Role of the fatty pancreatic infiltration in pancreatic oncogenesis.

Although pancreatic precancerous lesions are known to be related to obesity and fatty pancreatic infiltration, the mechanisms remain unclear. We assessed the role of fatty infiltration in the process of pancreatic oncogenesis and obesity. A combined transcriptomic, lipidomic and pathological approach was used to explore neoplastic transformations. Intralobular (ILF) and extralobular (ELF) lipidomic profiles were analyzed to search for lipids associated with pancreatic intraepithelial neoplasia (PanINs) and obesity; the effect of ILF and ELF on acinar tissue and the histopathological aspects of pancreatic parenchyma changes in obese (OB) and non-obese patients. This study showed that the lipid composition of ILF was different from that of ELF. ILF was related to obesity and ELF-specific lipids were correlated to PanINs. Acinar cells were shown to have different phenotypes depending on the presence and proximity to ILF in OB patients. Several lipid metabolic pathways, oxidative stress and inflammatory pathways were upregulated in acinar tissue during ILF infiltration in OB patients. Early acinar transformations, called acinar nodules (AN) were linked to obesity but not ELF or ILF suggesting that they are the first reversible precancerous pancreatic lesions to occur in OB patients. On the other hand, the number of PanINs was higher in OB patients and was positively correlated to ILF and ELF scores as well as to fibrosis. Our study suggests that two types of fat infiltration must be distinguished, ELF and ILF. ILF plays a major role in acinar modifications and the development of precancerous lesions associated with obesity, while ELF may play a role in the progression of PDAC.

Primary liver cancer classification from routine tumour biopsy using weakly supervised deep learning.

Background & Aims: The diagnosis of primary liver cancers (PLCs) can be challenging, especially on biopsies and for combined hepatocellular-cholangiocarcinoma (cHCC-CCA). We automatically classified PLCs on routine-stained biopsies using a weakly supervised learning method. Method: We selected 166 PLC biopsies divided into training, internal and external validation sets: 90, 29 and 47 samples, respectively. Two liver pathologists reviewed each whole-slide hematein eosin saffron (HES)-stained image (WSI). After annotating the tumour/non-tumour areas, tiles of 256x256 pixels were extracted from the WSIs and used to train a ResNet18 neural network. The tumour/non-tumour annotations served as labels during training, and the network's last convolutional layer was used to extract new tumour tile features. Without knowledge of the precise labels of the malignancies, we then applied an unsupervised clustering algorithm. Results: Pathological review classified the training and validation sets into hepatocellular carcinoma (HCC, 33/90, 11/29 and 26/47), intrahepatic cholangiocarcinoma (iCCA, 28/90, 9/29 and 15/47), and cHCC-CCA (29/90, 9/29 and 6/47). In the two-cluster model, Clusters 0 and 1 contained mainly HCC and iCCA histological features. The diagnostic agreement between the pathological diagnosis and the two-cluster model predictions (major contingent) in the internal and external validation sets was 100% (11/11) and 96% (25/26) for HCC and 78% (7/9) and 87% (13/15) for iCCA, respectively. For cHCC-CCA, we observed a highly variable proportion of tiles from each cluster (cluster 0: 5-97%; cluster 1: 2-94%). Conclusion: Our method applied to PLC HES biopsy could identify specific morphological features of HCC and iCCA. Although no specific features of cHCC-CCA were recognized, assessing the proportion of HCC and iCCA tiles within a slide could facilitate the identification of cHCC-CCA. Impact and implications: The diagnosis of primary liver cancers can be challenging, especially on biopsies and for combined hepatocellular-cholangiocarcinoma (cHCC-CCA). We automatically classified primary liver cancers on routine-stained biopsies using a weakly supervised learning method. Our model identified specific features of hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Despite no specific features of cHCC-CCA being recognized, the identification of hepatocellular carcinoma and intrahepatic cholangiocarcinoma tiles within a slide could facilitate the diagnosis of primary liver cancers, and particularly cHCC-CCA.

A new NRF2 activator for the treatment of human metabolic dysfunction-associated fatty liver disease.

