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Additional file 2: Figure S2. PD-L1 staining on liver biopsies is shown at low (left—×100), moderate (middle—200×) and high magnification (right—400×), with scattered expression in the cytoplasm of some hepatocytes and sinusoïdal expression.

International audience; Adrenocortical carcinoma (ACC) is a tumor with poor prognosis in which overexpression of a panel of microRNAs has been associated with malignancy but a very limited number of investigations on their role in ACC pathogenesis have been conducted. We examined the involvement of miR-483-5p and miR-139-5p in adrenocortical cancer aggressiveness. Using bioinformatics predictions and mRNA/miRNA expression profiles, we performed an integrated analysis to identify inversely correlated miRNA-mRNA pairs in ACC. We identified N-myc downstream-regulated gene family members 2 and 4 (NDRG2 and NDRG4) as targets of miR-483-5p and miR-139-5p, respectively. NDRG2 and NDRG4 expressions were inversely correlated respectively with miR-483-5p and miR-139-5p levels in aggressive ACC samples from two independent cohorts of 20 and 44 ACC. Moreover, upregulation of miR-139-5p and downregulation of NDRG4 demonstrated a striking prognostic value. A direct interaction between miR-483-5p or miR-139-5p and their targets was demonstrated in reporter assays. Downregulation of miR-483-5p or miR-139-5p in the ACC cell lines NCI-H295R and SW13 increased NDRG2 or NDRG4 mRNA and protein expression, compromised adrenocortical cancer cell invasiveness and anchorage-independent growth. MiR-483-5p or miR-139-5p overexpression and NDRG2 or NDRG4 inhibition produce similar changes, which are rescued by NDRG2 or NDRG4 ectopic expression. We established that key factors mediating epithelial-to-mesenchymal transition are downstream effectors of miR-483-5p/NDRG2 and miR-139-5p/NDRG4 pathways. Collectively, our data show for the first time that miR-483-5p/NDRG2 and miR-139-5p/NDRG4 axes promote ACC aggressiveness, with potential implications for prognosis and therapeutic interventions in adrenocortical malignancies.

International audience; BACKGROUND & AIMS:Hepatocellular adenomas (HCAs) are benign liver tumors that can be assigned to molecular subtypes based on inactivating mutations in hepatocyte nuclear factor 1A, activating mutations in β-catenin, or activation of inflammatory signaling pathways. We aimed to update the classification system for HCA and associate the subtypes with disease risk factors and complications.METHODS:We analyzed expression levels of 20 genes and sequenced exon regions of 8 genes (HNF1A, IL6ST, CTNNB1, FRK, STAT3, GNAS, JAK1, and TERT) in 607 samples of 533 HCAs from 411 patients, collected from 28 centers mainly in France from 2000 and 2014. We performed gene expression profile, RNA sequence, whole-exome and genome sequence, and immunohistochemical analyses of select samples. Molecular data were associated with risk factors, histopathology, bleeding, and malignant transformation.RESULTS:Symptomatic bleeding occurred in 14% of the patients (85% of cases were female, median age, 38 years); 7% of the nodules were borderline between HCA and hepatocellular carcinoma, and 3% of patients developed hepatocellular carcinoma from HCA. Based on molecular features, we classified HCA into 8 subgroups. One new subgroup, composed of previously unclassified HCA, represented 4% of HCAs overall and was associated with obesity and bleeding. These tumors were characterized by activation of sonic hedgehog signaling, due to focal deletions that fuse the promoter of INHBE with GLI1. Analysis of genetic heterogeneity among multiple HCAs, from different patients, revealed a molecular subtype field effect; multiple tumors had different mutations that deregulated similar pathways. Specific molecular subtypes of HCA associated with various HCA risk factors, including imbalances in estrogen or androgen hormones. Specific molecular subgroup of HCA with β-catenin and sonic hedgehog activation associated with malignant transformation and bleeding, respectively.CONCLUSIONS:Using sequencing and gene expression analyses, we identified a subgroup of HCA characterized by fusion of the INHBE and GLI1 genes and activation of sonic hedgehog pathway. Molecular subtypes of HCAs associated with different patients' risk factors for HCA, disease progression, and pathology features of tumors. This classification system might be used to select treatment strategies for patients with HCA.

