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BACKGROUND AND AIMS: Outcomes after Kasai portoenterostomy (KPE) for biliary atresia remain highly variable for unclear reasons. As reliable early biomarkers predicting KPE outcomes are lacking, we studied the prognostic value of FGF19. APPROACH AND RESULTS: Serum and liver specimens, obtained from biliary atresia patients (N=87) at KPE or age-matched cholestatic controls (N=26) were included. Serum concentration of FGF19 and bile acids, liver mRNA expression of FGF19 , and key regulators of bile acid synthesis were related to KPE outcomes and liver histopathology. Immunohistochemistry and in situ hybridization were used for the localization of liver FGF19 expression. Serum levels (223 vs. 61 pg/mL, p <0.001) and liver mRNA expression of FGF19 were significantly increased in biliary atresia. Patients with unsuccessful KPE (419 vs. 145 pg/mL, p =0.047), and those subsequently underwent liver transplantation (410 vs. 99 pg/mL, p =0.007) had significantly increased serum, but not liver, FGF19, which localized mainly in hepatocytes. In Cox hazard modeling serum FGF19 <109 pg/mL predicted native liver survival (HR: 4.31, p <0.001) also among patients operated <60 days of age (HR: 8.77, p =0.004) or after successful KPE (HR: 6.76, p =0.01). Serum FGF19 correlated positively with increased serum primary bile acids ( R =0.41, p =0.004) and ductular reaction ( R =0.39, p =0.004). CONCLUSIONS: Increased serum FGF19 at KPE predicted inferior long-term native liver survival in biliary atresia and was associated with unsuccessful KPE, elevated serum primary bile acids, and ductular reaction.
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Atresia Biliar , Humanos , Lactente , Atresia Biliar/complicações , Portoenterostomia Hepática , Prognóstico , Ácidos e Sais Biliares , RNA Mensageiro , Resultado do Tratamento , Fatores de Crescimento de FibroblastosRESUMO
Many hereditary cancer syndromes are associated with an increased risk of small and large intestinal adenocarcinomas. However, conditions bearing a high risk to both adenocarcinomas and neuroendocrine tumors are yet to be described. We studied a family with 16 individuals in four generations affected by a wide spectrum of intestinal tumors, including hyperplastic polyps, adenomas, small intestinal neuroendocrine tumors, and colorectal and small intestinal adenocarcinomas. To assess the genetic susceptibility and understand the novel phenotype, we utilized multiple molecular methods, including whole genome sequencing, RNA sequencing, single cell sequencing, RNA in situ hybridization and organoid culture. We detected a heterozygous deletion at the cystic fibrosis locus (7q31.2) perfectly segregating with the intestinal tumor predisposition in the family. The deletion removes a topologically associating domain border between CFTR and WNT2, aberrantly activating WNT2 in the intestinal epithelium. These consequences suggest that the deletion predisposes to small intestinal neuroendocrine tumors and small and large intestinal adenocarcinomas, and reveals the broad tumorigenic effects of aberrant WNT activation in the human intestine.
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Adenocarcinoma , Adenoma , Neoplasias Colorretais , Tumores Neuroendócrinos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/patologia , Neoplasias Colorretais/genética , Humanos , Mucosa Intestinal/patologia , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Proteína Wnt2RESUMO
RNA in situ hybridization (RNA-ISH) is a powerful spatial transcriptomics technology to characterize target RNA abundance and localization in individual cells. This allows analysis of tumor heterogeneity and expression localization, which are not readily obtainable through transcriptomic data analysis. RNA-ISH experiments produce large amounts of data and there is a need for automated analysis methods. Here we present QuantISH, a comprehensive open-source RNA-ISH image analysis pipeline that quantifies marker expressions in individual carcinoma, immune, and stromal cells on chromogenic or fluorescent in situ hybridization images. QuantISH is designed to be modular and can be adapted to various image and sample types and staining protocols. We show that in chromogenic RNA in situ hybridization images of high-grade serous carcinoma (HGSC) QuantISH cancer cell classification has high precision, and signal expression quantification is in line with visual assessment. We further demonstrate the power of QuantISH by showing that CCNE1 average expression and DDIT3 expression variability, as captured by the variability factor developed herein, act as candidate biomarkers in HGSC. Altogether, our results demonstrate that QuantISH can quantify RNA expression levels and their variability in carcinoma cells, and thus paves the way to utilize RNA-ISH technology.
