RESUMO
Abberrant DNA methylation is one of the hallmarks of cancerogenesis. Our study aims to delineate differential DNA methylation in cirrhosis and hepatic cancerogenesis. Patterns of methylation of 27,578 individual CpG loci in 12 hepatocellular carcinomas (HCCs), 15 cirrhotic controls and 12 normal liver samples were investigated using an array-based technology. A supervised principal component analysis (PCA) revealed 167 hypomethylated loci and 100 hypermethylated loci in cirrhosis and HCC as compared to normal controls. Thus, these loci show a "cirrhotic" methylation pattern that is maintained in HCC. In pairwise supervised PCAs between normal liver, cirrhosis and HCC, eight loci were significantly changed in all analyses differentiating the three groups (p < 0.0001). Of these, five loci showed highest methylation levels in HCC and lowest in control tissue (LOC55908, CELSR1, CRMP1, GNRH2, ALOX12 and ANGPTL7), whereas two loci showed the opposite direction of change (SPRR3 and TNFSF15). Genes hypermethylated between normal liver to cirrhosis, which maintain this methylation pattern during the development of HCC, are depleted for CpG islands, high CpG content promoters and polycomb repressive complex 2 (PRC2) targets in embryonic stem cells. In contrast, genes selectively hypermethylated in HCC as compared to nonmalignant samples showed an enrichment of CpG islands, high CpG content promoters and PRC2 target genes (p < 0.0001). Cirrhosis and HCC show distinct patterns of differential methylation with regards to promoter structure, PRC2 targets and CpG islands.
Assuntos
Carcinoma Hepatocelular/genética , Metilação de DNA , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Ilhas de CpG , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Humanos , Proteínas do Grupo Polycomb , Análise de Componente Principal/métodos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Reprodutibilidade dos TestesRESUMO
Members of the CCN [cystein-rich 61 (Cyr61)/connective tissue growth factor (CTGF)/nephroblastoma (NOV)] protein family are involved in the regulation of cellular proliferation, apoptosis, and migration and are also assumed to play a role in carcinogenesis. Therefore, we performed a retrospective study to investigate the immunohistochemical expression of both Cyr61 and CTGF in 92 borderline tumors (BOTs) and 107 invasive carcinomas of the ovary (IOCs). To determine their diagnostic and prognostic value, we correlated protein expression with clinicopathologic factors including overall and disease-free survival. Cyr61 and CTGF were found to be inversely expressed in both BOTs and IOCs, with a stronger expression of Cyr61 in IOCs. Moreover, Cyr61 was found to be preferentially expressed in high-grade serous carcinomas, whereas CTGF was found more frequently in low-grade serous carcinomas. Weak Cyr61 levels correlated with both low estrogen receptor and p53 expression (P=0.038, P=0.04, respectively). However, no association was observed between CTGF, estrogen receptor, and p53 expression levels in IOCs. Regarding prognosis, Cyr61 was found to be of no value, but the loss of CTGF was found to be associated with a poor prognosis in multivariate analysis of overall (relative risk 2.8; P=0.050) and disease-free (relative risk 2.3; P=0.031) survival. Cyr61 and CTGF are inversely expressed in BOTs and IOCs, and loss of CTGF independently indicates poor prognosis in IOCs.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/análise , Proteína Rica em Cisteína 61/análise , Neoplasias Epiteliais e Glandulares/química , Neoplasias Ovarianas/química , Adulto , Idoso , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Fator de Crescimento do Tecido Conjuntivo/fisiologia , Proteína Rica em Cisteína 61/fisiologia , Tubas Uterinas/química , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Ovarianas/mortalidade , Ovário/química , Prognóstico , Estudos RetrospectivosAssuntos
Adenoma/complicações , Micetoma/complicações , Doenças dos Seios Paranasais/complicações , Neoplasias Hipofisárias/complicações , Adenoma/patologia , Adenoma/fisiopatologia , Adenoma/terapia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Micetoma/patologia , Micetoma/fisiopatologia , Micetoma/terapia , Doenças dos Seios Paranasais/patologia , Doenças dos Seios Paranasais/fisiopatologia , Doenças dos Seios Paranasais/terapia , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/fisiopatologia , Neoplasias Hipofisárias/terapia , Seio EsfenoidalRESUMO
We investigated various phenotypic and genotypic biomarkers of gastric cancer (GC) testing the following hypotheses: are these biomarkers suitable for the identification of GC subtypes, are they of prognostic significance, and should any of these biomarkers be considered to tailor patient treatment in the future. The study cohort consisted of 482 patients. pTNM-stage was based on surgical pathologic examination. The Laurén and mucin phenotype was assessed. Helicobacter pylori and Epstein-Barr virus infections were documented. The following biomarkers were determined: BRAF, KRAS, NRAS, and PIK3CA genotype, microsatellite instability, mucin 1, mucin 2, mucin 5, and mucin 6, CD10, E-cadherin, ß-catenin, and lysozyme. The histologic phenotype correlated with 10/13 (77%) clinicopathologic patient characteristics and 6/13 (46%) immunohistochemical/molecular biological biomarkers. Inversely, immunohistochemical biomarkers (mucin phenotype, E-cadherin, ß-catenin, and lysozyme) were unsuitable for subclassification of GC. It showed too much overlap between the different subtypes. Among the genotypes, only microsatellite instability correlated with tumor type being more prevalent in intestinal and unclassified GCs. Patient survival correlated significantly with 8 (62%) clinicopathologic and 5 (36%) immunohistochemical/molecular biomarkers. Interestingly, in proximal GCs, KRAS mutation was associated with worse prognosis, as was persistent H. pylori infection in unclassified GCs. Mucin 2 (all patients, proximal GCs) and PIK3CA (exon 20; intestinal type GC) prognosticated independently patient survival. The biomarkers examined herein are unsuitable to aid histologic classification of GC. However, several of them show a correlation with either phenotype and/or prognosis and may be considered to tailor patient treatment in the future, such as KRAS, PIK3CA, MSI, and H. pylori status.