Chronic hepatitis C is associated with the failure of the immune system. We were interested to NK cells and Treg cells, partners in the innate immune response. The number of NK, particularly the CD56+dim, is significantly reduced in infected patients, in the liver more than in the blood, and increases during the process of fibrogenesis. The number of circulating CD3- CD56+brightNKG2A+ correlates with the severity of inflammation and fibrosis and that of CD3- CD56+dimNKG2A+ inversely correlates with viral load. The NK functional capacity to produce IFN-γ and initiate a process of cytolysis is maintened. The CD158 expression is significantly reduced on the surface of intrahepatic NK, whereas NKG2A,C,D expression in circulating and hepatic NK is identical to that of non-infected patients. The intrahepatic Treg FoxP3+ are almost exclusively CD4+ phenotype. In multivariate analysis, the number of FoxP3+ is independently associated with that of CD8+, especially in necroinflammatory lesions and a strong correlation is observed between CD8, FoxP3, IL-10 and TGF-β, suggesting that Treg could inhibit CD8 expansion and cytotoxicity by cell contact or through immunosuppressive cytokines. The balance between FoxP3 and CD8 is broken in the most severe stages of the disease (METAVIR A>2 and F>3), which results in a drop in the FoxP3/CD8 ratio. Chronic inflammation is accompanied by liver fibrosis, leading to cirrhosis, the main cause of HCC. In viral C cirrhosis with or without HCC, CD3+, CD4+, CD8+, CD20+, CD56+, TCR γδ+, FoxP3+ lymphocytes are more numerous in fibrosis than in parenchyma. The number of CD3+, CD4+, CD8+, CD20+ and the expression of IFN-γ and RANTES were higher in cirrhosis developing HCC. In multivariate analysis, CD8 is the only independent predictor of tumor recurrence and is associated with a significant decrease in the 5 years disease free survival. The CD3+, CD4+, CD8+, CD20+, CD56+, TCR γδ+, FoxP3+ tumor infiltrating lymphocytes were significantly lower than in distant cirrhosis. However, FoxP3+ are significantly higher and CD56+ significantly lower in HCC than in parenchymatous nodules, without LT changes, leading to an increase in the FoxP3/CD8 ratio into the tumor. The number of CD56+ decreases from cirrhosis to HCC. No correlation was found between the density of studied tumor infiltrating lymphocytes and HCC recurrence. Conclusion. A dense inflammatory infiltrate in viral C cirrhosis, particularly rich in CD8, promotes HCC development and/or recurrence.; L'hépatite chronique virale C est associée à une défaillance du système immunitaire. Nous nous sommes intéressés aux cellules NK et aux lymphocytes Treg, partenaires de la réponse immunitaire innée. Le nombre des NK, particulièrement les CD56dim, est significativement diminué chez les patients infectés, dans le foie plus que dans le sang, et s'accentue avec la fibrogenèse. Le nombre de CD3-CD56+brightNKG2A+ circulantes est corrélé à la sévérité de l'inflammation et de la fibrose et celui des CD3-CD56+dimNKG2A+ inversement corrélé à la charge virale. Les NK sont fonctionnelles, en capacité de produire de l'IFN-γ et d'engager un processus de cytolyse. L'expression de CD158 est significativement diminuée à la surface des NK hépatiques mais conservée dans les NK circulantes. L'expression de NKG2A,C,D dans les NK circulantes et hépatiques est identique à celles de patients non infectés. Les Treg intrahépatiques FoxP3+ sont quasi-exclusivement de phénotype CD4+. En analyse multivariée, le nombre de FoxP3+ est indépendamment associé à celui de CD8+, surtout dans les lésions nécrotico-inflammatoires et une corrélation forte est observée entre les transcrits CD8, FoxP3, IL-10 et TGF-β, suggérant que les Treg bloquent l'expansion et la cytotoxicité des TCD8 par contact cellulaire ou par le biais de cytokines immunosuppressives. L'équilibre entre FoxP3 et CD8 est rompu dans les grades et stades Métavir A>2 et F>3, avec un effondrement du rapport FoxP3/CD8. L'inflammation hépatique chronique s'accompagne de fibrose, aboutissant à la cirrhose, principale cause de CHC. Dans les cirrhoses virales C avec ou sans CHC, les lymphocytes CD3+, CD4+, CD8+, CD20+, CD56+, TCRγδ +, FoxP3+ sont plus nombreux dans la fibrose que dans le parenchyme. Le nombre de CD20+, CD3+, CD4+, CD8+ et l'expression d'IFN-γ et RANTES sont plus élevés dans les cirrhoses qui développent un CHC. En analyse multivariée, CD8 est le seul facteur indépendament associé à la récidive tumorale et à une diminution de la survie sans récidive à 5 ans. Les CD20+, CD3+, CD4+, CD8+, CD56+, TCRγδ+, FoxP3+ sont significativement moins nombreux dans le CHC que dans la cirrhose. Mais les FoxP3+ sont significativement plus nombreux et les CD56+ moins nombreux dans le CHC que dans le nodule parenchymateux, sans modification des LT, conduisant à une augmentation du rapport FoxP3/CD8 dans la tumeur. Les CD56+ diminuent de la cirrhose au CHC. Aucune corrélation n'est observée entre la densité intra-tumorale des lymphocytes étudiés et la récidive carcinomateuse. Conclusion. Un infiltrat inflammatoire dense au sein de la cirrhose C, particulièrement riche en CD8, favorise le développement et/ou la récidive du CHC.