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Biomarcadores Tumorais , RNA , Biomarcadores Tumorais/metabolismo , Perfilação da Expressão Gênica , Hibridização In Situ , Hibridização in Situ Fluorescente/métodos , RNA/genéticaRESUMO
Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3' mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2 (FOXL2) mutation. We compared the transcriptomic profiles of 14 non-relapsed archival primary AGCTs (follow-up time 17-26 years after diagnosis) with 13 relapsed primary AGCTs (follow-up time 1.7-18 years) and eight relapsed tumors (follow-up time 2.8-18.9 years). Non-relapsed and relapsed primary AGCTs had similar transcriptomic profiles. In relapsed tumors three genes were differentially expressed: plasmalemma vesicle associated protein (PLVAP) was upregulated (p = 0.01), whereas argininosuccinate synthase 1 (ASS1) (p = 0.01) and perilipin 4 (PLIN4) (p = 0.02) were downregulated. PLVAP upregulation was validated using tissue microarray RNA in situ hybridization. In our patient cohort with extremely long follow-up, we observed similar gene expression patterns in both primary AGCT groups, suggesting that relapse is not driven by transcriptomic changes. These results reinforce earlier findings that molecular markers do not predict AGCT behavior or risk of relapse.
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Defects in the mRNA export scaffold protein GANP, encoded by the MCM3AP gene, cause autosomal recessive early-onset peripheral neuropathy with or without intellectual disability. We extend here the phenotypic range associated with MCM3AP variants, by describing a severely hypotonic child and a sibling pair with a progressive encephalopathic syndrome. In addition, our analysis of skin fibroblasts from affected individuals from seven unrelated families indicates that disease variants result in depletion of GANP except when they alter critical residues in the Sac3 mRNA binding domain. GANP depletion was associated with more severe phenotypes compared with the Sac3 variants. Patient fibroblasts showed transcriptome alterations that suggested intron content-dependent regulation of gene expression. For example, all differentially expressed intronless genes were downregulated, including ATXN7L3B, which couples mRNA export to transcription activation by association with the TREX-2 and SAGA complexes. Our results provide insight into the molecular basis behind genotype-phenotype correlations in MCM3AP-associated disease and suggest mechanisms by which GANP defects might alter RNA metabolism.
