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Background: Autoimmune hepatitis (AIH) is a relatively rare autoimmune disease with a strong genetic background. The patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148 M (rs738409 C/G) variant has been associated with hepatic inflammation and fibrosis in chronic hepatic diseases beyond metabolic dysfunction-associated steatotic liver disease (MASLD). Aim: Our aim was to investigate the significance of PNPLA3 I148 M variant in AIH. Method: Two hundred AIH patients, followed in our centre, were evaluated while 100 healthy subjects served as controls. Genotyping was performed with allelic discrimination end-point polymerase chain reaction (PCR). Results: The I148 M variant was present in 95/200 (47.5 %) AIH patients compared to 47/100 (47 %) healthy controls (p = 1.000). Patients with GG/CG genotypes were more likely to present with decompensated cirrhosis at diagnosis (GG/CG 6.3 % vs. CC 1 %, p = 0.039). Comorbidity with cardiometabolic risk factors and concurrence of MASLD was similar across genotypes. Simple steatosis was present in 37/186 (19.9 %) and steatohepatitis in 14/186 (7.5 %) patients with available liver biopsy without correlation with PNPLA3 genotype. Fibrosis stage and grade of inflammation were not correlated with any genotype. Response to treatment was also independent of the presence of the I148 M variant, even though a longer time was needed to achieve complete biochemical response in those carrying the GG/CG genotypes (p = 0.07). On Kaplan Meier analysis homozygosity for the G allele corelated with reduced survival free of decompensation (p = 0.006), cirrhotic events (decompensation, liver transplantation, hepatocellular carcinoma; p = 0.001) and liver-related death or liver transplantation (p = 0.011) in treated patients. Conclusions: The PNPLA3 I148 M variant in AIH patients is associated with increased risk of advanced disease at diagnosis and reduced survival free of cirrhotic events and liver-related death or liver transplantation, regardless of the presence of MASLD. This signifies a potential role for the PNPLA3 I148 M variant as a new AIH biomarker allowing to identify patients at increased risk of disease progression.
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BACKGROUND AND AIMS: Metabolomics is used to predict, diagnose, and monitor metabolic disorders but altered metabolomic signatures have also been reported in diverse diseases, including autoimmune disorders. However, the metabolomic profile in autoimmune hepatitis (AIH) has not been investigated in depth. Therefore, we investigated the metabolomic signature of AIH and its significance as a diagnostic and pathogenetic tool. APPROACH AND RESULTS: Metabolites in plasma samples from 50 patients with AIH at diagnosis, 43 healthy controls, 72 patients with primary biliary cholangitis (PBC), 26 patients with metabolic dysfunction-associated liver disease, and 101 patients with chronic viral hepatitis were determined by 1 H NMR (nuclear magnetic resonance) spectroscopy. Fifty-two metabolites were quantified, and metabolic pathway analysis was performed. Multivariate analysis revealed that AIH could be differentiated from healthy controls and each of the disease controls ( p <0.001). Fifteen metabolites differentiated AIH from disease controls (PBC+chronic viral hepatitis+metabolic dysfunction-associated liver disease) (95% sensitivity and 92% specificity). Ten distinct metabolic pathways were altered in AIH compared to disease controls. The metabolic pathway of branched-chain amino acids (lower valine, leucine, and isoleucine levels and their catabolic intermediates in PBC), methionine (lower methionine, 2-aminobutyrate, and 2-hydroxybutyrate levels in PBC), alanine-aspartate-glutamate (lower metabolites in PBC), and that of metabolites associated with gut microbiota (lower choline, betaine, and dimethylamine levels in PBC) were significantly different between AIH and PBC ( p <0.01). CONCLUSIONS: 1 H NMR spectroscopy could be a promising novel tool to diagnose and study AIH pathogenesis as there is no need for much sample handling, is highly reproducible with high sensitivity and specificity, and low cost.
