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•Autophagy maintains liver endothelial cell homeostasis.•Autophagy deficiency in LSEC increases oxidative stress.•Autophagy regulates nitric oxide bioavailability and maintains LSEC ...phenotype.•Impairment of endothelial autophagy enhances endothelial dysfunction and exacerbates fibrosis.
Endothelial dysfunction plays an essential role in liver injury, yet the phenotypic regulation of liver sinusoidal endothelial cells (LSECs) remains unknown. Autophagy is an endogenous protective system whose loss could undermine LSEC integrity and phenotype. The aim of our study was to investigate the role of autophagy in the regulation of endothelial dysfunction and the impact of its manipulation during liver injury.
We analyzed primary isolated LSECs from Atg7control and Atg7endo mice as well as rats after CCl4 induced liver injury. Liver tissue and primary isolated stellate cells were used to analyze liver fibrosis. Autophagy flux, microvascular function, nitric oxide bioavailability, cellular superoxide content and the antioxidant response were evaluated in endothelial cells.
Autophagy maintains LSEC homeostasis and is rapidly upregulated during capillarization in vitro and in vivo. Pharmacological and genetic downregulation of endothelial autophagy increases oxidative stress in vitro. During liver injury in vivo, the selective loss of endothelial autophagy leads to cellular dysfunction and reduced intrahepatic nitric oxide. The loss of autophagy also impairs LSECs ability to handle oxidative stress and aggravates fibrosis.
Autophagy contributes to maintaining endothelial phenotype and protecting LSECs from oxidative stress during early phases of liver disease. Selectively potentiating autophagy in LSECs during early stages of liver disease may be an attractive approach to modify the disease course and prevent fibrosis progression.
Liver endothelial cells are the first liver cell type affected after any kind of liver injury. The loss of their unique phenotype during injury amplifies liver damage by orchestrating the response of the liver microenvironment. Autophagy is a mechanism involved in the regulation of this initial response and its manipulation can modify the progression of liver damage.
Cholangiocarcinoma (CCA), a deadly malignancy of the bile ducts, can be classified based on its anatomical location into either intrahepatic (iCCA) or extrahepatic (eCCA), each with different ...pathogenesis and clinical management. There is limited understanding of the molecular landscape of eCCA and no targeted therapy with clinical efficacy has been approved. We aimed to provide a molecular classification of eCCA and identify potential targets for molecular therapies.
An integrative genomic analysis of an international multicenter cohort of 189 eCCA cases was conducted. Genomic analysis included whole-genome expression, targeted DNA-sequencing and immunohistochemistry. Molecular findings were validated in an external set of 181 biliary tract tumors from the ICGC.
KRAS (36.7%), TP53 (34.7%), ARID1A (14%) and SMAD4 (10.7%) were the most prevalent mutations, with ∼25% of tumors having a putative actionable genomic alteration according to OncoKB. Transcriptome-based unsupervised clustering helped us define 4 molecular classes of eCCA. Tumors classified within the Metabolic class (19%) showed a hepatocyte-like phenotype with activation of the transcription factor HNF4A and enrichment in gene signatures related to bile acid metabolism. The Proliferation class (23%), more common in patients with distal CCA, was characterized by enrichment of MYC targets, ERBB2 mutations/amplifications and activation of mTOR signaling. The Mesenchymal class (47%) was defined by signatures of epithelial-mesenchymal transition, aberrant TGFβ signaling and poor overall survival. Finally, tumors in the Immune class (11%) had a higher lymphocyte infiltration, overexpression of PD-1/PD-L1 and molecular features associated with a better response to immune checkpoint inhibitors.
An integrative molecular characterization identified distinct subclasses of eCCA. Genomic traits of each class provide the rationale for exploring patient stratification and novel therapeutic approaches.
Targeted therapies have not been approved for the treatment of extrahepatic cholangiocarcinoma. We performed a multi-platform molecular characterization of this tumor in a cohort of 189 patients. These analyses revealed 4 novel transcriptome-based molecular classes of extrahepatic cholangiocarcinoma and identified ∼25% of tumors with actionable genomic alterations, which has potential prognostic and therapeutic implications.