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Acetiltransferases/genética , Flavoproteínas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Doenças do Sistema Nervoso/genética , Proteínas Nucleares/genética , Monoéster Fosfórico Hidrolases/genética , Fatores de Transcrição/genética , Acetiltransferases/química , Acetiltransferases/ultraestrutura , Idade de Início , Antígenos de Superfície/genética , Núcleo Celular/genética , Criança , Pré-Escolar , Exodesoxirribonucleases/genética , Feminino , Regulação da Expressão Gênica/genética , Glicoproteínas/genética , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Íntrons/genética , Masculino , Doenças do Sistema Nervoso/patologia , Proteínas Nucleares/ultraestrutura , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia , Fenótipo , Fosfoproteínas/genética , Conformação Proteica , Transporte de RNA/genética , RNA Mensageiro/genéticaRESUMO
MOTIVATION: Fusion genes are both useful cancer biomarkers and important drug targets. Finding relevant fusion genes is challenging due to genomic instability resulting in a high number of passenger events. To reveal and prioritize relevant gene fusion events we have developed FUsionN Gene Identification toolset (FUNGI) that uses an ensemble of fusion detection algorithms with prioritization and visualization modules. RESULTS: We applied FUNGI to an ovarian cancer dataset of 107 tumor samples from 36 patients. Ten out of 11 detected and prioritized fusion genes were validated. Many of detected fusion genes affect the PI3K-AKT pathway with potential role in treatment resistance. AVAILABILITYAND IMPLEMENTATION: FUNGI and its documentation are available at https://bitbucket.org/alejandra_cervera/fungi as standalone or from Anduril at https://www.anduril.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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MOTIVATION: A major challenge in analyzing cancer patient transcriptomes is that the tumors are inherently heterogeneous and evolving. We analyzed 214 bulk RNA samples of a longitudinal, prospective ovarian cancer cohort and found that the sample composition changes systematically due to chemotherapy and between the anatomical sites, preventing direct comparison of treatment-naive and treated samples. RESULTS: To overcome this, we developed PRISM, a latent statistical framework to simultaneously extract the sample composition and cell-type-specific whole-transcriptome profiles adapted to each individual sample. Our results indicate that the PRISM-derived composition-free transcriptomic profiles and signatures derived from them predict the patient response better than the composite raw bulk data. We validated our findings in independent ovarian cancer and melanoma cohorts, and verified that PRISM accurately estimates the composition and cell-type-specific expression through whole-genome sequencing and RNA in situ hybridization experiments. AVAILABILITYAND IMPLEMENTATION: https://bitbucket.org/anthakki/prism. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Neoplasias Ovarianas , Transcriptoma , Feminino , Humanos , RNA-Seq , Estudos Prospectivos , Análise de Sequência de RNA/métodos , RNA/genética , Perfilação da Expressão Gênica , SoftwareRESUMO
Platinum-resistance is a major limitation to effective chemotherapy regimens in high-grade serous ovarian cancer (HGSOC). To better understand the mechanisms involved we characterized the proteome and phosphoproteome in cisplatin sensitive and resistant HGSOC primary cells using a mass spectrometry-based proteomic strategy. PCA analysis identified a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines. The most phosphorylated protein in cisplatin resistant cells was sequestosome-1 (p62/SQSTM1). Changes in expression of apoptosis and autophagy related proteins Caspase-3 and SQSTM1, respectively, were validated by Western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while SQSTM1 revealed increased expression in the resistant cell line relative to sensitive cell line. Furthermore, site-specific phosphorylation on 20 amino acid residues of SQSTM1 was detected indicating a hyper-phosphorylation phenotype. This elevated hyper-phosphorylation of SQSTM1 in resistant HGSOC cell lines was validated with Western blot analysis. Immunofluoresence staining of s28-pSQSTM1 showed inducible localization to autophagosomes upon cisplatin treatment in the sensitive cell line while being constitutively expressed to autophagosomes in the resistant cell. Furthermore, SQSTM1 expression was localized in cancer cells of clinical high-grade serous tumors. Here, we propose hyper-phosphorylation of SQSTM1 as a marker and a key proteomic change in cisplatin resistance development in ovarian cancers by activating the autophagy pathway and influencing down-regulation of apoptosis.
