Development of a novel non-invasive biomarker panel for hepatic fibrosis in MASLD.

Accurate non-invasive biomarkers to diagnose metabolic dysfunction-associated steatotic liver disease (MASLD)-related fibrosis are urgently needed. This study applies a translational approach to develop a blood-based biomarker panel for fibrosis detection in MASLD. A molecular gene expression signature identified from a diet-induced MASLD mouse model (LDLr-/-.Leiden) is translated into human blood-based biomarkers based on liver biopsy transcriptomic profiles and protein levels in MASLD patient serum samples. The resulting biomarker panel consists of IGFBP7, SSc5D and Sema4D. LightGBM modeling using this panel demonstrates high accuracy in predicting MASLD fibrosis stage (F0/F1: AUC = 0.82; F2: AUC = 0.89; F3/F4: AUC = 0.87), which is replicated in an independent validation cohort. The overall accuracy of the model outperforms predictions by the existing markers Fib-4, APRI and FibroScan. In conclusion, here we show a disease mechanism-related blood-based biomarker panel with three biomarkers which is able to identify MASLD patients with mild or advanced hepatic fibrosis with high accuracy.

Inhibition of hepatic p63 ameliorates steatohepatitis with fibrosis in mice.

OBJECTIVE: p63 is a transcription factor involved in multiple biological functions. In the liver, the TAp63 isoform induces lipid accumulation in hepatocytes. However, the role of liver TAp63 in the progression of metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis is unknown. METHODS: We evaluated the hepatic p63 levels in different mouse models of steatohepatitis with fibrosis induced by diet. Next, we used virogenetic approaches to manipulate the expression of TAp63 in adult mice under diet-induced steatohepatitis with fibrosis and characterized the disease condition. Finally, we performed proteomics analysis in mice with overexpression and knockdown of hepatic TAp63. RESULTS: Levels of TAp63, but not of ΔN isoform, are increased in the liver of mice with diet-induced steatohepatitis with fibrosis. Both preventive and interventional strategies for the knockdown of hepatic TAp63 significantly ameliorated diet-induced steatohepatitis with fibrosis in mice fed a methionine- and choline-deficient diet (MCDD) and choline deficient and high fat diet (CDHFD). The overexpression of hepatic TAp63 in mice aggravated the liver condition in mice fed a CDHFD. Proteomic analysis in the liver of these mice revealed alteration in multiple proteins and pathways, such as oxidative phosphorylation, antioxidant activity, peroxisome function and LDL clearance. CONCLUSIONS: These results indicate that liver TAp63 plays a critical role in the progression of diet-induced steatohepatitis with fibrosis, and its inhibition ameliorates the disease.

Kinase activities in pancreatic ductal adenocarcinoma with prognostic and therapeutic avenues.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read-out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)-based proteomics and phosphoproteomics were applied to 42 PDAC tumors. Data encompassed over 19 936 phosphoserine or phosphothreonine (pS/T; in 5412 phosphoproteins) and 1208 phosphotyrosine (pY; in 501 phosphoproteins) sites and a total of 3756 proteins. Proteome data identified three distinct subtypes with tumor intrinsic and stromal features. Subsequently, three phospho-subtypes were apparent: two tumor intrinsic (Phos1/2) and one stromal (Phos3), resembling known PDAC molecular subtypes. Kinase activity was analyzed by the Integrative iNferred Kinase Activity (INKA) scoring. Phospho-subtypes displayed differential phosphorylation signals and kinase activity, such as FGR and GSK3 activation in Phos1, SRC kinase family and EPHA2 in Phos2, and EGFR, INSR, MET, ABL1, HIPK1, JAK, and PRKCD in Phos3. Kinase activity analysis of an external PDAC cohort supported our findings and underscored the importance of PI3K/AKT and ERK pathways, among others. Interestingly, unfavorable patient prognosis correlated with higher RTK, PAK2, STK10, and CDK7 activity and high proliferation, whereas long survival was associated with MYLK and PTK6 activity, which was previously unknown. Subtype-associated activity profiles can guide therapeutic combination approaches in tumor and stroma-enriched tissues, and emphasize the critical role of parallel signaling pathways. In addition, kinase activity profiling identifies potential disease markers with prognostic significance.