Assuntos
Biomarcadores/análise , Patologia/métodos , Medicina de Precisão/métodos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Análise em Microsséries/métodos , Pessoa de Meia-Idade , PrognósticoRESUMO
BACKGROUND AND AIMS: High frequency electrosurgery has a key role in the broadening application of liver surgery. Its molecular signature, i.e. the metabolites evolving from electrocauterization which may inhibit hepatic wound healing, have not been systematically studied. METHODS: Human liver samples were thus obtained during surgery before and after electrosurgical dissection and subjected to a two-stage metabolomic screening experiment (discovery sample: N = 18, replication sample: N = 20) using gas chromatography/mass spectrometry. RESULTS: In a set of 208 chemically defined metabolites, electrosurgical dissection lead to a distinct metabolic signature resulting in a separation in the first two dimensions of a principal components analysis. Six metabolites including glycolic acid, azelaic acid, 2-n-pentylfuran, dihydroactinidiolide, 2-butenal and n-pentanal were consistently increased after electrosurgery meeting the discovery (p<2.0 × 10(-4)) and the replication thresholds (p<3.5 × 10(-3)). Azelaic acid, a lipid peroxidation product from the fragmentation of abundant sn-2 linoleoyl residues, was most abundant and increased 8.1-fold after electrosurgical liver dissection (preplication = 1.6 × 10(-4)). The corresponding phospholipid hexadecyl azelaoyl glycerophosphocholine inhibited wound healing and tissue remodelling in scratch- and proliferation assays of hepatic stellate cells and cholangiocytes, and caused apoptosis dose-dependently in vitro, which may explain in part the tissue damage due to electrosurgery. CONCLUSION: Hepatic electrosurgery generates a metabolic signature with characteristic lipid peroxidation products. Among these, azelaic acid shows a dose-dependent toxicity in liver cells and inhibits wound healing. These observations potentially pave the way for pharmacological intervention prior liver surgery to modify the metabolic response and prevent postoperative complications.
Assuntos
Ácidos Dicarboxílicos/farmacologia , Eletrocirurgia , Fígado/metabolismo , Metaboloma , Fosforilcolina/análogos & derivados , Cicatrização/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Linhagem Celular , Ácidos Dicarboxílicos/isolamento & purificação , Ácidos Dicarboxílicos/metabolismo , Dissecação/métodos , Relação Dose-Resposta a Droga , Feminino , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos , Fígado/patologia , Fígado/cirurgia , Masculino , Pessoa de Meia-Idade , Fosforilcolina/isolamento & purificação , Fosforilcolina/metabolismo , Fosforilcolina/farmacologia , Análise de Componente PrincipalRESUMO
Gastric cancer (GC) is one of the most common causes of cancer-related deaths worldwide. We investigated the differential expression and putative tumor biological significance of five G-protein-coupled receptors (GPCRs) in GC, i.e., LGR4, LGR6, GPR34, GPR160, and GPR171. Based on our previous microarray analyses, we identified five candidate genes in human GC samples. Real-time RT-PCR was carried out to validate their expression in malignant and non-malignant tissues on an independent collective comprising 32 GC patients with and without lymph node metastases. Selected protein targets LGR4 and LGR6 were further validated on paraffin-embedded sections of ten intestinal and ten poorly cohesive (diffuse)-type GCs and their corresponding non-malignant tissue using immunohistochemistry. Additionally, the putative tumor biological significance of LGR4 and LGR6 was studied using tissue microarrays obtained from a cohort of 481 GC patients. On transcriptional level, GPR34, GPR160, and GPR171 were not differentially expressed in GC compared with non-neoplastic mucosa. LGR4 and LGR6 were up-regulated on transcriptional (real-time RT-PCR) and translational (immunohistochemistry) levels in GC. Furthermore, in tissue microarray analysis, LGR6 expression was significantly associated with local tumor growth (T-category; p = 0.04) and correlated with patient survival. LGR4 expression was significantly correlated with nodal spread (N-category; p = 0.025). Our systematic analysis indicates that LGR4 and LGR6 may play a role in GC biology. Future studies will have to demonstrate whether these are also putative diagnostic, prognostic, and/or therapeutic targets for GC.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Mucosa Gástrica/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Biópsia , Estudos de Casos e Controles , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Receptores de Lisofosfolipídeos/metabolismo , Estômago/patologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Taxa de Sobrevida , Regulação para CimaRESUMO
In most adult humans, hepatitis B is a self-limiting disease leading to life-long protective immunity, which is the consequence of a robust adaptive immune response occurring weeks after hepatitis B virus (HBV) infection. Notably, HBV-specific T cells can be detected shortly after infection, but the mechanisms underlying this early immune priming and its consequences for subsequent control of viral replication are poorly understood. Using primary human and mouse hepatocytes and mouse models of transgenic and adenoviral HBV expression, we show that HBV-expressing hepatocytes produce endoplasmic reticulum (ER)-associated endogenous antigenic lipids including lysophospholipids that are generated by HBV-induced secretory phospholipases and that lead to activation of natural killer T (NKT) cells. The absence of NKT cells or CD1d or a defect in ER-associated transfer of lipids onto CD1d results in diminished HBV-specific T and B cell responses and delayed viral control in mice. NKT cells may therefore contribute to control of HBV infection through sensing of HBV-induced modified self-lipids.
