, treatment, and outcome-related data. Tumor regression grade was scored per CAP criteria for gastrointestinal carcinomas. RESULTS: 47 iCCA patients received neoadjuvant therapy and 72 did not. Neoadjuvant treatment led to objective response and tumor regression by CAP score. After adjustment for age, clinical stage, and tumor size, the outcomes of patients who had neoadjuvant therapy followed by surgery were not significantly different from those patients who had surgery first. DISCUSSION: In conclusion, neoadjuvant therapy in iCCA facilitated surgical care. The progression-free and overall survival for surgical patients with and without neoadjuvant therapy were not significantly different suggesting this approach needs further exploration as an effective treatment paradigm.

WITHDRAWN: Neoadjuvant therapy leads to objective response in intrahepatic cholangiocarcinoma.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.hpb.2024.04.011. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/policies/article-withdrawal.

Artificial intelligence-based pathology as a biomarker of sensitivity to atezolizumab-bevacizumab in patients with hepatocellular carcinoma: a multicentre retrospective study.

BACKGROUND: Clinical benefits of atezolizumab plus bevacizumab (atezolizumab-bevacizumab) are observed only in a subset of patients with hepatocellular carcinoma and the development of biomarkers is needed to improve therapeutic strategies. The atezolizumab-bevacizumab response signature (ABRS), assessed by molecular biology profiling techniques, has been shown to be associated with progression-free survival after treatment initiation. The primary objective of our study was to develop an artificial intelligence (AI) model able to estimate ABRS expression directly from histological slides, and to evaluate if model predictions were associated with progression-free survival. METHODS: In this multicentre retrospective study, we developed a model (ABRS-prediction; ABRS-P), which was derived from the previously published clustering-constrained attention multiple instance learning (or CLAM) pipeline. We trained the model fit for regression analysis using a multicentre dataset from The Cancer Genome Atlas (patients treated by surgical resection, n=336). The ABRS-P model was externally validated on two independent series of samples from patients with hepatocellular carcinoma (a surgical resection series, n=225; and a biopsy series, n=157). The predictive value of the model was further tested in a series of biopsy samples from a multicentre cohort of patients with hepatocellular carcinoma treated with atezolizumab-bevacizumab (n=122). All samples in the study were from adults (aged ≥18 years). The validation sets were sampled between Jan 1, 2008, to Jan 1, 2023. For the multicentre validation set, the primary objective was to assess the association of high versus low ABRS-P values, defined relative to cross-validation median split thresholds in the first biopsy series, with progression-free survival after treatment initiation. Finally, we performed spatial transcriptomics and matched prediction heatmaps with in situ expression profiles. FINDINGS: Of the 840 patients sampled, 641 (76%) were male and 199 (24%) were female. Across the development and validation datasets, hepatocellular carcinoma risk factors included alcohol intake, hepatitis B and C virus infections, and non-alcoholic steatohepatitis. Using cross-validation in the development series, the mean Pearson's correlation between ABRS-P values and ABRS score (mean expression of ABRS genes) was r=0·62 (SD 0·09; mean p<0·0001, SD<0·0001). The ABRS-P generalised well on the external validation series (surgical resection series, r=0·60 [95% CI 0·51-0·68], p<0·0001; biopsy series, r=0·53 [0·40-0·63], p<0·0001). In the 122 patients treated with atezolizumab-bevacizumab, those with ABRS-P-high tumours (n=74) showed significantly longer median progression-free survival than those with ABRS-P-low tumours (n=48) after treatment initiation (12 months [95% CI 7-not reached] vs 7 months [4-9]; p=0·014). Spatial transcriptomics showed significantly higher ABRS score, along with upregulation of various other immune effectors, in tumour areas with high ABRS-P values versus areas with low ABRS-P values. INTERPRETATION: Our study indicates that AI applied on hepatocellular carcinoma digital slides is able to serve as a biomarker for progression-free survival in patients treated with atezolizumab-bevacizumab. This approach could be used in the development of inexpensive and fast biomarkers for targeted therapies. The combination of AI heatmaps with spatial transcriptomics provides insight on the molecular features associated with predictions. This methodology could be applied to other cancers or diseases and improve understanding of the biological mechanisms that drive responses to treatments. FUNDING: Institut National du Cancer, Fondation ARC, China Scholarship Council, Ligue Contre le Cancer du Val de Marne, Fondation de l'Avenir, Ipsen, and Fondation Bristol Myers Squibb Pour la Recherche en Immuno-Oncologie.