The Neo-Open Reading Frame Peptides That Comprise the Tumor Framome Are a Rich Source of Neoantigens for Cancer Immunotherapy.

Identification of immunogenic cancer neoantigens as targets for therapy is challenging. Here, we integrate the whole-genome and long-read transcript sequencing of cancers to identify the collection of neo-open reading frame peptides (NOP) expressed in tumors. We termed this collection of NOPs the tumor framome. NOPs represent tumor-specific peptides that are different from wild-type proteins and may be strongly immunogenic. We describe a class of hidden NOPs that derive from structural genomic variants involving an upstream protein coding gene driving expression and translation of noncoding regions of the genome downstream of a rearrangement breakpoint, i.e., where no gene annotation or evidence for transcription exists. The entire collection of NOPs represents a vast number of possible neoantigens particularly in tumors with many structural genomic variants and a low number of missense mutations. We show that NOPs are immunogenic and epitopes derived from NOPs can bind to MHC class I molecules. Finally, we provide evidence for the presence of memory T cells specific for hidden NOPs in peripheral blood from a patient with lung cancer. This work highlights NOPs as a major source of possible neoantigens for personalized cancer immunotherapy and provides a rationale for analyzing the complete cancer genome and transcriptome as a basis for the detection of NOPs.

Anaerobutyricum soehngenii Reduces Hepatic Lipogenic Pathways and Increases Intestinal Gluconeogenic Gene Expression in Metabolic-Dysfunction-Associated Steatotic Liver Disease (MASLD) Mice.

Metabolic-dysfunction-associated steatotic liver disease (MASLD) is a growing health problem for which no therapy exists to date. The modulation of the gut microbiome may have treatment potential for MASLD. Here, we investigated Anaerobutyricum soehngenii, a butyrate-producing anaerobic bacterium with beneficial effects in metabolic syndrome, in a diet-induced MASLD mouse model. Male C57BL/6J mice received a Western-type high-fat diet and water with 15% fructose (WDF) to induce MASLD and were gavaged with A. soehngenii (108 or 109 colony-forming units (CFU) 3 times per week) or a placebo for 6 weeks. The A. soehngenii gavage increased the cecal butyrate concentrations. Although there was no effect on histological MASLD scores, A. soehngenii improved the glycemic response to insulin. In the liver, the WDF-associated altered expression of three genes relevant to the MASLD pathophysiology was reversed upon treatment with A. soehngenii: Lipin-1 (Lpin1), insulin-like growth factor binding protein 1 (Igfbp1) and Interleukin 1 Receptor Type 1 (Il1r1). A. soehngenii administration also increased the intestinal expression of gluconeogenesis and fructolysis genes. Although these effects did not translate into significant histological improvements in MASLD, these results provide a basis for combined gut microbial approaches to induce histological improvements in MASLD.

Cancer cell genetics shaping of the tumor microenvironment reveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma.

Myeloid cells are abundant and plastic immune cell subsets in the liver, to which pro-tumorigenic, inflammatory and immunosuppressive roles have been assigned in the course of tumorigenesis. Yet several aspects underlying their dynamic alterations in hepatocellular carcinoma (HCC) progression remain elusive, including the impact of distinct genetic mutations in shaping a cancer-permissive tumor microenvironment (TME). Here, in newly generated, clinically-relevant somatic female HCC mouse models, we identify cancer genetics' specific and stage-dependent alterations of the liver TME associated with distinct histopathological and malignant HCC features. Mitogen-activated protein kinase (MAPK)-activated, NrasG12D-driven tumors exhibit a mixed phenotype of prominent inflammation and immunosuppression in a T cell-excluded TME. Mechanistically, we report a NrasG12D cancer cell-driven, MEK-ERK1/2-SP1-dependent GM-CSF secretion enabling the accumulation of immunosuppressive and proinflammatory monocyte-derived Ly6Clow cells. GM-CSF blockade curbs the accumulation of these cells, reduces inflammation, induces cancer cell death and prolongs animal survival. Furthermore, GM-CSF neutralization synergizes with a vascular endothelial growth factor (VEGF) inhibitor to restrain HCC outgrowth. These findings underscore the profound alterations of the myeloid TME consequential to MAPK pathway activation intensity and the potential of GM-CSF inhibition as a myeloid-centric therapy tailored to subsets of HCC patients.