Chronic hepatitis C is associated with the failure of the immune system. We were interested to NK cells and Treg cells, partners in the innate immune response. The number of NK, particularly the CD56+dim, is significantly reduced in infected patients, in the liver more than in the blood, and increases during the process of fibrogenesis. The number of circulating CD3- CD56+brightNKG2A+ correlates with the severity of inflammation and fibrosis and that of CD3- CD56+dimNKG2A+ inversely correlates with viral load. The NK functional capacity to produce IFN-γ and initiate a process of cytolysis is maintened. The CD158 expression is significantly reduced on the surface of intrahepatic NK, whereas NKG2A,C,D expression in circulating and hepatic NK is identical to that of non-infected patients. The intrahepatic Treg FoxP3+ are almost exclusively CD4+ phenotype. In multivariate analysis, the number of FoxP3+ is independently associated with that of CD8+, especially in necroinflammatory lesions and a strong correlation is observed between CD8, FoxP3, IL-10 and TGF-β, suggesting that Treg could inhibit CD8 expansion and cytotoxicity by cell contact or through immunosuppressive cytokines. The balance between FoxP3 and CD8 is broken in the most severe stages of the disease (METAVIR A>2 and F>3), which results in a drop in the FoxP3/CD8 ratio. Chronic inflammation is accompanied by liver fibrosis, leading to cirrhosis, the main cause of HCC. In viral C cirrhosis with or without HCC, CD3+, CD4+, CD8+, CD20+, CD56+, TCR γδ+, FoxP3+ lymphocytes are more numerous in fibrosis than in parenchyma. The number of CD3+, CD4+, CD8+, CD20+ and the expression of IFN-γ and RANTES were higher in cirrhosis developing HCC. In multivariate analysis, CD8 is the only independent predictor of tumor recurrence and is associated with a significant decrease in the 5 years disease free survival. The CD3+, CD4+, CD8+, CD20+, CD56+, TCR γδ+, FoxP3+ tumor infiltrating lymphocytes were significantly lower than in distant cirrhosis. However, FoxP3+ are significantly higher and CD56+ significantly lower in HCC than in parenchymatous nodules, without LT changes, leading to an increase in the FoxP3/CD8 ratio into the tumor. The number of CD56+ decreases from cirrhosis to HCC. No correlation was found between the density of studied tumor infiltrating lymphocytes and HCC recurrence. Conclusion. A dense inflammatory infiltrate in viral C cirrhosis, particularly rich in CD8, promotes HCC development and/or recurrence., L'hépatite chronique virale C est associée à une défaillance du système immunitaire. Nous nous sommes intéressés aux cellules NK et aux lymphocytes Treg, partenaires de la réponse immunitaire innée. Le nombre des NK, particulièrement les CD56dim, est significativement diminué chez les patients infectés, dans le foie plus que dans le sang, et s'accentue avec la fibrogenèse. Le nombre de CD3-CD56+brightNKG2A+ circulantes est corrélé à la sévérité de l'inflammation et de la fibrose et celui des CD3-CD56+dimNKG2A+ inversement corrélé à la charge virale. Les NK sont fonctionnelles, en capacité de produire de l'IFN-γ et d'engager un processus de cytolyse. L'expression de CD158 est significativement diminuée à la surface des NK hépatiques mais conservée dans les NK circulantes. L'expression de NKG2A,C,D dans les NK circulantes et hépatiques est identique à celles de patients non infectés. Les Treg intrahépatiques FoxP3+ sont quasi-exclusivement de phénotype CD4+. En analyse multivariée, le nombre de FoxP3+ est indépendamment associé à celui de CD8+, surtout dans les lésions nécrotico-inflammatoires et une corrélation forte est observée entre les transcrits CD8, FoxP3, IL-10 et TGF-β, suggérant que les Treg bloquent l'expansion et la cytotoxicité des TCD8 par contact cellulaire ou par le biais de cytokines immunosuppressives. L'équilibre entre FoxP3 et CD8 est rompu dans les grades et stades Métavir A>2 et F>3, avec un effondrement du rapport FoxP3/CD8. L'inflammation hépatique chronique s'accompagne de fibrose, aboutissant à la cirrhose, principale cause de CHC. Dans les cirrhoses virales C avec ou sans CHC, les lymphocytes CD3+, CD4+, CD8+, CD20+, CD56+, TCRγδ +, FoxP3+ sont plus nombreux dans la fibrose que dans le parenchyme. Le nombre de CD20+, CD3+, CD4+, CD8+ et l'expression d'IFN-γ et RANTES sont plus élevés dans les cirrhoses qui développent un CHC. En analyse multivariée, CD8 est le seul facteur indépendament associé à la récidive tumorale et à une diminution de la survie sans récidive à 5 ans. Les CD20+, CD3+, CD4+, CD8+, CD56+, TCRγδ+, FoxP3+ sont significativement moins nombreux dans le CHC que dans la cirrhose. Mais les FoxP3+ sont significativement plus nombreux et les CD56+ moins nombreux dans le CHC que dans le nodule parenchymateux, sans modification des LT, conduisant à une augmentation du rapport FoxP3/CD8 dans la tumeur. Les CD56+ diminuent de la cirrhose au CHC. Aucune corrélation n'est observée entre la densité intra-tumorale des lymphocytes étudiés et la récidive carcinomateuse. Conclusion. Un infiltrat inflammatoire dense au sein de la cirrhose C, particulièrement riche en CD8, favorise le développement et/ou la récidive du CHC.