Background & Aims: Oxidative stress triggers metabolic-associated fatty liver disease (MAFLD) and fibrosis. Previous animal studies demonstrated that the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2), the master regulator of antioxidant response, protects against MAFLD and fibrosis. S217879, a next generation NRF2 activator has been recently shown to trigger diet-induced steatohepatitis resolution and to reduce established fibrosis in rodents. Our aim was to evaluate the therapeutic potential of S217879 in human MAFLD and its underlying mechanisms using the relevant experimental 3D model of patient-derived precision cut liver slices (PCLS). Methods: We treated PCLS from 12 patients with varying stages of MAFLD with S217879 or elafibranor (peroxisome proliferator-activated receptor [PPAR]α/δ agonist used as a referent molecule) for 2 days. Safety and efficacy profiles, steatosis, liver injury, inflammation, and fibrosis were assessed as well as mechanisms involved in MAFLD pathophysiology, namely antioxidant response, autophagy, and endoplasmic reticulum-stress. Results: Neither elafibranor nor S217879 had toxic effects at the tested concentrations on human PCLS with MAFLD. PPARα/δ and NRF2 target genes (pyruvate dehydrogenase kinase 4 [PDK4], fibroblast growth factor 21 [FGF21], and NAD(P)H quinone dehydrogenase 1 [NQO1], heme oxygenase 1 [HMOX1], respectively) were strongly upregulated in PCLS in response to elafibranor and S217879, respectively. Compared with untreated PCLS, elafibranor and S217879-treated slices displayed lower triglycerides and reduced inflammation (IL-1ß, IL-6, chemokine (C-C motif) ligand 2 [CCL2]). Additional inflammatory markers (chemokine (C-C motif) ligand 5 [CCL5], stimulator of interferon genes [STING], intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) were downregulated by S217879. S217879 but not elafibranor lowered DNA damage (phospho-Histone H2A.X [p-H2A.X], RAD51, X-ray repair cross complementing 1 [XRCC1]) and apoptosis (cleaved caspase-3), and inhibited fibrogenesis markers expression (alpha smooth muscle actin [α-SMA], collagen 1 alpha 1 [COL1A1], collagen 1 alpha 2 [COL1A2]). Such effects were mediated through an improvement of lipid metabolism, activated antioxidant response and enhanced autophagy, without effect on endoplasmic reticulum-stress. Conclusions: This study highlights the therapeutic potential of a new NRF2 activator for MAFLD using patient-derived PCLS supporting the evaluation of NRF2 activating strategies in clinical trials. Impact and implications: Oxidative stress is a major driver of metabolic-associated fatty liver disease (MAFLD) development and progression. Nuclear factor (erythroid-derived 2)-like 2, the master regulator of the antioxidative stress response, is an attractive therapeutic target for the treatment of MAFLD. This study demonstrates that S217879, a new potent and selective nuclear factor (erythroid-derived 2)-like 2 activator, displays antisteatotic effects, lowers DNA damage, apoptosis, and inflammation and inhibits fibrogenesis in human PCLS in patients with MAFLD.

Endothelial autophagy is not required for liver regeneration after partial hepatectomy in mice with fatty liver.

BACKGROUND & AIMS: Patients with non-alcoholic fatty liver disease (NAFLD) have impaired liver regeneration. Liver endothelial cells play a key role in liver regeneration. In non-alcoholic steatohepatitis (NASH), liver endothelial cells display a defect in autophagy, contributing to NASH progression. We aimed to determine the role of endothelial autophagy in liver regeneration following liver resection in NAFLD. METHODS: First, we assessed autophagy in primary endothelial cells from wild type mice fed a high fat diet and subjected to partial hepatectomy. Then, we assessed liver regeneration after partial hepatectomy in mice deficient (Atg5lox/lox ;VE-cadherin-Cre+ ) or not (Atg5lox/lox ) in endothelial autophagy and fed a high fat diet. The role of endothelial autophagy in liver regeneration was also assessed in ApoE-/- hypercholesterolemic mice and in mice with NASH induced by methionine- and choline-deficient diet. RESULTS: First, autophagy (LC3II/protein) was strongly increased in liver endothelial cells following hepatectomy. Then, we observed at 40 and 48 h and at 7 days after partial hepatectomy, that Atg5lox/lox ;VE-cadherin-Cre+ mice fed a high fat diet had similar liver weight, plasma AST, ALT and albumin concentration, and liver protein expression of proliferation (PCNA), cell-cycle (Cyclin D1, BrdU incorporation, phospho-Histone H3) and apoptosis markers (cleaved Caspase-3) as Atg5lox/lox mice fed a high fat diet. Same results were obtained in ApoE-/- and methionine- and choline-deficient diet fed mice, 40 h after hepatectomy. CONCLUSION: These results demonstrate that the defect in endothelial autophagy occurring in NASH does not account for the impaired liver regeneration occurring in this setting.