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Hepatite Autoimune , Metabolômica , Humanos , Hepatite Autoimune/diagnóstico , Hepatite Autoimune/sangue , Hepatite Autoimune/metabolismo , Masculino , Pessoa de Meia-Idade , Feminino , Metabolômica/métodos , Adulto , Espectroscopia de Ressonância Magnética/métodos , Idoso , Estudos de Casos e ControlesRESUMO
BACKGROUND & AIMS: Calprotectin reflects neutrophil activation and is increased in various inflammatory conditions including severe COVID-19. However, serial serum calprotectin measurements in COVID-19 patients are limited. We assessed prospectively, calprotectin levels as biomarker of severity/outcome of the disease and a COVID-19 monitoring parameter in a large cohort of consecutive COVID-19 patients. METHODS: Calprotectin serum levels were measured in 736 patients (58.2 % males; median age 63-years; moderate disease, n = 292; severe, n = 444, intubated and/or died, n = 50). Patients were treated with combined immunotherapies according to our published local algorithm. The endpoint was the composite event of intubation due to severe respiratory failure (SRF)/COVID-19-related mortality. RESULTS: Median (interquartile range) calprotectin levels were significantly higher in patients with severe disease [7(8.2) vs. 6.1(8.1)µg/mL, p = 0.015]. Calprotectin on admission was the only independent risk factor for intubation/death (HR=1.473, 95 %CI=1.003-2.165, p = 0.048) even after adjustment for age, sex, body mass index, comorbidities, neutrophils, lymphocytes, neutrophil to lymphocytes ratio, ferritin, and CRP. The area under the curve (AUC, 95 %CI) of calprotectin for prediction of intubation/death was 0.619 (0.531-0.708), with an optimal cut-off at 13 µg/mL (sensitivity: 44 %, specificity: 79 %, positive and negative predictive values: 13 % and 95 %, respectively). For intubated/died patients, paired comparisons from baseline to middle of hospitalization and subsequently to intubation/death showed significant increase of calprotectin (p = 0.009 and p < 0.001, respectively). Calprotectin alteration had the higher predictive ability for intubation/death [AUC (95 %CI):0.803 (0.664-0.943), p < 0.001]. CONCLUSIONS: Calprotectin levels on admission and their subsequent dynamic alterations could serve as indicator of COVID-19 severity and predict the occurrence of SRF and mortality.
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COVID-19 , Complexo Antígeno L1 Leucocitário , Masculino , Humanos , Pessoa de Meia-Idade , Feminino , Estudos Prospectivos , Seguimentos , COVID-19/terapia , Biomarcadores , Estudos RetrospectivosRESUMO
Background: Hepatitis E virus (HEV) infection incidence is increasing in Europe, accounting for the majority of acute hepatitis cases. We investigated the prevalence and clinical characteristics of acute hepatitis E (AHE) in patients with acute non-A/B/C hepatitis from central Greece, their differences from acute autoimmune hepatitis (AIH) patients and the molecular similarity of human strains to local HEV strains in wild boars. Methods: Sera from 20 patients with non-A/B/C acute hepatitis (2015-2017) were tested prospectively for anti-HEV IgM, IgG antibodies and HEV-RNA. Sera from patients diagnosed with acute AIH (2000-2015; n=56) were tested retrospectively. Liver tissue samples from 40 wild boars were tested for HEV-RNA. Positive wild boar and patients' samples were sequenced and phylogenetically analyzed. Results: Twelve of the 76 (16%) patients were diagnosed with AHE: HEV-RNA 11.5x104 (38.7-39.7x106) IU/mL; 11/20 (55%) acute non-A/B/C hepatitis and 1/56 (2%) AIH patients. Patients with AHE were older than those without, predominately men, with higher alanine aminotransferase but lower IgG levels (P=0.005 and P=0.002, respectively), and had high titers of smooth muscle antibodies. Liver biopsies, performed in 6/12 patients with HEV infection, revealed histology compatible with AIH. HEV strains from both patients and wild boars belonged to genotype 3. Conclusions: Approximately one sixth of patients with acute non-A/B/C hepatitis had autochthonous HEV infection with AIH features. Therefore, a careful workup to exclude HEV should be carried out in all acute hepatitis cases before a definite diagnosis of AIH is established. Wild boars seem to be an important reservoir of HEV in Greece.