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Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína Sequestossoma-1/metabolismo , Autofagossomos/metabolismo , Caspase 1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Espectrometria de Massas , Gradação de Tumores , Fosforilação , Estudos Prospectivos , Proteômica/métodos , Proteína Sequestossoma-1/químicaRESUMO
Biliary atresia (BA), a neonatal liver disease, is characterized by obstruction of extrahepatic bile ducts with subsequent cholestasis, inflammation, and progressive liver fibrosis. To gain insights into the pathophysiology of BA, we focused attention on GATA6, a transcription factor implicated in biliary development. Early in fetal development GATA6 expression is evident in cholangiocytes and hepatocytes, but by late gestation it is extinguished in hepatocytes. Utilizing a unique set of BA liver samples collected before and after successful portoenterostomy (PE), we found that GATA6 expression is markedly upregulated in hepatocytes of patients with BA compared with healthy and cholestatic disease controls. This upregulation is recapitulated in two murine models simulating bile duct obstruction and intrahepatic bile ductule expansion. GATA6 expression in BA livers correlates with two established negative prognostic indicators (age at PE, degree of intrahepatic bile ductule expansion) and decreases after normalization of serum bilirubin by PE. GATA6 expression in BA livers correlates with expression of known regulators of cholangiocyte differentiation ( JAGGED1, HNF1ß, and HNF6). These same genes are upregulated after enforced expression of GATA6 in human hepatocyte cell models. In conclusion, GATA6 is a novel marker and a putative driver of hepatocyte-cholangiocyte metaplasia in BA, and its expression in hepatocytes is downregulated after successful PE. NEW & NOTEWORTHY A pathological hallmark in the liver of patients with biliary atresia is ductular reaction, an expansion of new bile ductules that are thought to arise from conversion of mature hepatocytes. Here, we show that transcription factor GATA6 is a marker and potential driver of hepatocyte ductal metaplasia in biliary atresia. Hepatocyte GATA6 expression is elevated in biliary atresia, correlates with bile duct expansion, and decreases after successful portoenterostomy.
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Ductos Biliares Extra-Hepáticos/patologia , Atresia Biliar , Fator de Transcrição GATA6/metabolismo , Hepatócitos/metabolismo , Fígado/patologia , Animais , Atresia Biliar/metabolismo , Atresia Biliar/patologia , Atresia Biliar/cirurgia , Biomarcadores/metabolismo , Transdiferenciação Celular/fisiologia , Colestase/metabolismo , Modelos Animais de Doenças , Células Hep G2 , Humanos , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Portoenterostomia Hepática/métodosRESUMO
GATA4, a transcription factor crucial for early liver development, has been implicated in the pathophysiology of hepatoblastoma, an embryonal tumor of childhood. However, the molecular and phenotypic consequences of GATA4 expression in hepatoblastoma are not fully understood. We surveyed GATA4 expression in 24 hepatoblastomas using RNA in situ hybridization and immunohistochemistry. RNA interference was used to inhibit GATA4 in human HUH6 hepatoblastoma cells, and changes in cell migration were measured with wound healing and transwell assays. RNA microarray hybridization was performed on control and GATA4 knockdown HUH6 cells, and differentially expressed genes were validated by quantitative polymerase chain reaction or immunostaining. Plasmid transfection was used to overexpress GATA4 in primary human hepatocytes and ensuring changes in gene expression were measured by quantitative polymerase chain reaction. We found that GATA4 expression was high in most hepatoblastomas but weak or negligible in normal hepatocytes. GATA4 gene silencing impaired HUH6 cell migration. We identified 106 differentially expressed genes (72 downregulated, 34 upregulated) in knockdown versus control HUH6 cells. GATA4 silencing altered the expression of genes associated with cytoskeleton organization, cell-to-cell adhesion, and extracellular matrix dynamics (e.g. ADD3, AHNAK, DOCK8, RHOU, MSF, IGFBP1, COL4A2). These changes in gene expression reflected a more epithelial (less malignant) phenotype. Consistent with this notion, there was reduced F-actin stress fiber formation in knockdown HUH6 cells. Forced expression of GATA4 in primary human hepatocytes triggered opposite changes in the expression of genes identified by GATA4 silencing in HUH6 cells. In conclusion, GATA4 is highly expressed in most hepatoblastomas and correlates with a mesenchymal, migratory phenotype of hepatoblastoma cells.