MBOAT7-driven lysophosphatidylinositol acylation in adipocytes contributes to systemic glucose homeostasis.

We previously demonstrated that antisense oligonucleotide-mediated knockdown of Mboat7, the gene encoding membrane bound O-acyltransferase 7, in the liver and adipose tissue of mice promoted high fat diet-induced hepatic steatosis, hyperinsulinemia, and systemic insulin resistance. Thereafter, other groups showed that hepatocyte-specific genetic deletion of Mboat7 promoted striking fatty liver and NAFLD progression in mice but does not alter insulin sensitivity, suggesting the potential for cell autonomous roles. Here, we show that MBOAT7 function in adipocytes contributes to diet-induced metabolic disturbances including hyperinsulinemia and systemic insulin resistance. We generated Mboat7 floxed mice and created hepatocyte- and adipocyte-specific Mboat7 knockout mice using Cre-recombinase mice under the control of the albumin and adiponectin promoter, respectively. Here, we show that MBOAT7 function in adipocytes contributes to diet-induced metabolic disturbances including hyperinsulinemia and systemic insulin resistance. The expression of Mboat7 in white adipose tissue closely correlates with diet-induced obesity across a panel of ∼100 inbred strains of mice fed a high fat/high sucrose diet. Moreover, we found that adipocyte-specific genetic deletion of Mboat7 is sufficient to promote hyperinsulinemia, systemic insulin resistance, and mild fatty liver. Unlike in the liver, where Mboat7 plays a relatively minor role in maintaining arachidonic acid-containing PI pools, Mboat7 is the major source of arachidonic acid-containing PI pools in adipose tissue. Our data demonstrate that MBOAT7 is a critical regulator of adipose tissue PI homeostasis, and adipocyte MBOAT7-driven PI biosynthesis is closely linked to hyperinsulinemia and insulin resistance in mice.

Heterozygous midnolin knockout attenuates severity of nonalcoholic fatty liver disease in mice fed a Western-style diet high in fat, cholesterol, and fructose.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that the knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this antitumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. Although the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.NEW & NOTEWORTHY We have developed and now report on the first functional global midnolin knockout mouse model. We found that global heterozygous midnolin knockout attenuated the severity of nonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism.

Characteristics of Immune Checkpoint Inhibitor-Associated Gastritis: Report from a Major Tertiary Care Center.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have increased our ability to treat an ever-expanding number of cancers. We describe a case series of 25 patients who were diagnosed with gastritis following ICI therapy. MATERIALS AND METHODS: This was a retrospective study involving 1712 patients treated for malignancy with immunotherapy at Cleveland Clinic from January 2011 to June 2019 (IRB 18-1225). We searched electronic medical records using ICD-10 codes for gastritis diagnosis confirmed on endoscopy and histology within 3 months of ICI therapy. Patients with upper gastrointestinal tract malignancy or documented Helicobacter pylori-associated gastritis were excluded. RESULTS: Twenty-five patients were found to meet the criteria for diagnosis of gastritis. Of these 25 patients, most common malignancies were non-small cell lung cancer (52%) and melanoma (24%). Median number of infusions preceding symptoms was 4 (1-30) and time to symptom onset 2 (0.5-12) weeks after last infusion. Symptoms experienced were nausea (80%), vomiting (52%), abdominal pain (72%), and melena (44%). Common endoscopic findings were erythema (88%), edema (52%), and friability (48%). The most common diagnosis of pathology was chronic active gastritis in 24% of patients. Ninety-six percent received acid suppression treatment and 36% of patients also received steroids with an initial median dose of prednisone 75 (20-80) mg. Within 2 months, 64% had documented complete resolution of symptoms and 52% were able to resume immunotherapy. CONCLUSION: Patients presenting with nausea, vomiting, abdominal pain, or melena following immunotherapy should be assessed for gastritis and if other causes are excluded, may require treatment as consideration for complication of immunotherapy.