Diagnosis and treatment of patients with suspected mucinous cystic neoplasms of the liver: a retrospective cohort study.

PURPOSE: Mucinous cystic neoplasms of the liver (MCN-L) are hepatic cysts with a low malignant potential. The recent European Association for the Study of the Liver (EASL) guidelines provide guidance on the imaging features and surgical management of MCN-L, yet are hampered by a lack of studies adhering to the revised World Health Organization (WHO) criteria. This study attempted to validate the new 2022 EASL-guidelines in a retrospective cohort study of patients who underwent surgery for suspected MCN-L. METHODS: Patients undergoing surgery for suspected MCN-L in a single center between 2010 and 2020 were included. Imaging features were assessed according to the EASL guidelines and were compared to final pathological diagnoses, according to the WHO criteria. RESULTS: In total, 35 patients were included. In three patients, there were no worrisome imaging features, yet final pathological diagnosis showed MCN-L. Contrarily, six patients with worrisome imaging features did not have MCN-L. Five patients were diagnosed with MCN-L on final pathology. The sensitivity of the EASL-guidelines for the diagnosis of MCN-L was 40% (95%CI: 5.3-85%) and the specificity was 80% (95% CI: 61-92%). CONCLUSION: Although the new EASL-guidelines provide some guidance, they could not reliably distinguish MCN-L from other cysts in our series. Thus, preoperative diagnosis of MCN-L remains challenging and we should be careful in selecting surgical strategies based on these criteria.

Intrapancreatic fat deposition is unrelated to liver steatosis in metabolic dysfunction-associated steatotic liver disease.

Background & Aims: Individuals with obesity may develop intrapancreatic fat deposition (IPFD) and fatty pancreas disease (FPD). Whether this causes inflammation and fibrosis and leads to pancreatic dysfunction is less established than for liver damage in metabolic dysfunction-associated steatotic liver disease (MASLD). Moreover, the interrelations of FPD and MASLD are poorly understood. Therefore, we aimed to assess IPFD and fibro-inflammation in relation to pancreatic function and liver disease severity in individuals with MASLD. Methods: Seventy-six participants from the Amsterdam MASLD-MASH cohort (ANCHOR) study underwent liver biopsy and multiparametric MRI of the liver and pancreas, consisting of proton-density fat fraction sequences, T1 mapping and intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI). Results: The prevalence of FPD was 37.3%. There was a clear correlation between pancreatic T1 relaxation time, which indicates fibro-inflammation, and parameters of glycemic dysregulation, namely HbA1c (R = 0.59; p <0.001), fasting glucose (R = 0.51; p <0.001) and the presence of type 2 diabetes (mean 802.0 ms vs. 733.6 ms; p <0.05). In contrast, there was no relation between IPFD and hepatic fat content (R = 0.03; p = 0.80). Pancreatic IVIM diffusion (IVIM-D) was lower in advanced liver fibrosis (p <0.05) and pancreatic perfusion (IVIM-f), reflecting vessel density, inversely correlated to histological MASLD activity (p <0.05). Conclusions: Consistent relations exist between pancreatic fibro-inflammation on MRI and endocrine function in individuals with MASLD. However, despite shared dysmetabolic drivers, our study suggests IPFD is a separate pathophysiological process from MASLD. Impact and implications: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide and 68% of people with type 2 diabetes have MASLD. However, fat infiltration and inflammation in the pancreas are understudied in individuals with MASLD. In this cross-sectional MRI study, we found no relationship between fat accumulation in the pancreas and liver in a cohort of patients with MASLD. However, our results show that inflammatory and fibrotic processes in the pancreas may be interrelated to features of type 2 diabetes and to the severity of liver disease in patients with MASLD. Overall, the results suggest that pancreatic endocrine dysfunction in individuals with MASLD may be more related to glucotoxicity than to lipotoxicity. Clinical trial number: NTR7191 (Dutch Trial Register).