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Autoimmune hepatitis (AIH) is an acute or chronic inflammatory disease of the liver caused by an immune response of unknown origin. It affects people from all ethnic groups irrespective of age or sex. AIH is characterized by hyperglobulinemia, presence of circulating autoantibodies, and liver inflammation. The clinical picture of the disease varies from asymptomatic or mild to severe acute hepatitis or liver failure. A timely and prompt diagnosis is of utmost importance to prevent progression to advanced liver disease by immediate initiation of immunosuppressive treatment. So far, several diagnostic scoring systems have been proposed, which incorporated demographic data as well as biochemical, clinical, and histological characteristics of the disease. However, due to the high heterogeneity of the disease presentation, diagnosis of AIH remains challenging. Most patients initially respond to first line treatment, which consists of corticosteroids combined with azathioprine or mycophenolate mofetil. However, insufficient response to the treatment and intolerance due to side effects are common, so a significant proportion of patients require second- and / or third line therapies. Herein, we review the challenges and recent advances in AIH diagnosis and management.
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Hepatite Autoimune , Autoanticorpos , Azatioprina/uso terapêutico , Hepatite Autoimune/diagnóstico , Hepatite Autoimune/tratamento farmacológico , Humanos , Imunossupressores/uso terapêutico , Ácido Micofenólico/uso terapêuticoRESUMO
HBV RNA is considered as a promising predictor in patients who discontinue nucleos(t)ide analogues (NAs). We determined HBV RNA levels in non-cirrhotic HBeAg-negative patients who discontinued NAs and assessed their predictability for 12-month outcomes. Fifty-seven patients of DARING-B study were included. HBV RNA levels were determined in stored monthly serum samples drawn at 0-3 months after end of therapy (EOT). Other markers previously determined in the same cohort including hepatitis B core-related antigen (HBcrAg) were also assessed. HBV RNA at EOT was detectable in 7% of patients, who developed virological/clinical relapse and required retreatment at month 2; in patients with undetectable EOT HBV RNA, 12-month cumulative rates of virological relapse, clinical relapse and retreatment were 68%, 28% and 21%, respectively (p ≤ 0.008). HBV RNA at month-1 after EOT was detectable in 19% of patients being associated with higher probability only of virological relapse (p = 0.001). HBV RNA levels correlated significantly to HBV DNA, HBcrAg, ALT and interferon-induced protein-10, but not HBsAg levels. Combined EOT HBV RNA and HBcrAg detection and/or HBsAg >1000 IU/ml was associated only with higher probability of retreatment having higher sensitivity and lower specificity than HBV RNA alone. In conclusion, serum HBV RNA is detectable in a minority of non-cirrhotic HBeAg-negative patients under effective long-term NAs therapy offering low sensitivity but 100% specificity for early retreatment due to severe clinical relapses after NA discontinuation. The combinations of EOT HBV RNA with HBcrAg and/or high HBsAg levels increase sensitivity but decrease specificity for prediction of retreatment after NAs withdrawal.