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•Integrative molecular characterization of extrahepatic cholangiocarcinoma was performed.•Targeted DNA-sequencing and whole-genome expression was conducted.•∼25% of extrahepatic cholangiocarcinoma have a putative actionable genomic alteration.•Four distinct molecular classes of extrahepatic cholangiocarcinoma were identified.
We previously reported a characterisation of the hepatocellular carcinoma (HCC) immune contexture and described an immune-specific class. We now aim to further delineate the immunogenomic ...classification of HCC to incorporate features that explain responses/resistance to immunotherapy.
We performed RNA and whole-exome sequencing, T-cell receptor (TCR)-sequencing, multiplex immunofluorescence and immunohistochemistry in a novel cohort of 240 HCC patients and validated our results in other cohorts comprising 660 patients.
Our integrative analysis led to define: (1) the inflamed class of HCC (37%), which includes the previously reported immune subclass (22%) and a new immune-like subclass (15%) with high interferon signalling, cytolytic activity, expression of immune-effector cytokines and a more diverse T-cell repertoire. A 20-gene signature was able to capture ~90% of these tumours and is associated with response to immunotherapy. Proteins identified in liquid biopsies recapitulated the inflamed class with an area under the ROC curve (AUC) of 0.91; (2) The intermediate class, enriched in
mutations (49% vs 29%, p=0.035), and chromosomal losses involving immune-related genes and; (3) the excluded class, enriched in
mutations (93% vs 27%, p<0.001) and
overexpression due to gene amplification and promoter hypomethylation.
mutations outside the excluded class led to weak activation of the Wnt-βcatenin pathway or occurred in HCCs dominated by high interferon signalling and type I antigen presenting genes.
We have characterised the immunogenomic contexture of HCC and defined inflamed and non-inflamed tumours. Two distinct
patterns associated with a differential role in immune evasion are described. These features may help predict immune response in HCC.
Background and Aims
Lenvatinib is an effective drug in advanced HCC. Its combination with the anti‐PD1 (programmed cell death protein 1) immune checkpoint inhibitor, pembrolizumab, has generated ...encouraging results in phase Ib and is currently being tested in phase III trials. Here, we aimed to explore the molecular and immunomodulatory effects of lenvatinib alone or in combination with anti‐PD1.
Approach and Results
We generated three syngeneic models of HCC in C57BL/6J mice (subcutaneous and orthotopic) and randomized animals to receive placebo, lenvatinib, anti‐PD1, or combination treatment. Flow cytometry, transcriptomic, and immunohistochemistry analyses were performed in tumor and blood samples. A gene signature, capturing molecular features associated with the combination therapy, was used to identify a subset of candidates in a cohort of 228 HCC patients who might respond beyond what is expected for monotherapies. In mice, the combination treatment resulted in tumor regression and shorter time to response compared to monotherapies (P < 0.001). Single‐agent anti‐PD1 induced dendritic and T‐cell infiltrates, and lenvatinib reduced the regulatory T cell (Treg) proportion. However, only the combination treatment significantly inhibited immune suppressive signaling, which was associated with the TGFß pathway and induced an immune‐active microenvironment (P < 0.05 vs. other therapies). Based on immune‐related genomic profiles in human HCC, 22% of patients were identified as potential responders beyond single‐agent therapies, with tumors characterized by Treg cell infiltrates, low inflammatory signaling, and VEGFR pathway activation.
Conclusions
Lenvatinib plus anti‐PD1 exerted unique immunomodulatory effects through activation of immune pathways, reduction of Treg cell infiltrate, and inhibition of TGFß signaling. A gene signature enabled the identification of ~20% of human HCCs that, although nonresponding to single agents, could benefit from the proposed combination.