Association Between Gadoxetic Acid-Enhanced Magnetic Resonance Imaging, Organic Anion Transporters, and Farnesoid X Receptor in Benign Focal Liver Lesions.

The organic anion uptake and efflux transporters [organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and multidrug resistance-associated protein (MRP)2 and MRP3] that mediate the transport of the hepatobiliary-specific contrast agent gadoxetate (Gd-EOB-DTPA) are direct or indirect targets of the farnesoid X receptor (FXR), a key regulator of bile acid and lipid homeostasis. In benign liver tumors, FXR expression and activation is not yet characterized. We investigated the expression and activation of FXR and its targets in hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH) and their correlation with Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI). Gd-EOB-DTPA MRI patterns were assessed by an expert radiologist. The intensity of the lesions on the hepatobiliary phase was correlated to mRNA expression levels of OATP1B1, OATP1B3, MRP2, MRP3, FXR, and small heterodimer partner (SHP) in fresh surgical specimens of patients with FNH or HCA subtypes. Normal and tumor sample pairs of 43 HCA and 14 FNH were included. All FNH (14/14) were hyperintense. Of the 34 HCA with available Gd-EOB-DTPA-enhanced MRI, 6 were hyperintense and 28 HCA were hypointense. OATP1B3 was downregulated in the hypointense tumors compared with normal surrounding liver tissue (2.77±3.59 vs. 12.9±15.6, P < 0.001). A significant positive correlation between FXR expression and activation and OATP1B3 expression level was found in the HCA cohort. SHP showed a trend toward downregulation in hypointense HCA. In conclusion, this study suggests that the MRI relative signal in HCA may reflect expression level and/or activity of SHP and FXR. Moreover, our data confirms the pivotal role of OATP1B3 in Gd-EOB-DTPA uptake in HCA. SIGNIFICANCE STATEMENT: FXR represents a valuable target for the treatment of liver disease and metabolic syndrome. Currently, two molecules, ursodeoxycholate and obeticholate, are approved for the treatment of primary biliary cirrhosis and cholestasis, with several compounds in clinical trials for the treatment of metabolic dysfunction-associated fatty liver disease. Because FXR expression and activation is associated with gadoxetate accumulation in HCA, an atypical gadoxetate-enhanced MRI pattern might arise in patients under FXR-targeted therapy, thereby complicating the differential diagnosis.

The Influence of Hepatic Steatosis and Fibrosis on Postoperative Outcomes After Major Liver Resection of Perihilar Cholangiocarcinoma.