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Hepatite B Crônica , Antivirais/uso terapêutico , DNA Viral , Antígenos do Núcleo do Vírus da Hepatite B , Antígenos de Superfície da Hepatite B , Antígenos E da Hepatite B , Vírus da Hepatite B/genética , Humanos , Interferons/uso terapêutico , RNA , RecidivaRESUMO
Risk stratification of coronavirus disease-19 (COVID-19) patients by simple markers is critical to guide treatment. We studied the predictive value of soluble interleukin-2 receptor (sIL-2R) for the early identification of patients at risk of developing severe clinical outcomes. sIL-2R levels were measured in 197 patients (60.9% males; median age 61 years; moderate disease, n = 65; severe, n = 132, intubated and/or died, n = 42). All patients received combined immunotherapies (anakinra ± corticosteroids ± intravenous immunoglobulin ± tocilizumab) according to our local treatment algorithm. The endpoint was the composite event of intubation due to severe respiratory failure (SRF) or mortality. Median (interquartile range) sIL-2R levels were significantly higher in patients with severe disease, compared with those with moderate disease (6 (6.2) vs. 5.2 (3.4) ng/mL, p = 0.017). sIL-2R was the strongest laboratory predictive factor for intubation/death (hazard ratio 1.749, 95%CI 1.041-2.939, p = 0.035) after adjustment for other known risk factors. Youden's index revealed optimal sIL-2R cut-off for predicting intubation/death at 9 ng/mL (sensitivity: 67%; specificity: 86%; positive and negative predictive value: 57% and 91%, respectively). Delta sIL-2R between the day of event or discharge minus admission date was higher in patients that intubated/died than in those who did not experience an event (2.91 (10.42) vs. 0.44 (2.88) ng/mL; p = 0.08)). sIL-2R on admission and its dynamic changes during follow-up may reflect disease severity and predict the development of SRF and mortality.
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COVID-19 , Receptores de Interleucina-2 , Insuficiência Respiratória , Biomarcadores , COVID-19/metabolismo , COVID-19/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Receptores de Interleucina-2/sangue , Receptores de Interleucina-2/metabolismo , Insuficiência Respiratória/diagnóstico , Insuficiência Respiratória/metabolismoRESUMO
BACKGROUND AND AIMS: Little is known about the impact of DNA methylation modifications on autoimmune hepatitis (AIH) pathogenesis and therapeutic response. We investigated the potential alterations of DNA methylation in AIH peripheral lymphocytes at diagnosis and remission. METHODS: Ten AIH patients at diagnosis (time-point 1; AIH-tp1), 8/10 following biochemical response (time-point 2; AIH-tp2), 9 primary biliary cholangitis (PBC) and 10 healthy controls (HC) were investigated. Peripheral CD19(+) and CD4(+) cells were isolated. Global DNA methylation (5m C)/hydroxymethylation (5hm C) was studied by ELISAs. mRNA of DNA methylation (DNMT1/3A/3B) and their counteracting hydroxymethylation enzymes (TET1/2/3) was determined by quantitative RT-PCR. Epigenome wide association study (EWAS) was performed in CD4(+) cells (Illumina HumanMethylation 850 K array) in AIH and HC. Total 5m C/5hm C was also assessed by immunohistochemistry (IHC) on paraffin-embedded liver sections. RESULTS: Reduced TET1 and increased DNMT3A mRNA levels characterized CD19(+) and CD4(+)-lymphocytes from AIH-tp1 compared to HC and PBC, respectively, without affecting global DNA 5m C/5hm C. In AIH-tp1, CD4(+) DNMT3A expression was negatively correlated with serum IgG (P = .03). In remission, DNMT3A decreased in both CD19(+) and CD4(+) cells compared to AIH-tp1 (P = .02, P = .03 respectively). EWAS in CD4(+) cells from AIH patients confirmed important modifications in genes implicated in immune responses (HLA-DP, TNF, lnRNAs and CD86). IHC showed increased 5hm C staining of periportal infiltrating lymphocytes in AIH-tp1 compared to HC and PBC. CONCLUSION: Altered TET1 and DNMT3A expressions, characterize peripheral lymphocytes in AIH. DNMT3A was associated with disease activity and decreased following remission. Gene DNA methylation modifications affect immunological pathways that may play an important role in AIH pathogenesis.