International audience; BACKGROUND & AIMS: In chronic hepatitis C (CHC), HCV-specific T-cell responses are often dysfunctionnal. In vitro data point out that regulatory T cells (Treg) are able to suppress HCV-specific lymphocyte proliferation and cytokine secretion but their implication in this pathology is still debated. METHODS: Three complementary approaches were performed to investigate phenotype, frequency or localization of intra-hepatic Treg in treatment naïve CHC patients. Double immunohistochemical analysis was performed in 20 formalin-fixed biopsies with CD8/FoxP3 and CD4/FoxP3 antibodies. Cellular markers and cytokines were investigated by quantitative RT-PCR in 27 additional frozen biopsies. Eight other fresh liver biopsies were selected for complementary analysis of immunophenotyping and frequency of intra-hepatic Treg. RESULTS: Immunohistochemical analyses showed the presence of intra-hepatic CD4(+)FoxP3(+)T cells while CD8(+)FoxP3(+)T cells were very scarce. CD4(+)FoxP3(+)T cells were located in necro-inflammatory areas in contact with CD8(+)T cells, suggesting that Treg-mediated inhibition of CD8(+)T cell proliferation may occur by cell-cell contact. RT-PCR analyses showed strong correlations between CD8, FoxP3, and IL-10 with emergence of four distinct gene clusters, CD8-FoxP3, CD8-IL-10, TGF-beta-IL-10, and TNF-alpha-TGF-beta. No correlation was found between serum viral load and any immune markers. Interestingly, the FoxP3(+)/CD8(+) cells ratio significantly decreased in severe fibrosis (F>3) due to the dramatic decline of FoxP3 cells. CONCLUSIONS: This study provides new insights into the histological localization of Treg within HCV-infected liver, with a special accumulation of CD4(+)FoxP3(+)Treg cells in necro-inflammatory areas, in contact with CD8(+)T cells. Our results suggest a link between Treg, CD8, and IL-10 which altogether could balance immune responses against the virus to avoid immunopathogenesis.

International audience; BACKGROUND/AIMS: The fate of intrahepatic NK cell subsets in the course of HCV and HBV infections is not clearly understood. METHODS: Blood and intrahepatic CD56(+) NK cell subsets (expressing NKG2A, CD158a,h or CD158b,j receptors) from HCV or HBV patients were quantified by flow cytometry and localized by immunohistochemistry in liver biopsies. RESULTS: A significant reduction in NK cell frequency and a quantitative imbalance between CD56(bright) and CD56(dim) subsets were observed in chronic HCV patients as compared to HBV patients, underlining that the inflammatory environment is not the only cause of these phenomena. The proportions of intrahepatic NK cells expressing either NKG2A, and/or CD158a,h, CD158b,j differed significantly between HCV and HBV patients. A higher frequency of perforin among intrahepatic CD56(+)CD3(-) cells was observed in HCV compared to HBV patients. Double immunohistochemical staining showed that CD56(+)CD3(-) cells were localized within necrotic areas. Immune monitoring of circulating CD56 subsets revealed that CD3(-)CD56(bright)NKG2A(+) and CD3(-)CD56(dim)NKG2A(+) cells were positively correlated with the necroinflammatory score and inversely correlated with viral load, respectively, in HCV patients. CONCLUSIONS: HCV and HBV affect NK cell subsets according to the status of the diseases, especially CD3(-)CD56(dim)NKG2A(+) and CD3(-)CD56(bright)NKG2A(+) cells, may be of interest for disease monitoring.