Pathological overview of steatohepatitic hepatocellular carcinoma in a surgical series.

AIMS: According to the last WHO classification, steatohepatitic hepatocellular carcinoma (SH-HCC) is recognized as a distinct HCC subtype, even though a consensual definition is still lacking. The objectives of the study were to carefully describe the morphological features of SH-HCC and evaluate its impact on prognosis. METHODS AND RESULTS: We conducted a single-centre retrospective study including 297 surgically resected HCC. Pathological features including SH criteria (steatosis, ballooning, Mallory-Denk bodies, fibrosis, and inflammation) were assessed. SH-HCC was defined by the presence of at least four of the five SH criteria and the SH component represented >50% of the tumour area. According to this definition, 39 (13%) HCC cases corresponded to SH-HCC and 30 cases (10%) corresponded to HCC with an SH component (<50%). SH criteria in SH-HCC and non-SH-HCC were distributed as follows: ballooning (100% versus 11%), fibrosis (100% versus 81%), inflammation (100% versus 67%), steatosis (92% versus 8%), and Mallory-Denk bodies (74% versus 3%). Inflammation markers (c-reactive protein [CRP] and serum amyloid A [SAA]) were significantly more expressed in SH-HCC compared to non-SH-HCC (82% versus 14%, P = <0.001). Five-year recurrence-free survival (RFS) and 5-year overall survival (OS) were similar for SH-HCC and non-SH-HCC (P = 0.413 and P = 0.866, respectively). The percentage of SH component does not impact OS and RFS. CONCLUSION: We confirm in a large cohort the relatively high prevalence (13%) of SH-HCC. Ballooning is the most specific criteria for this subtype. The percentage of the SH component does not impact prognosis.

MALDI Imaging, a Powerful Multiplex Approach to Decipher Intratumoral Heterogeneity: Combined Hepato-Cholangiocarcinomas as Proof of Concept.

Combined hepato-cholangiocarcinomas (cHCC-CCA) belong to the spectrum of primary liver carcinomas, which include hepatocellular carcinomas (HCC) and intrahepatic cholangiocarcinomas (iCCA) at both ends of the spectrum. Mainly due to the high intratumor heterogeneity of cHCC-CCA, its diagnosis and pathological description remain challenging. Taking advantage of in situ non-targeted molecular mapping provided by MALDI (Matrix Assisted Laser Desorption Ionization) imaging, we sought to develop a multiscale and multiparametric morphological approach, integrating molecular and conventional pathological analysis. MALDI imaging was applied to five representative cases of resected cHCC-CCA. Principal component analysis and segmentations with MALDI imaging techniques identified areas related to either iCCA or HCC and also hidden tumor areas not visible microscopically. In addition, the overlap between MALDI segmentation and immunostaining provided a comprehensive description of cHCC-CCA tumor heterogeneity by identifying transitional and micro-metastatic areas. Moreover, a list of peptides derived from in silico digestion was obtained for each immunohistochemical marker and was matched within the peptide peak list acquired by MALDI. Comparison of immunostaining images with ions from in silico digestion revealed an accurate identification of iCCA and HCC areas. Our study provides further evidence on the performance of MALDI imaging in exploring intratumor heterogeneity and offering virtual multiplex immunostaining through a single acquisition.

The Appendix Orchestrates T-Cell Mediated Immunosurveillance in Colitis-Associated Cancer.

BACKGROUND & AIMS: Although appendectomy may reduce colorectal inflammation in patients with ulcerative colitis (UC), this surgical procedure has been suggested to be associated with an increased risk of colitis-associated cancer (CAC). Our aim was to explore the mechanism underlying the appendectomy-associated increased risk of CAC. METHODS: Five-week-old male BALB/c mice underwent appendectomy, appendicitis induction, or sham laparotomy. They were then exposed to azoxymethane/dextran sodium sulfate (AOM/DSS) to induce CAC. Mice were killed 12 weeks later, and colons were taken for pathological analysis and immunohistochemistry (CD3 and CD8 staining). Human colonic tumors from 21 patients with UC who underwent surgical resection for CAC were immunophenotyped and stratified according to appendectomy status. RESULTS: Whereas appendectomy significantly reduced colitis severity and increased CAC number, appendicitis induction without appendectomy led to opposite results. Intratumor CD3+ and CD8+ T-cell densities were lower after appendectomy and higher after appendicitis induction compared with the sham laparotomy group. Blocking lymphocyte trafficking to the colon with the anti-α4ß7 integrin antibody or a sphingosine-1-phosphate receptor agonist suppressed the inducing effect of the appendectomy on tumors' number and on CD3+/CD8+ intratumoral density. CD8+ or CD3+ T cells isolated from inflammatory neo-appendix and intravenously injected into AOM/DSS-treated recipient mice increased CD3+/CD8+ T-cell tumor infiltration and decreased tumor number. In UC patients with a history of appendectomy, intratumor CD3+ and CD8+ T-cell densities were decreased compared with UC patients without history of appendectomy. CONCLUSIONS: In UC, appendectomy could suppress a major site of T-cell priming, resulting in a less efficient CAC immunosurveillance.