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Hepatite Autoimune , Cirrose Hepática Biliar , Linfócitos T CD4-Positivos , Metilação de DNA , Hepatite Autoimune/diagnóstico , Humanos , Cirrose Hepática Biliar/complicações , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , RNA MensageiroRESUMO
Autoimmune hepatitis (AIH) is a chronic non-resolving liver disease characterized by diffuse hypergammaglobulinemia, the presence of autoantibodies and characteristic histological findings. The disease can have catastrophic outcome with the development of end-stage liver disease if misdiagnosed/undiagnosed and left untreated. AIH pathogenesis remains obscure and the main hypothesis supports its development in genetically predisposed individuals after being exposed to certain environmental triggers. Genetic predisposition is linked to the presence of certain HLA alleles, mainly HLA-DR3 and HLA-DR4. However, a wide number of non-HLA epitopes have also been associated with the disease although data vary significantly among different ethnic groups. Therefore, it is likely that epigenetic alterations may also play a crucial role in disease's pathogenesis, although not yet extensively studied. The aim of this review was to summarize the genetic and environmental factors that have been associated with AIH, but also to open new insights towards the role of epigenetic modifications in the etiology of the disease.
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BACKGROUND: Autoimmune hepatitis (AIH) is a relatively rare chronic liver disease of unknown etiology. The genetic background affects susceptibility, clinical phenotype, and prognosis. The programmed cell death-1 rs11568821 polymorphism (PD1.3) has been associated with susceptibility to autoimmune diseases. The interleukin-28B (IL28B) rs12979860 polymorphism has been associated with steatosis, inflammation, and fibrosis in liver diseases. AIM: Our aim was to investigate for the first time the incidence and clinical significance of PD1.3 and IL28B rs12979860 in AIH. METHODS: Two hundred patients with AIH were evaluated, while 100 healthy subjects were used as controls. Genotyping was performed with in-house allelic discrimination End-Point PCR. RESULTS: The SNP PD1.3/A was present in 36/200 (18%) AIH patients compared to 28/100 (28%) healthy controls (p = 0.065). The AA/GA genotypes were not associated with the mode of presentation of AIH, the histological grade or stage, the presence of cirrhosis, risk of disease progression, response to treatment and survival. The IL28B rs12979860 genotype distribution was CC 79/200 (39.5%), TT 36/200 (18%) and CT 85/200 (42.5%), in similar rates with healthy controls (p = 0.878). Inflammatory activity and fibrosis stage did not differ between CC homozygotes and CT/TT carriers. LDL cholesterol was significantly higher in CC than CT/TT patients (P = 0.027), though no differences was found regarding the presence of steatosis or steatohepatitis. On-treatment response to immunosuppressive treatment was not affected by the IL28B rs12979860 polymorphism. However, CC homozygotes AIH patients achieved treatment withdrawal in significantly higher rates (OR 2.3, 95%CI: 1.1-4.7, P = 0.02) irrespective of the presence of steatosis or steatohepatitis. CONCLUSIONS: The PD1.3 and IL28B rs12979860 variants are unlikely to contribute to AIH susceptibility, disease presentation and prognosis. The IL28B rs12979860 is not associated with the presence of concurrent steatosis or steatohepatitis. However, although on-treatment response rates to immunosuppression were not affected by the IL28B rs12979860 polymorphism, AIH patients with CC homozygosity were more likely to achieve complete treatment withdrawal. This novel finding needs validation and further clarification from larger multicenter studies.