BACKGROUND: Surgical resection for perihilar cholangiocarcinoma (pCCA) is associated with high operative risks. Impaired liver regeneration in patients with pre-existing liver disease may contribute to posthepatectomy liver failure (PHLF) and postoperative mortality. This study aimed to determine the incidence of hepatic steatosis and fibrosis and their association with PHLF and 90-day postoperative mortality in pCCA patients. METHODS: Patients who underwent a major liver resection for pCCA were included in the study between 2000 and 2021 from three tertiary referral hospitals. Histopathologic assessment of hepatic steatosis and fibrosis was performed. The primary outcomes were PHLF and 90-day mortality. RESULTS: Of the 401 included patients, steatosis was absent in 334 patients (83.3%), mild in 58 patients (14.5%) and moderate to severe in 9 patients (2.2%). There was no fibrosis in 92 patients (23.1%), periportal fibrosis in 150 patients (37.6%), septal fibrosis in 123 patients (30.8%), and biliary cirrhosis in 34 patients (8.5%). Steatosis (≥ 5%) was not associated with PHLF (odds ratio [OR] 1.36; 95% confidence interval [CI] 0.69-2.68) or 90-day mortality (OR 1.22; 95% CI 0.62-2.39). Neither was fibrosis (i.e., periportal, septal, or biliary cirrhosis) associated with PHLF (OR 0.76; 95% CI 0.41-1.41) or 90-day mortality (OR 0.60; 95% CI 0.33-1.06). The independent risk factors for PHLF were preoperative cholangitis (OR 2.38; 95% CI 1. 36-4.17) and future liver remnant smaller than 40% (OR 2.40; 95% CI 1.31-4.38). The independent risk factors for 90-day mortality were age of 65 years or older (OR 2.40; 95% CI 1.36-4.23) and preoperative cholangitis (OR 2.25; 95% CI 1.30-3.87). CONCLUSION: In this study, no association could be demonstrated between hepatic steatosis or fibrosis and postoperative outcomes after resection of pCCA.

Prognostic and predictive value of human equilibrative nucleoside transporter 1 (hENT1) in extrahepatic cholangiocarcinoma: a translational study.

Introduction: Effective (neo) adjuvant chemotherapy for cholangiocarcinoma is lacking due to chemoresistance and the absence of predictive biomarkers. Human equilibrative nucleoside transporter 1 (hENT1) has been described as a potential prognostic and predictive biomarker. In this study, the potential of rabbit-derived (SP120) and murine-derived (10D7G2) antibodies to detect hENT1 expression was compared in tissue samples of patients with extrahepatic cholangiocarcinoma (ECC), and the predictive value of hENT1 was investigated in three ECC cell lines. Methods: Tissues of 71 chemonaïve patients with histological confirmation of ECC were selected and stained with SP120 or 10D7G2 to assess the inter-observer variability for both antibodies and the correlation with overall survival. Concomitantly, gemcitabine sensitivity after hENT1 knockdown was assessed in the ECC cell lines EGI-1, TFK-1, and SK-ChA-1 using sulforhodamine B assays. Results: Scoring immunohistochemistry for hENT1 expression with the use of SP120 antibody resulted in the highest interobserver agreement but did not show a prognostic role of hENT1. However, 10D7G2 showed a prognostic role for hENT1, and a potential predictive role for gemcitabine sensitivity in hENT1 in SK-ChA-1 and TFK-1 cells was found. Discussion: These findings prompt further studies for both preclinical validation of the role of hENT1 and histochemical standardization in cholangiocarcinoma patients treated with gemcitabine-based chemotherapy.

Nationwide treatment and outcomes of intrahepatic cholangiocarcinoma.

BACKGROUND: Most data on the treatment and outcomes of intrahepatic cholangiocarcinoma (iCCA) derives from expert centers. This study aimed to investigate the treatment and outcomes of all patients diagnosed with iCCA in a nationwide cohort. METHODS: Data on all patients diagnosed with iCCA between 2010 and 2018 were obtained from the Netherlands Cancer Registry. RESULTS: In total, 1747 patients diagnosed with iCCA were included. Resection was performed in 292 patients (17%), 548 patients (31%) underwent palliative systemic treatment, and 867 patients (50%) best supportive care (BSC). The OS median and 1-, and 3-year OS were after resection: 37.5 months (31.0-44.0), 79.2%, and 51.6%,; with systemic therapy, 10.0 months (9.2-10.8), 38.4%, and 5.1%, and with BSC 2.2 months (2.0-2.5), 10.4%, and 1.3% respectively. The resection rate for patients who first presented in academic centers was 33% (96/292) compared to 13% (195/1454) in non-academic centers (P < 0.001). DISCUSSION: Half of almost 1750 patients with iCCA over an 8 year period did not receive any treatment with a 1-year OS of 10.4%. Three-year survival was about 50% after resection, while long-term survival was rare after palliative treatment. The resection rate was higher in academic centers compared to non-academic centers.

