Inflammatory bowel diseases (IBD) and primary sclerosing cholangitis (PSC) are chronic inflammatory conditions with limited biomarker-driven diagnostic tools. Proteomic profiling offers a promising approach to uncover specific biomarkers that could refine diagnostic accuracy, monitor disease activity, and guide therapeutic strategies. Our primary aim is to identify novel biomarkers for PSC-IBD and conventional ulcerative colitis (UC) via proteomic approach. The secondary aim is to advance the etiopathogenic understanding of the diseases by linking specific proteomic profiles with disease phenotypes. This single-center, prospective, biomarker-discovery study will involve 50 participants with PSC-IBD, 50 with UC, and 50 healthy controls. Biopsy samples from five bowel segments will be analyzed for proteomic signatures by an untargeted approach. The findings will subsequently undergo multi-step external validation in separate cohorts of 30 patients with PSC-IBD and 30 with UC, utilizing targeted proteomics, immunohistochemistry, and ELISA in bowel mucosa and peripheral blood, respectively. This proposed study aims to identify novel biomarkers to improve the diagnostic accuracy of PSC-IBD and UC and refine the disease activity assessment. Its robust design and large sample size provide a strong foundation for successful biomarker identification, with the potential to enhance clinical management of patients.

BackgroundEpigenetic alterations play an increasingly recognized role in carcinogenesis and in the development of resistance to anticancer therapies. Epigenetic enzymes (writers and erasers) and effectors (readers) are largely influenced by the availability of metabolites generated through one-carbon metabolism (OCM), the tricarboxylic acid (TCA) cycle, and acetyl-CoA synthesis (ACS). In this study we examined the expression of epigenetic and metabolic genes to investigate their interplay in cholangiocarcinoma (CCA).MethodWe examined 257 epigenetic genes (EpiGs), 96 metabolic genes (MGs), and 189 rate-limiting enzymes (RLEs) in transcriptomic data from iCCA, eCCA, and normal bile ducts, healthy liver-derived organoids, and CCA tumoroids, alongside prognostic signatures. CRISPR-Cas9 DepMap data evaluated the impact of EpiGs and MGs on cell viability. HuCCT-1 iCCA cells were exposed to hypoxia (1% O2, 24 h) to assess EpiG responses. Transcriptomic deconvolution characterized EpiGs, MGs, and RLEs expression across four tumor microenvironment (TME) subtypes. Two mouse CCA models (TAZ/Akt and NICD1/Akt) underwent RNA-seq, complemented by multi-omic profiling (transcriptomic, proteomic, metabolomic) in TAZ/Akt livers.ResultsSeveral EpiGs were upregulated in iCCA and eCCA, including writers (DNMT1, EZH2, SUZ12), readers (CBX3, PHF20L1, SMARCA4), and erasers (HDAC1, HDAC3, KDM5C). MGs in OCM, TCA, and ACS pathways were dysregulated (up: GART, IDH2, TYMS; down: ALDH1L1, MAT1A, SHMT1). Integrated analyses identified 27 EpiGs and 8 MGs whose overexpression predicted poor survival. Subsets of EpiGs, MGs, and RLEs were linked to proliferative, high-recurrence iCCA subclasses. CRISPR screens highlighted 50 EpiGs and 23 MGs essential for CCA viability. Tumor microenvironment (TME) analyses revealed distinct immune-stromal subclasses with coherent epigenetic-metabolic signatures. In CCA cells, hypoxia induced epigenetic programs that mirrored those in CCA patients. Transcriptomic analyses in human and multi-omic analyses (transcriptomic, metabolomic, and proteomic studies) in mouse CCA livers highlighted rewiring of nucleotide, one-carbon, lipid, and mitochondrial pathways, with evidence of metabolic-epigenetic crosstalk.ConclusionEpiGs and MGs are markedly altered in both human and experimental CCA, with several changes particularly enriched in aggressive molecular subclasses associated with poor prognosis. We observed substantial rewiring of epigenetic cofactor-related MG expression in CCAs. Functional assays validated new targets among EpiGs (e.g., CBX3, CHD4, DEK, SMARCA4, and TRIM28) and MGs (TYMS and IDH2) in CCA.