Genomic Predictors Associated With Exceptional Response to Systemic Therapy in Advanced Pancreatic Cancer.

INTRODUCTION: Exceptional response to therapy is rare in patients with advanced pancreatic cancer. This study explored potential genomic differences between typical and exceptional responses that could confer more favorable biology. METHODS: We included exceptional responders and controls with advanced pancreatic cancer from Cleveland Clinic from April 2013 to August 2017. Exceptional responders were defined as patients with an overall survival of more than 18 months for metastatic disease and more than 24 months for locally advanced disease. Clinical data were obtained, and next-generation sequencing was performed. Statistical analyses comparing the 2 groups were performed using descriptive statistics, the Kaplan-Meier method, and the log-rank test. RESULTS: The study comprised 4 exceptional responders and 6 controls. Both groups were well balanced in age, sex, race, and treatment regimens. Exceptional responders had significantly fewer nonsynonymous mutations than controls (2.25 vs 5.17; P = .014). A mutation count of less than 3 was associated with significantly better progression-free survival (17.2 vs 2.3 months; P = .002) and overall survival (29.4 vs 4.6 months; P = .013). Tumor mutational burden did not differ between exceptional responders and controls (4.88 vs 5.70 mut/Mb; P = .39). CONCLUSION: A lower number of nonsynonymous mutations may correlate with exceptional outcomes in patients with pancreatic cancer. These findings should encourage future studies into genomic signatures of exceptional response.

How reliable is restaging MRI after neoadjuvant therapy in rectal cancer?

BACKGROUND: Despite the pivotal role of magnetic resonance imaging (MRI) in rectal cancer staging and evaluation, the reliability of restaging MRI after neoadjuvant therapy is still debatable. This study aimed to assess the accuracy of restaging MRI by comparing post-neoadjuvant MRI findings with those of the final pathology. METHODS: This study was a retrospective review of the medical records of adult rectal cancer patients who had restaging MRI following neoadjuvant therapy and prior to rectal cancer resection in a NAPRC-certified rectal cancer centre between 2016 and 2021. The study compared findings of preoperative, post-neoadjuvant MRI with final pathology relative to T stage, N stage, tumour size, and circumferential resection margin (CRM) status. RESULTS: A total of 126 patients were included in the study. We found fair concordance (kappa -0.316) for T stage between restaging MRI and pathology report, and slight concordance for N stage and CRM status (kappa -0.11, kappa = 0.089, respectively). Concordance rates were lower for patients following total neoadjuvant treatment (TNT) or with a low rectal tumour. In total, 73% of patients with positive N pathology status had negative N status in the restaging MRI. Sensitivity and specificity regarding positive CRM in post-neoadjuvant treatment MRI were 45.45% and 70.4%, respectively. CONCLUSION: We found low concordance levels between restaging MRI and pathology regarding TN stage and CRM status. Concordance levels were even lower for patients after TNT regimen and with a low rectal tumour. In the era of TNT and watch-and-wait approach, we should not rely solely on restaging MRI to make post-neoadjuvant treatment decisions.

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.

BACKGROUND & AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs. METHODS: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling. RESULTS: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies. CONCLUSION: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. LAY SUMMARY: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.

Metallothionein immunohistochemistry has high sensitivity and specificity for detection of Wilson disease.