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Reliable predictors of outcomes after treatment discontinuation in HBeAg-negative chronic hepatitis B (CHB) patients have not been established. We investigated the role of hepatitis B surface antigen (HBsAg), interferon-inducible protein-10 (IP10) and hepatitis B core-related antigen (HBcrAg) serum levels as predictors of HBsAg loss, relapse and retreatment in noncirrhotic HBeAg-negative CHB patients who discontinued long-term antiviral therapy. All HBsAg-positive (n = 57) patients of the prospective DARING-B study were included and followed monthly for 3 months, every 2/3 months until month-12 and every 3/6 months thereafter. HBsAg, IP10 and HBcrAg levels were measured by enzyme immunoassays, and SCALE-B score was calculated. Twelve patients achieved HBsAg loss before retreatment with 18-month cumulative incidence of 25%. Independent predictors of HBsAg loss were baseline HBsAg and month-1 IP10 levels. Of 10 patients with baseline HBsAg ≤100 IU/mL, 70% cleared HBsAg and 10% required retreatment. Of 23 patients with baseline HBsAg >1000 IU/mL, 4% cleared HBsAg and 43% required retreatment. Of 24 patients with intermediate baseline HBsAg (100-1000 IU/mL), 17% cleared HBsAg and 21% required retreatment; in this subgroup, month-1 IP10 was significantly associated with HBsAg loss, which occurred in 30% and 7% of cases with IP10 >150 and ≤150 pg/mL, respectively. Baseline HBcrAg was undetectable in all patients who cleared HBsAg and was associated with retreatment. SCALE-B was associated with HBsAg loss but not with relapse or retreatment. In conclusion, HBsAg, IP10 and HBcrAg serum levels can be useful for the decisions and management of treatment discontinuation in noncirrhotic Caucasian patients with HBeAg-negative CHB.
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Antivirais/uso terapêutico , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/tratamento farmacológico , Idoso , Quimiocina CXCL10/sangue , Feminino , Antígenos do Núcleo do Vírus da Hepatite B/sangue , Humanos , Cirrose Hepática , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Recidiva , RetratamentoRESUMO
Sjögren's syndrome (SjS) and primary biliary cholangitis (PBC) can be classified as a model of generalized autoimmune epithelitis based on their frequent coexistence in clinical practice and the highly specific immune mediated injury of target epithelial cells. Both of these autoimmune diseases are characterized by female predominance, highly specific circulating autoantibodies, and immune-mediated destruction of the salivary and lachrymal glands and the biliary epithelial cells, respectively. Although the genetic predisposition has been well described for both diseases, genetic studies have failed to completely elucidate their pathogenesis. The recent integration of epigenetic data, analyzing the different cellular partners, opens new perspectives and allows for better understanding of these complex and still incurable diseases. Epigenetic studies on SjS have elucidated the role of DNA methylation alterations in disease pathogenesis, while epigenetic changes that influence expression of genes on the X chromosome have been implicated in the geo-variability and occurrence of PBC. The aim of this review is to describe the advances in epigenetics in the field of autoimmune epithelitis as well as to highlight how epigenetic changes could contribute to better understanding of disease pathogenesis and progression. These advances could yield insights on novel therapeutic interventions.
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Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function.
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Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/química , Proteínas de Ligação à Região de Interação com a Matriz/genética , Microscopia de Fluorescência , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Ratos , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Processamento de Serina-Arginina/química , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: We investigated the impact of PIK3CA and TP53 mutations and p53 protein status on the outcome of patients who had been treated with adjuvant anthracycline-taxane chemotherapy within clinical trials in the pre- and post-trastuzumab era. RESULTS: TP53 and PIK3CA mutations were found in 380 (21.5%) and 458 (25.9%) cases, respectively, including 104 (5.9%) co-mutated tumors; p53 immunopositivity was observed in 848 tumors (53.5%). TP53 mutations (p < 0.001) and p53 protein positivity (p = 0.001) were more frequent in HER2-positive and triple negative (TNBC) tumors, while PIK3CA mutations were more frequent in Luminal A/B tumors (p < 0.001). TP53 mutation status and p53 protein expression but not PIK3CA mutation status interacted with trastuzumab treatment for disease-free survival; patients with tumors bearing TP53 mutations or immunopositive for p53 protein fared better when treated with trastuzumab, while among patients treated with trastuzumab those with the above characteristics fared best (interaction p = 0.017 for mutations; p = 0.015 for IHC). Upon multivariate analysis the above interactions remained significant in HER2-positive patients; in the entire cohort, TP53 mutations were unfavorable in patients with Luminal A/B (p = 0.003) and TNBC (p = 0.025); p53 immunopositivity was strongly favorable in patients treated with trastuzumab (p = 0.009). MATERIALS AND METHODS: TP53 and PIK3CA mutation status was examined in 1766 paraffin tumor DNA samples with informative semiconductor sequencing results. Among these, 1585 cases were also informative for p53 protein status assessed by immunohistochemistry (IHC; 10% positivity cut-off). CONCLUSIONS: TP53 mutations confer unfavorable prognosis in patients with Luminal A/B and TNBC tumors, while p53 immunopositivity may predict for trastuzumab benefit in the adjuvant setting.