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Movimento Celular , Fator de Transcrição GATA4/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hepatoblastoma/patologia , Neoplasias Hepáticas/patologia , Mesoderma/patologia , Adolescente , Adulto , Estudos de Casos e Controles , Proliferação de Células , Criança , Pré-Escolar , Feminino , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Hepatoblastoma/genética , Humanos , Lactente , Neoplasias Hepáticas/genética , Masculino , Mesoderma/metabolismo , Pessoa de Meia-Idade , Prognóstico , RNA Interferente Pequeno/genética , Adulto JovemRESUMO
OBJECTIVE: Resistance to standard chemotherapy poses a major clinical problem in the treatment of ovarian cancer patients. Adult-type granulosa cell tumor (AGCT) is a unique ovarian cancer subtype for which efficient treatment options are lacking in advanced disease. To this end, systematic drug response and transcriptomics profiling were performed to uncover new therapy options for AGCTs. METHODS: The responses of three primary and four recurrent AGCTs to 230 anticancer compounds were screened in vitro using a systematic drug sensitivity and resistance testing (DSRT) platform, coupled with mRNA sequencing. The responses of the AGCTs were compared with those of human granulosa luteal cells and bone marrow mononuclear cells. RESULTS: Patient-derived AGCT cells showed selective sensitivity to the Src family tyrosine kinase inhibitor dasatinib. A combination of either dasatinib or an mTOR-inhibitor everolimus with paclitaxel resulted in synergistic inhibition of AGCT cell viability. The key kinase targets of dasatinib and members of the mTOR pathway were constantly expressed at mRNA and protein levels, indicating multikinase signal addictions in the AGCT cells. Transcriptomic characterization of the tumors revealed no known oncogenic mutations, suggesting that the drug sensitivity of AGCTs was rather conveyed by selective target expression. CONCLUSIONS: We used a systematic functional approach to reveal novel treatment options for a unique gynecological cancer. The selective synergy found between taxanes and dasatinib or mTOR inhibitors warrants further clinical investigations of these combinations in relapsed or aggressive AGCTs and demonstrate that high-throughput drug screening and molecular profiling can provide an effective approach to uncover new therapy options.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Dasatinibe/farmacologia , Tumor de Células da Granulosa/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Dasatinibe/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Tumor de Células da Granulosa/patologia , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagemRESUMO
FOXL2 is a transcription factor that is essential for ovarian function and maintenance, the germline mutations of which are responsible for the Blepharophimosis Ptosis Epicanthus-inversus Syndrome (BPES), often associated with premature ovarian failure. Recent evidence has linked FOXL2 downregulation or somatic mutation (p.Cys134Trp) to cancer, although underlying molecular mechanisms remain unclear. Using a functional genomic approach, we find that FOXL2 modulates cell-cycle regulators in a way which tends to induce G1 arrest. Indeed, FOXL2 upregulation promotes cell accumulation in G1 phase and protects cells from oxidative damage, notably by promoting oxidized DNA repair and by increasing the amounts of anti-oxidant agent glutathione. In agreement with clinical observations, we find that FOXL2-mutated versions leading to BPES along with ovarian dysfunction mostly fail to transactivate cell-cycle and DNA repair targets, whereas mutations leading to isolated craniofacial defects (and normal ovarian function) activate them correctly. Interestingly, these assays revealed a mild promoter-specific hypomorphy of the tumor-associated mutation (p.Cys134Trp). Finally, the SIRT1 deacetylase suppresses FOXL2 activity on targets linked to cell-cycle and DNA repair in a dose-dependent manner. Accordingly, we find that SIRT1 inhibition by nicotinamide limits proliferation, notably by increasing endogenous FOXL2 amount/activity. The body of evidence presented here supports the idea that FOXL2 plays a key role in granulosa cell homeostasis, the failure of which is central to ovarian ageing and tumorigenesis. As granulosa cell tumors respond poorly to conventional chemotherapy, our findings on the deacetylase inhibitor nicotinamide provide an interesting option for targeted therapy.