Diagnosis of Wilson disease (WD) can be difficult because of its protean clinical presentations, but early diagnosis is important because effective treatment is available and can prevent disease progression. Similarly, diagnosis of WD on liver biopsy specimens is difficult due to the wide range of histologic appearances. A stain that could help identify WD patients would be of great value. The goal of this study was to use mass spectrometry-based proteomics to identify potential proteins that are differentially expressed in WD compared to controls, and could serve as potential immunohistochemical markers for screening. Several proteins were differentially expressed in WD and immunohistochemical stains for two (metallothionein (MT) and cytochrome C oxidase copper chaperone (COX17)) were tested and compared to other methods of diagnosis in WD including copper staining and quantitative copper assays. We found diffuse metallothionein immunoreactivity in all liver specimens from patients with WD (n = 20); the intensity of the staining was moderate to strong. This staining pattern was distinct from that seen in specimens from the control groups (none of which showed strong, diffuse staining), which included diseases that may be in the clinical or histologic differential of WD (steatohepatitis (n = 51), chronic viral hepatitis (n = 40), autoimmune hepatitis (n = 50), chronic biliary tract disease (n = 42), and normal liver (n = 20)). COX17 immunostain showed no significant difference in expression between the WD and control groups. MT had higher sensitivity than rhodanine for diagnosis of WD. While the quantitative liver copper assays also had high sensitivity, they require more tissue, have a higher cost, longer turnaround time, and are less widely available than an immunohistochemical stain. We conclude that MT IHC is a sensitive immunohistochemical stain for the diagnosis of WD that could be widely deployed as a screening tool for liver biopsies in which WD is in the clinical or histologic differential diagnosis.

Gene Deconvolution Reveals Aberrant Liver Regeneration and Immune Cell Infiltration in Alcohol-Associated Hepatitis.

BACKGROUND AND AIMS: Acute liver damage causes hepatocyte stress and death, but in chronic liver disease impaired hepatocyte regeneration and immune cell infiltration prevents recovery. While the roles of both impaired liver regeneration and immune infiltration have been studied extensively in chronic liver diseases, the differential contribution of these factors is difficult to assess. APPROACH AND RESULTS: We combined single-cell RNA-sequencing (RNA-seq) data from healthy livers and peripheral immune cells to measure cell proportions in chronic liver diseases. Using bulk RNA-seq data from patients with early alcohol-associated hepatitis, severe AH (sAH), HCV, HCV with cirrhosis, and NAFLD, we performed gene deconvolution to predict the contribution of different cell types in each disease. Patients with sAH had the greatest change in cell composition, with increases in both periportal hepatocytes and cholangiocyte populations. Interestingly, while central vein hepatocytes were decreased, central vein endothelial cells were expanded. Endothelial cells are thought to regulate liver regeneration through WNT signaling. WNT2, important in central vein hepatocyte development, was down in sAH, while multiple other WNTs and WNT receptors were up-regulated. Immunohistochemistry revealed up-regulation of FZD6, a noncanonical WNT receptor, in hepatocytes in sAH. Immune cell populations also differed in disease. In sAH, a specific group of inflammatory macrophages was increased and distinct from the macrophage population in patients with HCV. Network and correlation analyses revealed that changes in the cell types in the liver were highly correlated with clinical liver function tests. CONCLUSIONS: These results identify distinct changes in the liver cell populations in chronic liver disease and illustrate the power of using single-cell RNA-seq data from a limited number of samples in understanding multiple different diseases.

Incidence of Complications from Percutaneous Biopsy in Chronic Liver Disease: A Systematic Review and Meta-Analysis.