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Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/genética , Análise Mutacional de DNA , Imuno-Histoquímica , Mutação , Trastuzumab/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Quimioterapia Adjuvante , Distribuição de Qui-Quadrado , Classe I de Fosfatidilinositol 3-Quinases/análise , Classe I de Fosfatidilinositol 3-Quinases/genética , Ensaios Clínicos como Assunto , Progressão da Doença , Intervalo Livre de Doença , Feminino , Predisposição Genética para Doença , Grécia , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Análise Multivariada , Seleção de Pacientes , Fenótipo , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Estudos Prospectivos , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/química , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/análise , Adulto JovemRESUMO
Hepcidin, a liver hormone, is important for both innate immunity and iron metabolism regulation. As dysfunction of the hepcidin pathway may contribute to liver pathology, we analysed liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases. Hepcidin mRNA levels were determined in liver biopsies obtained from 126 patients with HCV (n = 21), HBV (n = 23), autoimmune cholestatic disease (primary biliary cirrhosis and primary sclerosing cholangitis; PBC/PSC; n = 34), autoimmune hepatitis (AIH; n = 16) and non-alcoholic fatty liver disease (NAFLD; n = 32). Sera sampled on the biopsy day from the same patients were investigated for serum hepcidin levels. Hepatic hepcidin mRNA levels correlated positively with ferritin and negatively with serum γ-GT levels. However, no correlation was found between serum hepcidin and either ferritin or liver hepcidin mRNA. Both serum hepcidin and the serum hepcidin/ferritin ratio were significantly lower in AIH and PBC/PSC patients' sera compared to HBV, HCV or NAFLD (P<0.001 for each comparison) and correlated negatively with serum ALP levels. PBC/PSC and AIH patients maintained low serum hepcidin during the course of their two-year long treatment. In summary, parallel determination of liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases shows that circulating hepcidin and its respective ratio to ferritin are significantly diminished in patients with autoimmune liver diseases. These novel findings, once confirmed by follow-up studies involving bigger size and better-matched disease subgroups, should be taken into consideration during diagnosis and treatment of autoimmune liver diseases.
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Colangite Esclerosante/patologia , Hepatite Autoimune/patologia , Hepcidinas/sangue , Hepcidinas/genética , Cirrose Hepática Biliar/patologia , Adulto , Idoso , Colangite Esclerosante/sangue , Colangite Esclerosante/genética , Diagnóstico Diferencial , Regulação para Baixo , Feminino , Ferritinas/sangue , Hepatite B/sangue , Hepatite B/genética , Hepatite B/patologia , Hepatite C/sangue , Hepatite C/genética , Hepatite C/patologia , Hepatite Autoimune/sangue , Hepatite Autoimune/genética , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/genética , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
AIM: Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA. METHODS: Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible. RESULTS: Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman's rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8-99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1-95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3-76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality. CONCLUSIONS: Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Proteínas de Neoplasias/genética , Neoplasias da Mama/patologia , Feminino , Formaldeído , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Inclusão em Parafina , Projetos Piloto , Semicondutores , Fixação de TecidosRESUMO