Arterial Calcifications in Patients with Liver Cirrhosis Are Linked to Hepatic Deficiency of Pyrophosphate Production Restored by Liver Transplantation.

Liver fibrosis is associated with arterial calcification (AC). Since the liver is a source of inorganic pyrophosphate (PPi), an anti-calcifying compound, we investigated the relationship between plasma PPi ([PPi]pl), liver fibrosis, liver function, AC, and the hepatic expression of genes regulating PPi homeostasis. To that aim, we compared [PPi]pl before liver transplantation (LT) and 3 months after LT. We also assessed the expression of four key regulators of PPi in liver tissues and established correlations between AC, and scores of liver fibrosis and liver failure in these patients. LT candidates with various liver diseases were included. AC scores were assessed in coronary arteries, abdominal aorta, and aortic valves. Liver fibrosis was evaluated on liver biopsies and from non-invasive tests (FIB-4 and APRI scores). Liver functions were assessed by measuring serum albumin, ALBI, MELD, and Pugh−Child scores. An enzymatic assay was used to dose [PPi]pl. A group of patients without liver alterations from a previous cohort provided a control group. Gene expression assays were performed with mRNA extracted from liver biopsies and compared between LT recipients and the control individuals. [PPi]pl negatively correlated with APRI (r = −0.57, p = 0.001, n = 29) and FIB-4 (r = −0.47, p = 0.006, n = 29) but not with interstitial fibrosis index from liver biopsies (r = 0.07, p = 0.40, n = 16). Serum albumin positively correlated with [PPi]pl (r = 0.71; p < 0.0001, n = 20). ALBI, MELD, and Pugh−Child scores correlated negatively with [PPi]pl (r = −0.60, p = 0.0005; r = −0.56, p = 0.002; r = −0.41, p = 0.02, respectively, with n = 20). Liver fibrosis assessed on liver biopsies by FIB-4 and by APRI positively correlated with coronary AC (r = 0.51, p = 0.02, n = 16; r = 0.58, p = 0.009, n = 20; r = 0.41, p = 0.04, n = 20, respectively) and with abdominal aorta AC (r = 0.50, p = 0.02, n = 16; r = 0.67, p = 0.002, n = 20; r = 0.61, p = 0.04, n = 20, respectively). FIB-4 also positively correlated with aortic valve calcification (r = 0.40, p = 0.046, n = 20). The key regulator genes of PPi production in liver were lower in patients undergoing liver transplantation as compared to controls. Three months after surgery, serum albumin levels were restored to physiological levels (40 [37−44] vs. 35 [30−40], p = 0.009) and [PPi]pl was normalized (1.40 [1.07−1.86] vs. 0.68 [0.53−0.80] µmol/L, p = 0.0005, n = 12). Liver failure and/or fibrosis correlated with AC in several arterial beds and were associated with low plasma PPi and dysregulation of key proteins involved in PPi homeostasis. Liver transplantation normalized these parameters.