BACKGROUND: Approaches to liver biopsy have changed over the past decade in patients with chronic liver disease. AIMS: We conducted a systematic review and meta-analysis on the incidence of all complications and technical failure associated with percutaneous liver biopsy. METHODS: We systematically searched PubMed and the Cochrane Library for cohort studies reporting on complications resulting from liver biopsy published between 2010 and 2020. Studies on participants of any age and sex, who underwent any percutaneous biopsy for non-focal liver disease, were selected. All events except mild pain, minor hematoma, vasovagal episodes, fever and fistula were defined as major complications. Random-effect model meta-analyses with and without covariates were performed, to examine the effect of publication year, patient characteristics, outcome collection, and biopsy type on incidences. RESULTS: We identified 30 studies reporting on complications resulting from percutaneous liver biopsy procedures (n = 64,356). Incidence of major complications was 2.44% (95% CI 0.85, 6.75), with mortality at 0.01% (95% CI 0.00, 0.11), hospitalization at 0.65% (95% CI 0.38, 1.11), major bleeding at 0.48% (95% CI 0.22, 1.06), and moderate/severe pain at 0.34% (95% CI 0.08, 1.37). Minor complications at 9.53% (95% CI 3.68, 22.5) were mainly pain at 12.9% (95% CI 5.34, 27.9). Technical failure was high at 0.91% (95% CI 0.27, 3.00). Decreasing patient age significantly increased incidence of hospitalization and major bleeding (P < 0.0001). Hospitalization incidence also significantly increased with disease severity. CONCLUSIONS: Incidence of major (2.4%) and minor (9.5%) complications, and technical failure (0.91%) in percutaneous liver biopsies continues.

ADAMTSL5 is an epigenetically activated gene underlying tumorigenesis and drug resistance in hepatocellular carcinoma.

BACKGROUND & AIMS: The tumour microenvironment shapes tumour growth through cellular communications that include both direct interactions and secreted factors. The aim of this study was to characterize the impact of the secreted glycoprotein ADAMTSL5, whose role in cancer has not been previously investigated, on hepatocellular carcinoma (HCC). METHODS: ADAMTSL5 methylation status was evaluated through bisulfite sequencing, and publicly available data analysis. ADAMTSL5 RNA and protein expression were assessed in mouse models and HCC patient samples and compared to data from published datasets. Functional studies, including association of ADAMTSL5 depletion with responsiveness to clinically relevant drugs, were performed in cellular and in vivo models. Molecular alterations associated with ADAMTSL5 targeting were determined using proteomics, biochemistry, and reverse-transcription quantitative PCR. RESULTS: Methylome analysis revealed hypermethylated gene body CpG islands at the ADAMTSL5 locus in both mouse and human HCC, correlating with higher ADAMTSL5 expression. ADAMTSL5 targeting interfered with tumorigenic properties of HCC cells in vitro and in vivo, whereas ADAMTSL5 overexpression conferred tumorigenicity to pre-tumoural hepatocytes sensitized to transformation by a modest level of MET receptor expression. Mechanistically, ADAMTSL5 abrogation led to a reduction of several oncogenic inputs relevant to HCC, including reduced expression and/or phosphorylation levels of receptor tyrosine kinases MET, EGFR, PDGFRß, IGF1Rß, or FGFR4. This phenotype was associated with significantly increased sensitivity of HCC cells to clinically relevant drugs, namely sorafenib, lenvatinib, and regorafenib. Moreover, ADAMTSL5 depletion drastically increased expression of AXL, accompanied by a sensitization to bemcentinib. CONCLUSIONS: Our results point to a role for ADAMTSL5 in maintaining the function of key oncogenic signalling pathways, suggesting that it may act as a master regulator of tumorigenicity and drug resistance in HCC. LAY SUMMARY: The environment of cancer cells has profound effects on establishment, progression, and response of a tumour to treatment. Herein, we show that ADAMTSL5, a protein secreted by liver cancer cells and overlooked in cancer so far, is increased in this tumour type, is necessary for tumour formation and supports drug resistance. Adamtsl5 removal conferred sensitivity of liver cancer cells to drugs used in current treatment. This suggests ADAMTSL5 as a potential marker in liver cancer as well as a possible drug target.