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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., Competing Interests: Conflict of interest JC consults for Crosscope, KB is Crosscope Chief Technology Officer, JS is Crosscope Chief Executive Officer. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs)., Methods: We used high-dimensional single-cell technologies to characterize the T-cell and myeloid compartments of iCCA tissues, comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the iCCA-specific role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology., Results: We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ Tregs. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, enrichment of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of patients with iCCA., Conclusions: We observed abundant infiltration of hyperactivated CD4+ Tregs in iCCA tumors along with reduced CD8+ T-cell effector functions. Interfering with hyperactivated Tregs should be explored as an approach to enhance antitumor immunity in iCCA., Lay Summary: Immune cells have the potential to slow or halt the progression of tumors. However, some tumors, such as intrahepatic cholangiocarcinoma, are associated with very limited immune responses (and infiltration of cancer-targeting immune cells). Herein, we show that a specific population of regulatory T cells (a type of immune cell that actually suppresses the immune response) are hyperactivated in intrahepatic cholangiocarcinoma. Targeting these cells could enable cancer-targeting immune cells to act more effectively and should be looked at as a potential therapeutic approach to this aggressive cancer type., Competing Interests: Conflict of interest E.L. receives research grants from Bristol Myers Squibb and consulting fees from BD Biosciences. A.L. reports receiving consulting fees from Intercept Pharma, AlfaSigma, Takeda, AbbVie, Gilead, and MSD and travel expenses from Intercept Pharma, AlfaSigma and AbbVie. M.I. participates in advisory boards/consultancies for Gilead Sciences, Roche, Third Rock Ventures, Antios Therapeutics, Amgen, Asher Bio, Allovir, ENYO Pharma. The other authors have no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Despite recent translation of immunotherapies into clinical practice, the immunobiology of hepatocellular carcinoma (HCC), in particular the role and clinical relevance of exhausted and liver-resident T cells remain unclear. We therefore dissected the landscape of exhausted and resident T cell responses in the peripheral blood and tumor microenvironment of patients with HCC., Methods: Lymphocytes were isolated from the blood, tumor and tumor-surrounding liver tissue of patients with HCC (n = 40, n = 10 treated with anti-PD-1 therapy). Phenotype, function and response to anti-PD-1 were analyzed by mass and flow cytometry ex vivo and in vitro, tissue residence was further assessed by immunohistochemistry and imaging mass cytometry. Gene signatures were analyzed in silico., Results: We identified significant enrichment of heterogeneous populations of exhausted CD8+ T cells (TEX) in the tumor microenvironment. Strong enrichment of severely exhausted CD8 T cells expressing multiple immune checkpoints in addition to PD-1 was linked to poor progression-free and overall survival. In contrast, PD-1 was also expressed on a subset of more functional and metabolically active CD103+ tissue-resident memory T cells (TRM) that expressed few additional immune checkpoints and were associated with better survival. TEX enrichment was independent of BCLC stage, alpha-fetoprotein levels or age as a variable for progression-free survival in our cohort. These findings were in line with in silico gene signature analysis of HCC tumor transcriptomes from The Cancer Genome Atlas. A higher baseline TRM/TEX ratio was associated with disease control in anti-PD-1-treated patients., Conclusion: Our data provide information on the role of peripheral and intratumoral TEX-TRM dynamics in determining outcomes in patients with HCC. The dynamics between exhausted and liver-resident T cells have implications for immune-based diagnostics, rational patient selection and monitoring during HCC immunotherapies., Lay Summary: The role of the immune response in hepatocellular carcinoma (HCC) remains unclear. T cells can mediate protection against tumor cells but are frequently dysfunctional and exhausted in cancer. We found that patients with a predominance of exhausted CD8+ T cells (TEX) had poor survival compared to patients with a predominance of tissue-resident memory T cells (TRM). This correlated with the molecular profile, metabolic and functional status of these cell populations. The enrichment of TEX was independently associated with prognosis in addition to disease stage, age and tumor markers. A high TRM proportion was also associated with better outcomes following checkpoint therapy. Thus, these T-cell populations are novel biomarkers with relevance in HCC., Competing Interests: Conflict of interest The authors declare no conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA., Methods: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters., Results: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence., Conclusions: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features., Lay Summary: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma., Competing Interests: Conflicts of interest The authors have no conflicts of interest to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche., Methods: CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14 + with CCA-sphere conditioned medium., Results: CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14 + macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163 + set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect., Conclusion: CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease., Lay Summary: Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Human hepatocarcinogenesis in cirrhosis is thought to be multistep and characterized by a spectrum of nodular lesions, ranging from low to high grade dysplastic nodules (LGDN and HGDN) to early and progressed hepatocellular carcinoma (eHCC and pHCC). The aim of this study was to investigate the morphophenotypical changes of this sequence and their potential translational significance., Methods: We scored the vascular profile, ductular reaction/stromal invasion and overexpression of five biomarkers (GPC3, HSP70, GS, CHC, and EZH2), in a series of 100 resected nodules (13 LGDN, 16 HGDN, 42 eHCC and 29 small pHCC)., Results: The score separated the four groups of nodules as individual entities (p<0.01). In the sequence, biomarker's overexpression progressively increased with parallel decrease of ductular reaction; the vascular remodeling started very early (LGDN) but did not further develop in a proportion of HCC. eHCC was the most heterogeneous entity, with marginal overlap with HGDN and pHCC. Liver environment (fibrosis, etiology) did not impact on the phenotype of the different nodules. A subclass of eHCC (16/42) without evidence of stromal invasion was identified, suggesting a "preinvasive stage" (p<0.05). For diagnosis, the application of four and five biomarkers (rather than the usual three) improved the sensitivity of the assay for the detection of eHCC (76% and 93% vs. 52%); biomarkers in alternative combinations, and also increased the sensitivity of the assay (GS+CHC+EZH2: 76%; GS+CHC+EZH2+HSP70: 90%)., Conclusions: This study supports the multistep nature of human hepatocarcinogenesis, and suggests that eHCC is more heterogeneous than previously thought. This provides further information of the potential translational significance into clinical practice., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: The incidence of metabolic syndrome-related hepatocellular carcinoma (MS-HCC) is increasing worldwide. High resection risks are anticipated because of underlying steatohepatitis, but long-term results are unknown. To clarify the outcomes following liver resection in patients with MS-HCC and to compare the outcomes of MS-HCC to HCV-related HCC (HCV-HCC)., Methods: All the consecutive patients undergoing liver resection for HCC in six high-volume HPB units between 2000 and 2012 were retrospectively considered. The patients with MS-HCC were identified and matched one-to-one with HCV-HCC patients without metabolic syndrome. Matching was based on age, cirrhosis, Child-Pugh class, portal hypertension, HCC number and diameter and liver resection extension., Results: Among 1563 patients undergoing liver resection for HCC in the study period, 96 (6.1%) had MS-HCC. They were matched with 96 HCV-HCC patients. All patients were Child-Pugh class A, 22.9% had cirrhosis. Forty-one patients per group (42.7%) required major hepatectomy. The MS-HCC group had a higher prevalence of steatohepatitis (25.0% vs. 9.4%, p=0.004). Operative mortality was 2.1% (1 MS-HCC, 3 HCV-HCC, p=0.621). Morbidity and liver failure rates were similar between the two groups. In the multivariate analysis, cirrhosis, major hepatectomy, and MELD >8, but not steatohepatitis, impacted severe morbidity and liver failure rates. The MS-HCC group had better 5-year overall survival (65.6% vs. 61.4%, p=0.031) and recurrence-free survival (37.0% vs. 27.5%, p=0.077). Independent negative prognostic factors were HCV-HCC, multiple HCC, microvascular invasion, and satellite nodules., Conclusions: Liver resection is safe for MS-HCC, as for HCV-HCC. Cirrhosis, but not steatohepatitis, affects short-term outcomes. MS-HCC is associated with excellent long-term outcomes, better than HCV-HCC., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Although the growth suppressing Hippo pathway has been implicated in hepatocellular carcinoma (HCC) pathogenesis, it is unknown at which stage of hepatocarcinogenesis its dysregulation occurs. We investigated in rat and human preneoplastic lesions whether overexpression of the transcriptional co-activator Yes-associated protein (YAP) is an early event., Methods: The experimental model used is the resistant-hepatocyte (R-H) rat model. Gene expression was determined by qRT-PCR or immunohistochemistry. Forward genetic experiments were performed in human HCC cells and in murine oval cells., Results: All foci of preneoplastic hepatocytes, generated in rats 4weeks after diethylnitrosamine (DENA) treatment, displayed YAP accumulation. This was associated with down-regulation of the β-TRCP ligase, known to mediate YAP degradation, and of microRNA-375, targeting YAP. YAP accumulation was paralleled by the up-regulation of its target genes. Increased YAP expression was also observed in human early dysplastic nodules and adenomas. Animal treatment with verteporfin (VP), which disrupts the formation of the YAP-TEAD complex, significantly reduced preneoplastic foci and oval cell proliferation. In vitro experiments confirmed that VP-mediated YAP inhibition impaired cell growth in HCC and oval cells; notably, oval cell transduction with wild type or active YAP conferred tumorigenic properties in vitro and in vivo., Conclusions: These results suggest that (i) YAP overexpression is an early event in rat and human liver tumourigenesis; (ii) it is critical for the clonal expansion of carcinogen-initiated hepatocytes and oval cells, and (iii) VP-induced disruption of the YAP-TEAD interaction may provide an important approach for the treatment of YAP-overexpressing cancers., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background/aims: Liver biopsy for hepatocellular carcinoma (HCC) detection is largely restricted to small hepatocellular lesions, which are often morphologically challenging, requiring careful distinction between dysplastic nodules (high-grade) and well-differentiated HCC., Methods: We investigated the diagnostic accuracy of a panel of markers (HSP70 GPC3 and GS), previously tested in resection specimens, in a series of liver biopsies of large regenerative nodules (n=13), low-grade dysplastic nodules (n=21), high-grade dysplastic nodules (n=50), very well-differentiated (VWD) (n=17), well-differentiated (WD-G1) (n=40) and G2-3 (n=35) HCC., Results: Almost all cases of large regenerative and low-grade dysplastic nodules did not stain while high-grade dysplastic nodules showed 1 marker (22%) but never 2 or 3. For HCC detection the overall accuracy of marker combination was 60.8% (3 markers) and 78.4% (2 markers) with 100% specificity. When restricted to VWD+WD-G1 HCC the accuracy was 57% (3 markers) and 72.9% (2 markers) with 100% specificity., Conclusions: This panel proved useful to detect well-differentiated HCC in biopsy. Two immunoreactive markers (out of 3) are recommended as the most valuable diagnostic combination for HCC detection. The diagnostic accuracy of the panel could be improved using additional markers, as suggested by studies of expression profiling in other human models.