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Ciclo Celular , Regulação para Baixo , Fatores de Transcrição Forkhead/metabolismo , Células da Granulosa/citologia , Estresse Oxidativo , Sirtuína 1/metabolismo , Linhagem Celular , Reparo do DNA , Feminino , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Células da Granulosa/enzimologia , Células da Granulosa/metabolismo , Humanos , Sirtuína 1/genéticaRESUMO
Chemotherapy resistance is a critical contributor to cancer mortality and thus an urgent unmet challenge in oncology. To characterize chemotherapy resistance processes in high-grade serous ovarian cancer, we prospectively collected tissue samples before and after chemotherapy and analyzed their transcriptomic profiles at a single-cell resolution. After removing patient-specific signals by a novel analysis approach, PRIMUS, we found a consistent increase in stress-associated cell state during chemotherapy, which was validated by RNA in situ hybridization and bulk RNA sequencing. The stress-associated state exists before chemotherapy, is subclonally enriched during the treatment, and associates with poor progression-free survival. Co-occurrence with an inflammatory cancer-associated fibroblast subtype in tumors implies that chemotherapy is associated with stress response in both cancer cells and stroma, driving a paracrine feed-forward loop. In summary, we have found a resistant state that integrates stromal signaling and subclonal evolution and offers targets to overcome chemotherapy resistance.
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Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Análise de Sequência de RNA , Transcriptoma , Sequenciamento do ExomaRESUMO
Successful portoenterostomy (SPE) improves the short-term outcome of patients with biliary atresia (BA) by relieving cholestasis and extending survival with native liver. Despite SPE, hepatic fibrosis progresses in most patients, leading to cirrhosis and a deterioration of liver function. The goal of this study was to characterize the effects of SPE on the BA liver transcriptome. We used messenger RNA sequencing to analyze global gene-expression patterns in liver biopsies obtained at the time of portoenterostomy (n = 13) and 1 year after SPE (n = 8). Biopsies from pediatric (n = 2) and adult (n = 2) organ donors and other neonatal cholestatic conditions (n = 5) served as controls. SPE was accompanied by attenuation of inflammation and concomitant up-regulation of key extracellular matrix (ECM) genes. Highly overexpressed genes promoting biliary fibrosis and bile duct integrity, such as integrin subunit beta 6 and previously unreported laminin subunit alpha 3, emerged as candidates to control liver fibrosis after SPE. At a cellular level, the relative abundance of activated hepatic stellate cells and liver macrophages decreased following SPE, whereas portal fibroblasts (PFs) and cholangiocytes persisted. Conclusion: The attenuation of inflammation following SPE coincides with emergence of an ECM molecular fingerprint, a set of profibrotic molecules mechanistically connected to biliary fibrosis. The persistence of activated PFs and cholangiocytes after SPE suggests a central role for these cell types in the progression of biliary fibrosis.
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Interleukin (IL)-8 (CXCL8), a chemokine involved in neutrophil recruitment, has been implicated in ductular reaction and liver fibrogenesis. We studied liver and serum IL-8 expression in a large biliary atresia (BA) cohort and explored its prognostic and pathophysiological potential. IL-8 expression was assessed in liver utilizing quantitative polymerase chain reaction (qPCR), immunohistochemistry and in situ hybridization and in serum using an enzyme-linked immunosorbent assay, among 115 BA patients, 10 disease controls and 68 normal controls. Results were correlated to portoenterostomy (PE) outcomes, biochemical and histological liver injury, transcriptional markers of fibrosis and cholangiocytes, and expression of other related cytokines. IL-8 was markedly overexpressed in liver and serum of BA patients at PE (n = 88) and in serum samples obtained during postoperative follow-up (n = 40). IL-8 expression in the liver was predominantly in cholangiocytes within areas of ductular reaction. Liver IL-8 mRNA expression correlated positively with its serum concentration, bile ductular proliferation, Metavir fibrosis stage, and transcriptional markers of activated myofibroblasts (ACTA2) and cholangiocytes (KRT19). Taken together, IL-8 may mediate liver injury in BA by promoting ductular reaction and associated liver fibrogenesis. Prognostic value of serum IL-8 to predict native liver survival was limited and confined to the postoperative period after PE.