BACKGROUND & AIMS:Hepatoblastoma (HB) is the most common malignant liver tumour in children. Despite improved survival in low-risk disease, outcomes for advanced or relapsed HB remain poor, emphasising the need for new therapeutic targets. Hypoxia, a hallmark of aggressive tumours, has recently been implicated in HB pathogenesis, but the molecular mechanisms involved are unclear. This study aimed to characterise the hypoxia-driven transcriptomic landscape of HB and identify key mediators of tumour progression. METHODS:Transcriptomic analyses of HB cell lines cultured under normoxic and hypoxic conditions were combined with bioinformatic interrogation of public HB datasets, immunohistochemistry of human and murine tumours and plasma ELISA assays. Functional roles of trefoil factor 3 (TFF3) were evaluated through overexpression and shRNA-mediated knockdown in vitro and in a β-catenin/YAP-driven mouse model of HB. RESULTS:Hypoxia induced broad transcriptional reprogramming in HB cells, including significant upregulation of TFF3, a secreted oncogenic peptide. TFF3 expression was elevated in HB tissues and plasma, and colocalized with hypoxia marker carbonic anhydrase 9 (CA9). TFF3 promoted proliferation, anchorage-independent growth and cisplatin resistance under both normoxia and hypoxia. Knockdown of murine Tff3 suppressed tumour formation and angiogenesis in vivo. Transcriptomic and molecular analyses revealed that TFF3 sustains C-MYC expression and modulates mTOR/GSK3β signalling. CONCLUSIONS:TFF3 is a hypoxia-inducible factor that enhances HB cell proliferation, survival and chemoresistance. Its tumour-promoting activity through C-MYC and mTOR pathways identifies TFF3 as a potential therapeutic target and circulating biomarker in hepatoblastoma.

Abstract Cholangiocarcinoma (CCA) is a highly aggressive malignancy characterized by poor prognosis, limited therapeutic options, and a predominantly immunosuppressive tumor microenvironment. Protein arginine methyltransferase 1 (PRMT1), the major mediator of asymmetric arginine dimethylation, has been implicated in multiple oncogenic processes, although its role in CCA remains unknown. Here, we demonstrate that PRMT1 is frequently overexpressed in human CCA and is associated with aggressive molecular subtypes and immune-desert tumors. Genetic dependency analyses and pharmacological inhibition using type I PRMT inhibitors markedly impaired CCA cell proliferation, clonogenicity, and tumoroid growth. Transcriptomic profiling revealed that PRMT1 inhibition induces broad alterations in gene expression and alternative splicing, affecting pathways involved in proliferation, apoptosis, DNA damage response, metabolism, and immune signaling. Mechanistically, PRMT1 targeting promoted genomic stress, accumulation of cytosolic double-stranded DNA, and activation of the cGAS-STING-TBK1-IRF3 signaling axis, resulting in enhanced interferon signaling and increased expression of T cell-recruiting chemokines, including CXCL9 and CXCL10. PRMT1 inhibition also synergized with cisplatin, poly-ADP-ribose polymerase (PARP) inhibition, and PRMT5 blockade in vitro and in patient-derived tumoroids. Importantly, in an aggressive orthotopic murine model of intrahepatic CCA, combined treatment with the PRMT1 inhibitor GSK3368715 and anti-PD-1 antibodies significantly reduced tumor burden and increased CD4+ and CD8+ T-cell infiltration compared with monotherapies. Collectively, these findings identify PRMT1 as a critical regulator of CCA growth and immune evasion and support the therapeutic potential of PRMT1 inhibition, particularly in combination with immunotherapy.

Primary biliary cholangitis (PBC) is a chronic autoimmune cholestatic liver disease characterised by immune-mediated injury of small intrahepatic bile ducts, ultimately leading to progressive fibrosis, cirrhosis, and liver-related complications. Although diagnosis is typically established through cholestatic biochemical abnormalities and positivity of anti-mitochondrial antibodies, liver biopsy remains an important diagnostic tool in selected clinical settings, including atypical presentations, suspected overlap syndromes, and assessment of post-transplant liver dysfunction.Traditionally, the histopathology of PBC has been defined by chronic non-suppurative destructive cholangitis, progressive ductopenia, and biliary fibrosis. However, PBC encompasses a broader histopathological spectrum than traditionally recognised. In addition to classical lesions, contemporary liver biopsies may demonstrate distinct phenotypic patterns, including non-destructive ductopenia, inflammatory overlap-like changes, porto-sinusoidal vascular disease-associated lesions, and therapy-modified morphology. Recognition of these manifestations is important for accurate diagnosis, prognostic assessment, and avoidance of common diagnostic pitfalls.This review summarises the expanding histopathological spectrum of PBC in native and transplanted livers, with emphasis on emerging morphological phenotypes, treatment-related changes, recurrent disease after liver transplantation, and contemporary differential diagnostic challenges.

Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by chronic inflammation and progressive fibrosis of the biliary tree, leading to significant liver function impairment over time. There is a strong association with inflammatory bowel diseases (IBD), together representing a distinct and complex medical condition. Patients with PSC-IBD face a heightened risk of various cancers, particularly colorectal carcinoma (CRC) and cholangiocarcinoma (CCA) as the most common types. In this review, we aim to characterize the distinctive features of PSC-IBD-associated carcinomas. Cancer pathogenesis in PSC-IBD is shaped by various factors including dysregulated bile acid metabolism, gut dysbiosis, and unique immune responses. PSC-IBD-associated CRC is often right-sided and warrants vigilant monitoring due to its higher incidence and unique morphological features compared to CRC arising in the terrain of IBD alone. CCA shares substantial genetic similarities with extrahepatic CCA and poses diagnostic challenges since it is frequently detected at advanced stages due to symptom overlap with PSC. Besides, reliable predictive biomarkers for targeted therapy remain largely unexplored. The distinct molecular, genetic, and histopathological profiles of CRC and CCA in PSC-IBD underscore the complexity of these malignancies and highlight the need for continued research to develop precise therapeutic strategies.