Prognostic value of desmoplastic stroma in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinomas (iCCs) are primary tumors of the liver characterized by the presence of a desmoplastic stroma. While tumor stroma may have a protective or a pejorative value depending on the type of malignant disease, the precise role of the desmoplastic stroma in iCC remains poorly understood. The aim of the present study was to evaluate the prognostic value of stromal compartment in iCC through a multiparametric morphological analysis. Forty-nine surgically resected iCCs were included. For all cases, tumor paraffin blocks of iCCs were selected for stromal morphological characterization through quantitative and qualitative approaches using immunohistochemistry and second-harmonic generation imaging. Intratumor heterogeneity was also evaluated in regards with the different stromal features. High proportionated stromal area (PSA) (defined by stromal to tumor area ratio) was inversely correlated with vascular invasion (62.5% vs 95.7%, p = 0.006) and positively correlated with well-differentiated grade (60% vs 12.5%, p = 0.001). Patients with high PSA had a better disease-free survival (DFS) than patients with low stromal area (60% vs 10%, p = 0.077). Low activated stroma index (defined by cancer-associated fibroblasts number to stromal area ratio) was associated with a better DFS (60% vs 10%, p = 0.05). High collagen reticulation index (CRI), defined as the number of collagen fiber branches within the entire length of the collagen network, was associated with a poorer overall survival (42% vs NR, p = 0.026). Furthermore, we showed that CRI was also an homogeneous marker throughout the tumor. Based on morphological features, desmoplastic stroma seems to exert a protective effect in patients with iCC. Stromal collagen reticulation may provide additional clinically relevant information. In addition, these data support the potential value to evaluate CRI in biopsy specimen.

Transarterial chemoembolisation enhances programmed death-1 and programmed death-ligand 1 expression in hepatocellular carcinoma.

AIMS: Immunotherapies represent a new alternative therapeutic approach for hepatocellular carcinomas (HCCs), and have shown promising results when used in combination therapy. The aim of this study was to evaluate the potential of transarterial chemoembolisation (TACE) to modulate programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) expression profiles in a cohort of surgically treated HCCs. METHODS AND RESULTS: A total of 82 surgically treated HCCs from patients who had undergone (n = 32) or not undergone (n = 50) preoperative TACE were included in the study. Immunohistochemical expression of PD-1 and of PD-L1 were analysed and compared according to TACE treatment. Pretreatment biopsies, which were available for 30 cases (20 with TACE and 10 without), were similarly analysed. Follow-up data were retrieved from patients' charts. PD-1 expression (≥1%) in intratumoral inflammatory cells (ICs) was observed in 46% of HCCs, and PD-L1 expression (≥1%) in ICs and PD-L1 expression in tumour cells (TCs) were observed in 46% and 16% of HCCs, respectively. A low level of PD-1 expression (<1%) was associated with strong and diffuse glutamine synthetase overexpression (8% versus 27%, P = 0.024). HCCs from patients with TACE pretreatment showed significantly higher PD-L1 expression in TCs than those from patients without TACE pretreatment (2% versus 0.4%, P = 0.027). PD-1 expression in ICs and PD-L1 expression in both ICs and TCs were higher in TACE-resected tumours than in corresponding pre-TACE biopsies (respectively: 1.8% versus 8.1%, P = 0.034; 0.8% versus 7.1%, P = 0.032; and 0% versus 2.4%, P = 0.043). CONCLUSION: Our results, showing increases in PD-1 expression and PD-L1 expression in HCCs following TACE, support the use of TACE in combination with immunotherapy in selected cases to optimise tumour response.

The histone acetyltransferase hMOF promotes vascular invasion in hepatocellular carcinoma.

BACKGROUND & AIMS: Vascular invasion is a major prognostic factor in hepatocellular carcinoma (HCC). We previously identified histone H4 acetylated at lysine 16 (H4K16ac), a histone modification involved in transcription activation, as a biomarker of microvascular invasion (mVI) in HCC. This study aimed to investigate the role of hMOF, the histone acetyltransferase responsible for H4K16 acetylation, in the process of vascular invasion in HCC. METHODS: hMOF expression was assessed by RT-qPCR and immunohistochemistry in a retrospective series of HCC surgical samples, and correlated with the presence of mVI. The functional role of hMOF in HCC vascular invasion was investigated in vitro in HCC cell lines using siRNA, transcriptomic analysis and transwell invasion assay, and in vivo using a Zebrafish embryo xenograft model. RESULTS: We found that hMOF was significantly upregulated at the protein level in HCC with mVI, compared with HCC without mVI (P < .01). Transcriptomic analysis showed that hMOF downregulation in HCC cell line lead to significant downregulation of key genes and pathways involved in vascular invasion. These results were confirmed by transwell invasion assay, where hMOF downregulation significantly reduced HCC cells invasion. Finally, hMOF downregulation significantly reduced tumour cell intravasation and metastasis in vivo. CONCLUSIONS: Altogether, these results underpin a critical role for hMOF in vascular invasion in HCC, via transcription activation of key genes involved in this process. These data confirm the major role of epigenetic alterations in HCC progression, and pave the way for future therapies targeting hMOF in HCC.