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During late lung development, alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia with validation of some of the findings in lungs from BPD patients. We observe dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, stromal fibroblasts, capillary endothelium and macrophage populations. Pathway analysis and predicted dynamic cellular crosstalk suggest inflammatory signaling as the main driver of hyperoxia-induced changes. Our data provides a single-cell view of cellular changes associated with late lung development in health and disease.
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Hiperóxia/genética , Hiperóxia/fisiopatologia , Pulmão/metabolismo , Pulmão/patologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/patologia , Genótipo , Masculino , CamundongosRESUMO
The Ikaros family transcription factors regulate lymphocyte development. Loss-of-function variants in IKZF1 cause primary immunodeficiency, but Ikaros family members IKZF2 and IKZF3 have not yet been associated with immunodeficiency. Here, we describe a pedigree with a heterozygous truncating variant in IKZF2, encoding the transcriptional activator and repressor Helios, which is highly expressed in regulatory T cells and effector T cells, particularly of the CD8+ T cell lineage. Protein-protein interaction analysis revealed that the variant abolished heterodimerization of Helios with Ikaros and Aiolos and also prevented Helios binding to members of the Mi-2/NuRD chromatin remodeling complex. Patients carrying the IKZF2 variant presented with a combined immunodeficiency phenotype characterized by recurrent upper respiratory infections, thrush and mucosal ulcers, and chronic lymphadenopathy. With extensive immunophenotyping, functional assays, and transcriptional analysis, we show that reduced Helios expression was associated with chronic T cell activation and increased production of proinflammatory cytokines both in effector and regulatory T cells. Lymph node histology from patients indicated dysregulated germinal center reactions. Moreover, affected individuals displayed a profound reduction in circulating MAIT cell numbers. In summary, we show that this previously undescribed loss-of-function variant in Helios leads to an immunodeficiency with signs of immune overactivation.
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Fator de Transcrição Ikaros/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Adulto , Idoso , Feminino , Centro Germinativo/imunologia , Humanos , Fator de Transcrição Ikaros/sangue , Fator de Transcrição Ikaros/genética , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Granulosa cell tumors of the ovary represent â¼5% of malignant ovarian cancers. It has recently been reported that 95-97% of adult granulosa cell tumors carry a unique somatic mutation in the FOXL2 gene. We undertook this study to verify the presence of the FOXL2 Cys134Trp mutation in two geographically independent cohorts of granulosa cell tumors and to examine the expression pattern of FOXL2 in these tumors. A total of 56 tumors with the histological diagnosis of adult granulosa cell tumor from two centers, Melbourne and Helsinki, were examined for the presence of the mutation using direct sequence analysis. Two granulosa cell tumor-derived cell lines, COV434 and KGN, three juvenile granulosa cell tumors and control tissues were also examined. The expression of the FOXL2 gene was determined using quantitative RT-PCR and/or immunohistochemistry. We found that 52 of the 56 adult granulosa cell tumors harbor the mutation, of which three were hemi/homozygous. Of the four cases with wild-type FOXL2 sequence, reappraisal suggests that three may have been misclassified at primary diagnosis. The KGN cells were heterozygous for the mutation, whereas the COV434 cells had a wild-type FOXL2 genotype. The expression levels of FOXL2 were similar across the adult granulosa cell tumors and the normal ovary controls; one mutation-negative granulosa cell tumor had high FOXL2 mRNA levels, whereas the COV434 cells and two of the three juvenile granulosa cell tumors lacked the expression of FOXL2. Our data provide confirmation of the frequent presence of the FOXL2 C134W mutation in adult granulosa cell tumors and demonstrate that the mutation is not associated with altered FOXL2 expression. The mutation analysis may be a useful tool to differentiate particularly between cell-rich diffuse granulosa cell tumors and mitotically active sex cord-stromal tumors. This unique FOXL2 mutation appears to be characteristic of adult granulosa cell tumors.