Bone hypoxia resulting from impaired blood flow is the final pathway for the development of osteonecrosis (ON). The aim of this study was to evaluate if HIF-1α, the major transcription factor triggered by hypoxia, is genetically implicated in susceptibility to ON. For this we analyzed frequencies of three known HIF-1α polymorphisms: one in exon 2 (C111A) and two in exon 12 (C1772T and G1790A) and their association with ON in a Greek population. Genotype analysis was performed using PCR-RFLP and rare alleles were further confirmed with sequencing. We found that genotype and allele frequency of C1772T and G1790A SNP of HIF-1α (SNPs found in our cohort) were not significantly different in ON patients compared to control patients. Furthermore these SNPs could not be associated with the different subgroups of ON. At the protein level we observed that the corresponding mutations (P582S and A588T, respectively) are not significant for protein function since the activity, expression and localization of the mutant proteins is practically indistinguishable from wt in HEK293 and Saos-2 cells. These results suggest that these missense mutations in the HIF-1α gene are not important for the risk of developing ON.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Osteonecrose/genética , Adolescente , Adulto , Idoso , Linhagem Celular , Éxons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto JovemRESUMO
Hepcidin, a hepatic hormone, regulates serum iron levels by controlling both intestinal iron absorption and iron release from macrophages. Although transcription of hepcidin is controlled by diverse stimuli, it remains elusive if post-transcriptional steps of its production are also regulated. To address this issue, GFP was fused to the C-terminus of hepcidin and the chimeric hepcidin-GFP protein was expressed in hepatoma Huh7 cells. Expression and secretion of hepcidin-GFP were analyzed by fluorescence microscopy or western blotting and its activity was assessed by in vitro biological assays. Transient over-expression of hepcidin-GFP resulted in production and secretion of premature forms. On the other hand, stable low-level expression led to synthesis and secretion of a properly matured hepcidin-GFP. This form was biologically active since it affected appropriately the levels of IRP2 and ferritin in human THP1 monocytes and targeted ferroportin in mouse J774 macrophages. Treatment of hepcidin-GFP expressing cells with hypoxia (0.1% O2) altered the subcellular distribution of pro-hepcidin-GFP and significantly reduced the secretion of mature hepcidin-GFP. Our hepcidin-GFP expression system allows the investigation of post-transcriptional processing of hepcidin and implicates hypoxia in its secretion control.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Carcinoma Hepatocelular/metabolismo , Quimera/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Neoplasias Hepáticas/metabolismo , Oxigênio/metabolismo , Animais , Linhagem Celular Tumoral , Quimera/genética , Proteínas de Fluorescência Verde/genética , Hepcidinas , Humanos , Macrófagos , Camundongos , Proteínas Recombinantes/metabolismoRESUMO
Hypoxia inducible factor-1 (HIF-1), a dimeric transcription factor of the bHLH-PAS family, is comprised of HIF-1α, which is inducible by hypoxia and ARNT or HIF-1ß, which is constitutively expressed. HIF-1 is involved in cellular homeostasis under hypoxia, in development and in several diseases affected by oxygen availability, particularly cancer. Since its expression is positively correlated with poor outcome prognosis for cancer patients, HIF-1 is a target for pharmaceutical therapy. We have previously shown that male germ cell Rac GTPase activating protein (MgcRacGAP), a regulator of Rho proteins which are principally involved in cytoskeletal organization, binds to HIF-1α and inhibits its transcriptional activity. In this work, we have explored the mechanism of the MgcRacGAP-mediated HIF-1 inactivation. We show that the Myo domain of MgcRacGAP, which is both necessary and sufficient for HIF-1 repression, binds to the PAS-B domain of HIF-1α. Furthermore MgcRacGAP competes with ARNT for binding to the HIF-1α PAS-B domain, as shown by in vitro binding pull down assays. In mammalian cells, ARNT overexpression can overcome the MgcRacGAP-mediated inhibition and MgcRacGAP binding to HIF-1α in vivo inhibits its dimerization with ARNT. We additionally present results indicating that MgcRacGAP binding to HIF-1α is specific, since it does not affect the transcriptional activity of HIF-2, a close evolutionary relative of HIF-1 also involved in hypoxia regulation and cancer. Our results reveal a new mechanism for HIF-1 transcriptional activity regulation, suggest a novel hypoxia-cytoskeleton link and provide new tools for selective HIF-1 inhibition.