Biological response under treatment and prognostic value of protein induced by vitamin K absence or antagonist-II in a French cohort of patients with hepatocellular carcinoma.

OBJECTIVES: We have confirmed the diagnostic value of protein induced by vitamin K absence or antagonist-II (PIVKA-II) in a French cohort of patients with hepatocellular carcinoma (HCC). Herein, we aim to study the biological response under treatment and the prognostic value of PIVKA-II serum level in patients treated for HCC. METHODS: Patients with primary HCC developed chronic liver disease with serum PIVKA-II, and alpha-fetoprotein (AFP) levels available at baseline and after first HCC treatment [within 3 months (M1-M3) and/or within 6-9 months (M6-M9)] were included. RESULTS: A total of 94 patients were included. Median follow-up was 23 months (range 11-31 months). PIVKA-II levels significantly decreased from baseline to M1-M3 (P = 0.002) and to M6-M9 (P = 0.035). By multivariate analysis, biological response (M1-M3/baseline PIVKA-II ratio) independently and significantly predicted overall survival (OS). A ratio below 0.73 was able to identify patients with the better prognosis in the total population [OS: 27 months (range 17-31) vs. 17 (range 9-25); P = 0.008] and in patients who had transarterial chemoembolization or selective internal radiation therapy as first treatment approach [OS: 26 months (range 14-31) vs. 16 (range 9-25); P = 0.002 and 2-year OS of 73% vs. 30%; P = 0.009]. PIVKA-II serum levels at baseline and PIVKA-II biological response were significantly associated with radiological response. CONCLUSION: PIVKA-II serum level seems to be a good prognostic and promising biomarker for early monitoring treatment outcomes for patients with HCC.

Polyploidy spectrum: a new marker in HCC classification.

OBJECTIVES: Polyploidy is a fascinating characteristic of liver parenchyma. Hepatocyte polyploidy depends on the DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Which role can be assigned to polyploidy during human hepatocellular carcinoma (HCC) development is still an open question. Here, we investigated whether a specific ploidy spectrum is associated with clinical and molecular features of HCC. DESIGN: Ploidy spectra were determined on surgically resected tissues from patients with HCC as well as healthy control tissues. To define ploidy profiles, a quantitative and qualitative in situ imaging approach was used on paraffin tissue liver sections. RESULTS: We first demonstrated that polyploid hepatocytes are the major components of human liver parenchyma, polyploidy being mainly cellular (binuclear hepatocytes). Across liver lobules, polyploid hepatocytes do not exhibit a specific zonation pattern. During liver tumorigenesis, cellular ploidy is drastically reduced; binuclear polyploid hepatocytes are barely present in HCC tumours. Remarkably, nuclear ploidy is specifically amplified in HCC tumours. In fact, nuclear ploidy is amplified in HCCs harbouring a low degree of differentiation and TP53 mutations. Finally, our results demonstrated that highly polyploid tumours are associated with a poor prognosis. CONCLUSIONS: Our results underline the importance of quantification of cellular and nuclear ploidy spectra during HCC tumorigenesis.

Variation of fetuin-A in maternal and fetal serum during human parturition.

BACKGROUND: parturition involves multiple signalling pathways and most advances in research underline the importance of fetal development and maturation in the timing of labour, especially the fetal pituitary-adrenal axis. But, there is currently no consensual hypothesis on all the physiological processes responsible for human parturition. METHODS: sixty low-risk pregnant women were enrolled in a prospective cohort. Maternal blood was sampled regularly during consultations each week in last trimester, at delivery and at postnatal consultation. Cord blood was collected at delivery. We used proteomic analysis to identify maternal blood biomarkers of potential interest, focusing on serum proteins from 39 W G in pregnancies to delivery and postpartum. RESULTS: among 56 peaks we identified variation in the N-terminal part of fetuin-A in maternal serum. Fetuin-A is a natural antagonist of TGF-beta and is able to bind calcium. We found a significant decrease in maternal serum fetuin-A in the days preceding delivery, independently of the mode of delivery. Also, there does not appear to be significant influence of the different fetal parameters (sex, Z-score) on maternal serum variations at delivery. CONCLUSION: fetuin-A is described by the literature as a potential biomarker for organ dysfunction and metabolic syndrome disorders. The protein mineral homeostasis would be an interesting pathway to explore during pregnancy and particularly parturition.