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Fatores de Transcrição Forkhead/genética , Mutação , Neoplasias Ovarianas/genética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Pré-Escolar , Análise Mutacional de DNA , Feminino , Finlândia , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Genótipo , Tumor de Células da Granulosa/genética , Tumor de Células da Granulosa/metabolismo , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neoplasias Ovarianas/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VitóriaRESUMO
Adult-type granulosa cell tumors (AGCTs) are sex-cord derived neoplasms with a propensity for late relapse. Hormonal modulators have been used empirically in the treatment of recurrent AGCT, albeit with limited success. To provide a more rigorous foundation for hormonal therapy in AGCT, we used a multimodal approach to characterize the expressions of key hormone biomarkers in 175 tumor specimens and 51 serum samples using RNA sequencing, immunohistochemistry, RNA in situ hybridization, quantitative PCR, and circulating biomarker analysis, and correlated these results with clinical data. We show that FSH receptor and estrogen receptor beta (ERß) are highly expressed in the majority of AGCTs, whereas the expressions of estrogen receptor alpha (ERα) and G-protein coupled estrogen receptor 1 are less prominent. ERß protein expression is further increased in recurrent tumors. Aromatase expression levels show high variability between tumors. None of the markers examined served as prognostic biomarkers for progression-free or overall survival. In functional experiments, we assessed the effects of FSH, estradiol (E2), and the aromatase inhibitor letrozole on AGCT cell viability using 2 in vitro models: KGN cells and primary cultures of AGCT cells. FSH increased cell viability in a subset of primary AGCT cells, whereas E2 had no effect on cell viability at physiological concentrations. Letrozole suppressed E2 production in AGCTs; however, it did not impact cell viability. We did not find preclinical evidence to support the clinical use of aromatase inhibitors in AGCT treatment, and thus randomized, prospective clinical studies are needed to clarify the role of hormonal treatments in AGCTs.
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BACKGROUND: Sentinel node biopsy (SNB) is an important step in melanoma staging and prognostication. It is commonly performed for patients with intermediate thickness melanomas, based on clinicopathological features. However, only 20-25% of patients eventually demonstrate nodal involvement. The aim of this study was to evaluate whether tissue biomarkers with links to melanoma biology, together with clinicopathological parameters, could aid in the prediction of sentinel node involvement and improve selection of patients for SNB. In addition, we examined the role of these clinical or biological markers in disease outcome. METHODS: We collected a case-control cohort of 140 intermediate thickness (Breslow 0,9-4,0mm) melanoma patients with or without SNB involvement matched for age, gender, Breslow thickness and location. From this cohort, we tested the predictive value of common clinicopathological parameters (ulceration, mitotic count and tumor regression) and FMNL-2, ezrin and BRAF V600E immunoreactivity, for sentinel node involvement and survival. We further analyzed the correlations in the superficial spreading melanoma subtype. RESULTS: Based on our case control analysis, of the markers, BRAF V600E status (p = 0.010) and mitotic count (p = 0.036) correlated with SNB involvement. SNB status was a strong independent prognosticator for recurrence free survival (RFS p<0.001), melanoma specific survival (MSS p = 0.000) and overall survival (OS p = 0.029). In the superficially spreading melanoma subgroup, BRAF V600E positivity indicated poorer RFS (p = 0.039) and OS (p = 0.012). By combining the Breslow thickness, mitotic count and BRAF immunohistochemistry, we identified a group of superficially spreading melanomas with an excellent survival probability independent of SNB status. CONCLUSIONS: These results demonstrate that BRAF immunohistochemistry could serve as a useful addition to a marker panel for selecting intermediate thickness melanoma patients for SNB.