IgG4-related cholangitis - a mimicker of fibrosing and malignant cholangiopathies.

IgG4-related cholangitis (IRC) is the major hepatobiliary manifestation of IgG4-related disease (IgG4-RD), a systemic fibroinflammatory disorder. The pathogenesis of IgG4-RD and IRC is currently viewed as multifactorial, as there is evidence of a genetic predisposition while environmental factors, such as blue-collar work, are major risk factors. Various autoantigens have been described in IgG4-RD, including annexin A11 and laminin 511-E8, proteins which may exert a partially protective function in cholangiocytes by enhancing secretion and barrier function, respectively. For the other recently described autoantigens, galectin-3 and prohibitin 1, a distinct role in cholangiocytes appears less apparent. In relation to these autoantigens, oligoclonal expansions of IgG4+ plasmablasts are present in patients with IRC and disappear upon successful treatment. More recently, specific T-cell subtypes including regulatory T cells, follicular T helper 2 cells, peripheral T helper cells and cytotoxic CD8+ and CD4+ SLAMF7+ T cells have been implicated in the pathogenesis of IgG4-RD. The clinical presentation of IRC often mimics other biliary diseases such as primary sclerosing cholangitis or cholangiocarcinoma, which may lead to inappropriate medical and potentially invalidating surgical interventions. As specific biomarkers are lacking, diagnosis is made according to the HISORt criteria comprising histopathology, imaging, serology, other organ manifestations and response to therapy. Treatment of IRC aims to prevent or alleviate organ damage and to improve symptoms and consists of (i) remission induction, (ii) remission maintenance and (iii) long-term management. Glucocorticosteroids are highly effective for remission induction, after which immunomodulators can be introduced for maintenance of remission as glucocorticosteroid-sparing alternatives. Increased insight into the pathogenesis of IRC will lead to improved diagnosis and novel therapeutic strategies in the future.

Faecal Microbiota transplantation affects liver DNA methylation in Non-alcoholic fatty liver disease: a multi-omics approach.

Individuals with nonalcoholic fatty liver disease (NAFLD) have an altered gut microbiota composition. Moreover, hepatic DNA methylation may be altered in the state of NAFLD. Using a fecal microbiota transplantation (FMT) intervention, we aimed to investigate whether a change in gut microbiota composition relates to altered liver DNA methylation in NAFLD. Moreover, we assessed whether plasma metabolite profiles altered by FMT relate to changes in liver DNA methylation. Twenty-one individuals with NAFLD underwent three 8-weekly vegan allogenic donor (n = 10) or autologous (n = 11) FMTs. We obtained hepatic DNA methylation profiles from paired liver biopsies of study participants before and after FMTs. We applied a multi-omics machine learning approach to identify changes in the gut microbiome, peripheral blood metabolome and liver DNA methylome, and analyzed cross-omics correlations. Vegan allogenic donor FMT compared to autologous FMT induced distinct differential changes in I) gut microbiota profiles, including increased abundance of Eubacterium siraeum and potential probiotic Blautia wexlerae; II) plasma metabolites, including altered levels of phenylacetylcarnitine (PAC) and phenylacetylglutamine (PAG) both from gut-derived phenylacetic acid, and of several choline-derived long-chain acylcholines; and III) hepatic DNA methylation profiles, most importantly in Threonyl-TRNA Synthetase 1 (TARS) and Zinc finger protein 57 (ZFP57). Multi-omics analysis showed that Gemmiger formicillis and Firmicutes bacterium_CAG_170 positively correlated with both PAC and PAG. E siraeum negatively correlated with DNA methylation of cg16885113 in ZFP57. Alterations in gut microbiota composition by FMT caused widespread changes in plasma metabolites (e.g. PAC, PAG, and choline-derived metabolites) and liver DNA methylation profiles in individuals with NAFLD. These results indicate that FMTs might induce metaorganismal pathway changes, from the gut bacteria to the liver.