Proteomic Landscape of Cholangiocarcinomas Reveals Three Different Subgroups According to Their Localization and the Aspect of Non-Tumor Liver.

PURPOSE: Cholangiocarcinomas (CCs) define a heterogeneous entity based upon their anatomic localization (intra versus extrahepatic) and, for the intrahepatic CCs, the aspect of background liver (normal versus cirrhosis). The aim of the study was to characterize the molecular heterogeneity of CCs by a global proteomic approach. EXPERIMENTAL DESIGN: Thirty-three tumor samples from 17 intrahepatic CCs (iCC) (9 developed on normal (iCCN ) and 8 developed in cirrhotic liver (iCCC )); 5 hilar CCs (CCH ); 5 pancreatic CCs (CCP ) and 6 hepatocellular carcinomas (HCC), were submitted to label-free quantitative proteomic analysis. Differential proteins were analyzed by immunohistochemistry in a validation set of 30 CCs. RESULTS: Unsupervised analysis revealed two main clusters: cluster 1 contained most of the iCCC while cluster 2 was divided in 2 subgroups, one containing most of the iCCN and the other regrouping CCH and CCP . Compared to iCCN , iCCC displayed upregulation of molecules involved in cell adhesion, motility and angiogenesis. Epithelial markers associated with secretory pathway and fibroblast markers were overexpressed in CCH compared to iCCN CONCLUSION AND CLINICAL RELEVANCE: This study demonstrated that iCCC is a specific entity, suggesting a major impact of the background liver on tumor biology, and confirmed that extrahepatic CCs define a homogeneous subgroup.

Endothelial fatty liver binding protein 4: a new targetable mediator in hepatocellular carcinoma related to metabolic syndrome.

Metabolic syndrome (MS) is becoming the leading risk factor for hepatocellular carcinoma (HCC). HCC development related to MS may occur in advanced or non-advanced liver fibrosis, suggesting specific molecular pathways. Among these pathways, basal inflammatory state and adipokines production are involved. The aim of this study was to evaluate the role of fatty acid-binding protein 4 (FABP4). In this study, we demonstrate the specific overexpression of FABP4 in human HCC samples from patients with MS compared to other risk factors for chronic liver disease with FABP4 expression restricted to peritumoral endothelial cells. In vitro, glucose, insulin, VEGFA and hypoxia upregulated endothelial FABP4, which was reversed by metformin through mTOR pathway inhibition. FABP4 exerts oncogenic effects on hepatoma cell lines by upregulating the angiogenesis gene signature and pathways involved in the cell cycle, leading to increased cell proliferation and migration, and downregulating HIF1 pathway; effects were reversed in the presence of a specific FABP4 inhibitor (BMS309403). We showed the role of microvesicles as FABP4 vectors between endothelial and tumor cells. In vivo, BMS309403 significantly reduces tumor growth in heterotopic and orthotopic xenografted mice model. In conclusion, this study demonstrates the emerging oncogenic role of liver endothelial cells through FABP4 in HCC related to MS, and highlights new anti-neoplastic mechanism of metformin.

Mucosal-associated invariant T cells are a profibrogenic immune cell population in the liver.

Liver fibrosis is the common response to chronic liver injury, and leads to cirrhosis and its complications. Persistent inflammation is a driving force of liver fibrosis progression. Mucosal-associated invariant T (MAIT) cells are non-conventional T cells that display altered functions during chronic inflammatory diseases. Here, we show that circulating MAIT cells are reduced in patients with alcoholic or non-alcoholic fatty liver disease-related cirrhosis while they accumulate in liver fibrotic septa. Using two models of chronic liver injury, we demonstrate that MAIT cell-enriched mice show increased liver fibrosis and accumulation of hepatic fibrogenic cells, whereas MAIT cell-deficient mice are resistant. Co-culture experiments indicate that MAIT cells enhance the proinflammatory properties of monocyte-derived macrophages, and promote mitogenic and proinflammatory functions of fibrogenic cells, via distinct mechanisms. Our results highlight the profibrogenic functions of MAIT cells and suggest that targeting MAIT cells may constitute an attractive antifibrogenic strategy during chronic liver injury.

Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis.