Sustained virological response (SVR) improves survival in post‐liver transplant (LT) recurrent hepatitis C. However, the impact of SVR on fibrosis regression is not well defined. In addition, the ...performance of noninvasive methods to evaluate the presence of fibrosis and portal hypertension (PH) post‐SVR has been scarcely evaluated. We aimed to investigate the degree of fibrosis regression (decrease ≥1 METAVIR stage) after‐SVR and its associated factors in recurrent hepatitis C, as well as the diagnostic capacity of noninvasive methods in the assessment of liver fibrosis and PH after viral clearance. We evaluated 112 hepatitis C virus–infected LT recipients who achieved SVR between 2001 and 2015. A liver biopsy was performed before treatment and 12 months post‐SVR. Hepatic venous pressure gradient (HVPG), liver stiffness measurement (LSM), and Enhanced Liver Fibrosis (ELF) score were also determined at the same time points. Sixty‐seven percent of the cohort presented fibrosis regression: 43% in recipients with cirrhosis and 72%‐85% in the remaining stages (P = 0.002). HVPG, LSM, and ELF significantly decreased post‐SVR. Liver function significantly improved, and survival was significantly better in patients achieving fibrosis regression. Baseline HVPG and LSM as well as decompensations before therapy were independent predictors of fibrosis regression. One year post‐SVR, LSM had a high diagnostic accuracy to discard the presence of advanced fibrosis (AF) and clinically significant PH (AUROC, 0.902 and 0.888). Conclusion: In conclusion, SVR post‐LT induces fibrosis regression in most patients, leading to significant clinical benefits. Pretreatment HVPG and LSM are significant determinants of the likelihood of fibrosis regression. Finally, LSM accurately predicts the presence of AF and PH 1 year after SVR and thus can be used to determine monitoring strategies. (Hepatology 2018;67:1683‐1694).
The clinical utility of serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC) is widely recognised. However, a clear understanding of the mechanisms of AFP overexpression and ...the molecular traits of patients with AFP-high tumours are not known. We assessed transcriptome data, whole-exome sequencing data and DNA methylome profiling of 520 HCC patients from two independent cohorts to identify distinct molecular traits of patients with AFP-high tumours (serum concentration > 400 ng/ml), which represents an accepted prognostic cut-off and a predictor of response to ramucirumab. Those AFP-high tumours (18% of resected cases) were characterised by significantly lower AFP promoter methylation (p < 0.001), significant enrichment of progenitor-cell features (CK19, EPCAM), higher incidence of BAP1 oncogene mutations (8.5% vs 1.6%) and lower mutational rates of CTNNB1 (14% vs 30%). Specifically, AFP-high tumours displayed significant activation of VEGF signalling (p < 0.001), which might provide the rationale for the reported benefit of ramucirumab in this subgroup of patients.
Targeted NGS allows a fast and efficient multi-gene analysis and the detection of key gene aberrations in melanoma. In this study, we aim to describe the genetic alterations in a series of 87 ...melanoma cases using the oncomine focus assay (OFA), relate these results with the clinicopathological features of the patients, and compare them with our previous study results in which we used a smaller panel, the oncomine solid tumor (OST) DNA kit. Patients diagnosed with advanced melanoma at our center from 2020 to 2022 were included and DNA and RNA were extracted for sequencing. Common mutated genes were BRAF (29%), NRAS (28%), ALK, KIT, and MAP2K1 (5% each). Co-occurring mutations were detected in 29% of the samples, including BRAF with KIT, CTNNB1, EGFR, ALK, HRAS, or MAP2K1. Amplifications and rearrangements were detected in 5% of cases. Only BRAF mutation showed a significant statistical association with sun exposure. For patients with a given genetic profile, the melanoma survival and recurrence-free survival rates were equivalent, but not for stage and LDH values. This expanded knowledge of molecular alterations has helped to more comprehensively characterize our patients and has provided relevant information for deciding the best treatment strategy.