The most common primary malignancy of the liver, hepatocellular carcinoma (HCC), is a heterogeneous tumor entity with high metastatic potential and complex pathophysiology. Increasing evidence suggests that tissue mechanics plays a critical role in tumor onset and progression. Here we show that plectin, a major cytoskeletal crosslinker protein, plays a crucial role in mechanical homeostasis and mechanosensitive oncogenic signaling that drives hepatocarcinogenesis. Our expression analyses revealed elevated plectin levels in liver tumors, which correlated with poor prognosis for HCC patients. Using autochthonous and orthotopic mouse models we demonstrated that genetic and pharmacological inactivation of plectin potently suppressed the initiation and growth of HCC. Moreover, plectin targeting potently inhibited the invasion potential of human HCC cells and reduced their metastatic outgrowth in the lung. Proteomic and phosphoproteomic profiling linked plectin-dependent disruption of cytoskeletal networks to attenuation of oncogenic FAK, MAPK/Erk, and PI3K/AKT signatures. Importantly, by combining cell line-based and murine HCC models, we show that plectin inhibitor plecstatin-1 (PST) is well-tolerated and potently inhibits HCC progression. In conclusion, our study demonstrates that plectin-controlled cytoarchitecture is a key determinant of HCC development and suggests that pharmacologically induced disruption of mechanical homeostasis may represent a new therapeutic strategy for HCC treatment.

Accumulation of extracellular matrix (ECM) in liver fibrosis is associated with changes in protein abundance and composition depending upon etiology of the underlying liver disease. Current efforts to unravel etiology-specific mechanisms and pharmacological targets rely on several models of experimental fibrosis. Here, we characterize and compare dynamics of hepatic proteome remodeling during fibrosis development and spontaneous healing in experimental mouse models of hepatotoxic (carbon tetrachloride [CCl 4 ] intoxication) and cholestatic (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] feeding) injury. Using detergent-based tissue extraction and mass spectrometry, we identified compartment-specific changes in the liver proteome with detailed attention to ECM composition and changes in protein solubility. Our analysis revealed distinct time-resolved CCl 4 and DDC signatures, with identified signaling pathways suggesting limited healing and a potential for carcinogenesis associated with cholestasis. Correlation of protein abundance profiles with fibrous deposits revealed extracellular chaperone clusterin with implicated role in fibrosis resolution. Dynamics of clusterin expression was validated in the context of human liver fibrosis. Atomic force microscopy of fibrotic livers complemented proteomics with profiles of disease-associated changes in local liver tissue mechanics. This study determined compartment-specific proteomic landscapes of liver fibrosis and delineated etiology-specific ECM components, providing thus a foundation for future antifibrotic therapies.

PURPOSE:This prospective pilot study aims to evaluate the capabilities of novel quantitative ultrasound (QUS) methods based on attenuation (Att.PLUS) and sound speed (SSp.PLUS) for detecting liver fat. PATIENTS AND METHODS:The study included 56 individuals with biopsy-proven steatosis (percutaneous liver biopsy) ranging from 0 % to 90 % of hepatocytes containing intracellular lipid vacuoles. Histopathology was considered reference standard. Abdominal QUS examinations were conducted using Att.PLUS and SSp.PLUS techniques on the Aixplorer MACH 30 system. Comparative assessments were made using the results of liver biopsy and magnetic resonance spectroscopy (MRS) together with magnetic resonance imaging proton density fat fraction (MRI-PDFF). MR examinations were performed on the Siemens VIDA 3 T system. RESULTS:ROC analysis was conducted for two groups: (a) patients without steatosis (S0) versus those with steatosis (S1 + S2 + S3) yielded AUC values of 0.79 for Att.PLUS and 0.78 for SSp.PLUS, in contrast to an AUC > 0.95 for MRS and MRI-PDFF; and (b) patients without or with mild steatosis (S0 + S1) versus those with severe steatosis (S2 + S3), yielded AUC values of 0.93 for Att.PLUS and 0.89 for SSp.PLUS, in contrast to an AUC > 0.99 for MRS and MRI-PDFF. However, MR methods were superior in detecting liver fat content in obese patients and post-liver transplantation individuals. CONCLUSION:Both QUS parameters (Att.PLUS and SSp.PLUS) appear equivalent at differentiating S0 vs. (S1 + S2 + S3) patients, but the Att.PLUS parameter may be more effective at identifying advanced steatosis (S2 + S3). MR techniques outperformed QUS methods, making them more suitable for clinical studies.