Microvesicles (MVs) are extracellular vesicles released by cells following activation or apoptosis. Some MV subpopulations augment with cirrhosis severity and contribute to portal hypertension. This study aimed at determining if plasma MV levels can estimate the presence of hepatic venous pressure gradient (HVPG) ≥10 mm Hg and predict mortality in patients with advanced chronic liver disease. All patients with severe fibrosis or cirrhosis undergoing liver catheterization between 2013 and 2015 at two centers were prospectively included. We measured circulating levels of annexin V+ , platelet, leukocyte, endothelial, and hepatocyte MVs. The test cohort included 139 patients. Hepatocyte MV levels were 4.0-fold and 2.2-fold higher in patients with Child-Pugh C than in those with Child-Pugh A or B liver disease, respectively. Levels of other MV subpopulations were not influenced by liver disease severity. Hepatocyte MV levels correlated with HVPG but could not identify patients with HVPG ≥10 mm Hg. Hepatocyte MV level >65 U/L predicted 6-month mortality independently of Child-Pugh score and of Model for End-Stage Liver Disease (MELD). Patients with hepatocyte MV levels >65 U/L and MELD >15 had a higher 6-month mortality than other patients (23% versus 3%; P = 0.001). These findings were confirmed in a validation cohort including 103 patients. CONCLUSION: Circulating MV levels cannot identify patients with HVPG ≥10 mm Hg; by contrast, hepatocyte MV levels strongly improve prediction of 6-month mortality in patients with advanced chronic liver disease; therapies associated with decreased levels of circulating hepatocyte MV might be attractive strategies in patients with severe cirrhosis. (Hepatology 2018).

Renal parenchyma impairment characterization in partial unilateral ureteral obstruction in mice with intravoxel incoherent motion-MRI.

Ureteropelvic junction obstruction constitutes a major cause of progressive pediatric renal disease. The biological mechanisms underlying the renal response to obstruction can be investigated using a clinically relevant mouse model of partial unilateral ureteral obstruction (pUUO). Renal function and kidney morphology data can be evaluated using renal ultrasound, scintigraphy and uro-magnetic resonance imaging (uro-MRI), but these methods are poorly linked to histological change and not all are quantitative. Here, we propose to investigate pUUO for the first time using an intravoxel incoherent motion diffusion sequence. The aim of this study was to quantitatively characterize impairment of the kidney parenchyma in the pUUO model. This quantitative MRI method was able to assess the perfusion and microstructure of the kidney without requiring the injection of a contrast agent. The results suggest that a perfusion fraction (f) reduction is associated with a decrease in the volume of the renal parenchyma, which could be related to decreased renal vascularization. The latter may occur before impairment by fibrosis and the findings are in accordance with the literature using the UUO mice model and, more specifically, on pUUO. Further investigation is required before this technique can be made available for the diagnosis and management of children with antenatal hydronephrosis and to select the optimal timing of surgery if required.

Contribution of virtual biopsy to the screening of microvascular invasion in hepatocellular carcinoma: A pilot study.

BACKGROUND & AIMS: Microvascular invasion (mVI) is a major prognostic factor in hepatocellular carcinoma (HCC) that cannot be detected before surgery. Predictive biomarkers of mVI are thus urgently needed. We have developed an original approach of virtual biopsy to assess the performance of an immunohistochemical panel comprising three biomarkers of mVI (H4K16ac, H4K20me2, PIVKA-II) for the prediction of mVI in HCC core needle biopsies (CNB). METHODS: A test set of HCC surgical specimens (n = 64) and an independent validation set of HCC CNB (n = 42) were retrospectively constituted. Immunostainings were first quantified in the test set on the whole tissue section, to determine optimal cut-off values for each marker. From the digitised image of the whole section, three virtual biopsies were provided. Immunostainings and accuracy of the panel for the prediction of mVI were further assessed in virtual biopsies and in the validation set of CNB. RESULTS: In virtual biopsies, PIVKA-II/H4K16ac had the best performance for prediction of mVI, with sensitivity, specificity, predictive positive value (PPV), and predictive negative value (PNV) of 30%, 97%, 91%, 56%, respectively. In CNB, PIVKA-II/H4K20me2 showed the best accuracy for prediction of mVI, with sensitivity, specificity, PPV, and NPV of 43%, 95%, 90%, and 62%, respectively. The two panels were independent predictive factors of mVI (PIVKA-II/H4K16ac, P = .037; PIVKA-II/H4K20me2, P = .026). CONCLUSION: This study shows that a panel of two markers is able to predict mVI in HCC CNB, and pave the way for the future development of prognostic biomarkers in HCC that could guide the therapeutic strategy.