Elafibranor is a dual peroxisome proliferator-activated receptor (PPAR)α and β/δ agonist that has reached a phase III clinical trial for the treatment of metabolic dysfunction-associated steatotic ...liver disease (MASLD). Here, we examined the effects of elafibranor in mice fed a choline-deficient high-fat diet (CD-HFD), a model of metabolic dysfunction-associated steatohepatitis (MASH) that presents obesity and insulin resistance. Our findings revealed that elafibranor treatment ameliorated steatosis, inflammation, and fibrogenesis in the livers of CD-HFD-fed mice. Unexpectedly, elafibranor also increased the levels of the epithelial-mesenchymal transition (EMT)-promoting protein S100A4 via PPARβ/δ activation. The increase in S100A4 protein levels caused by elafibranor was accompanied by changes in the levels of markers associated with the EMT program. The S100A4 induction caused by elafibranor was confirmed in the BRL-3A rat liver cells and a mouse primary hepatocyte culture. Furthermore, elafibranor reduced the levels of ASB2, a protein that promotes S100A4 degradation, while ASB2 overexpression prevented the stimulating effect of elafibranor on S100A4. Collectively, these findings reveal an unexpected hepatic effect of elafibranor on increasing S100A4 and promoting the EMT program.
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•Elafibranor improves steatosis, inflammation, and fibrogenesis in the livers of CD-HFD-fed mice.•Elafibranor upregulates the EMT-promoting protein S100A4 via PPARβ/δ.•Elafibranor increases S100A4 by downregulating the S100A4-degrading E3 ubiquitin ligase ASB2.•ASB2 overexpression prevents the stimulating effect of elafibranor on S100A4.
Xanthogranulomatous cholecystitis(XGC) is an uncommon variant of chronic cholecystitis. The perioperative findings in aggressive cases may be indistinguishable from those of gallbladder or biliary ...tract carcinomas. Three patients presented mass lesions that infiltrated the hepatic hilum,provoked biliary dilatation and jaundice,and were indicative of malignancy. Surgical excision was performed following oncological principles and included extirpation of the gallbladder,extrahepatic bile duct,and hilar lymph nodes,as well as partial hepatectomy. Postoperative morbidity was minimal. Surgical pathology demonstrated XGC and absence of malignancy in all three cases. All three patients are alive and well after years of follow-up. XGC may have such an aggressive presentation that carcinoma may only be ruled out on surgical pathology. In such cases,the best option may be radical resection following oncological principles performed by expert surgeons,in order that postoperative complications may be minimized if not avoided altogether.
Background
Colorectal cancer (CRC) screening programs result in the detection of early-stage asymptomatic carcinomas suitable to be surgically cured. Lymph nodes (LN) from early CRC are usually small ...and may be difficult to collect. Still, at least 12 LNs should be analyzed from colectomies, to ensure a reliable pN0 stage. Presurgical endoscopic tattooing improves LN procurement. In addition, molecular detection of occult LN tumor burden in histologically pN0 CRC patients is associated with a decreased survival rate. We aimed to study the impact of presurgical endoscopic tattooing on the molecular detection of LN tumor burden in early colon neoplasms.
Methods
A prospective cohort study from a CRC screening-based population was performed at a tertiary academic hospital. LNs from colectomies with and without preoperative endoscopic tattooing were assessed by two methods, hematoxylin and eosin (HE), and RT-LAMP, to detect tumor cytokeratin 19 (CK19) mRNA. We compared the amount of tumor burden and LN yields from tattooed and non-tattooed specimens.
Results
HE and RT-LAMP analyses of 936 LNs were performed from 71 colectomies containing early carcinomas and endoscopically unresectable adenomas (8 pT0, 17 pTis, 27 pT1, 19 pT2); 47 out of 71 (66.2 %) were tattooed. Molecular positivity correlated with the presence of tattoo in LN
p
< 0.001; OR 3.1 (95 % CI 1.7–5.5). A significantly higher number of LNs were obtained in tattooed specimens (median 17 LN vs. 14.5 LN;
p
= 0.019).
Conclusions
Endoscopic tattooing enables the analysis of those LNs most prone to harbor tumor cells and improves the number of LN harvested.
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