Assuntos
Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/prevenção & controle , Imunidade/imunologia , Metabolismo dos Lipídeos/imunologia , Células T Matadoras Naturais/imunologia , Imunidade Adaptativa/imunologia , Adenoviridae , Animais , Antígenos CD1d/metabolismo , Biomarcadores , Proteínas de Transporte/metabolismo , Técnicas de Cocultura , Antígenos de Superfície da Hepatite B/imunologia , Hepatite B Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Lisofosfolipídeos/metabolismo , Lisossomos/metabolismo , Camundongos , Fosfolipases A2 Secretórias/metabolismoRESUMO
Evaluation of: Magnin S, Viel E, Baraquin A et al. A multiplex SNaPshot assay as a rapid method for detecting KRAS and BRAF mutations in advanced colorectal cancers. J. Mol. Diagn. 13(5), 485-492 (2011). Since mutations in the KRAS and BRAF genes are associated with resistance to therapy with anticancer drugs targeting the EGF receptor pathway, the analysis of KRAS and BRAF mutational status has become an important tool in the clinical management of patients with advanced colorectal cancer. To be useful in the clinical setting, a diagnostic assay has to address several issues related to the sensitivity and specificity of the method, the modularity of the assay, the turnaround time and the running costs. A variety of methods have been applied to the diagnosis of KRAS and BRAF mutational status. Although there is a good concordance between different methods, differences exist regarding sensitivity, multiplexing capacity and costs. In this article, we review a recently published assay for the simultaneous detection of diagnostically relevant KRAS and BRAF mutations and discuss this work in the context of conventional diagnostic methods.
RESUMO
Targeted therapy of advanced colorectal carcinoma (CRC) necessitates KRAS genotyping. Because we were interested in diagnostic and therapeutic consequences, we studied the KRAS, NRAS, PIK3CA exon 20, and BRAF genotypes in synchronous and metachronous primary CRCs; in addition, we studied their available metastases. We studied 21 patients with 43 synchronous and 2 metachronous adenocarcinomas of the colorectum (n = 20) and stomach (n = 1). Five patients had liver metastases and one had a distant lymph node metastasis. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed by Sanger sequencing and pyrosequencing. Fifty-seven different neoplastic lesions were genotyped, showing 18 (31.6%) KRAS, 2 (3.5%) NRAS, and 7 (12.3%) BRAF mutations, distributed among 10 (47.6%), 1 (4.8%), and 5 (23.8%) of the patients. An identical genotype of all synchronous primary CRCs was found only in 7 (35%) of the patients; the remainder had dissimilar genotypes in various combinations. Interestingly, a single patient had an unknown KRAS genotype (c.37_39dupGGC). Six patients with 13 primary carcinomas had distant metastases. In three of these patients, the metastasis shared the genotype only with one of the primary tumors, because the other primary tumors had another genotype. Synchronous and metachronous primary CRCs of the same patient have variable KRAS, NRAS, and BRAF genotypes. When metastases occur in these patients, the genotype has diagnostic and therapeutic implications and should be determined from the simultaneous or metachronous distant metastases.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Neoplasias Primárias Múltiplas/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/mortalidade , Adenocarcinoma/terapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Análise Mutacional de DNA , Éxons , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Primárias Múltiplas/diagnóstico , Neoplasias Primárias Múltiplas/mortalidade , Neoplasias Primárias Múltiplas/terapia , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Análise de Sobrevida , Adulto JovemRESUMO
Overexpression of MDM4 (also known as MDMX or HDMX) is thought to promote tumorigenesis by decreasing p53 tumor suppressor function. Even modest decrease in Mdm4 levels affects tumorigenesis in mice, suggesting that genetic variants of MDM4 might have similar effects in humans. We sequenced the MDM4 gene in a series of ovarian cancer cell lines and carcinomas to identify mutations and/or single nucleotide polymorphisms (SNPs). We identified an SNP (SNP34091) in the 3'-UTR of MDM4 that creates a putative target site for hsa-miR-191, a microRNA that is highly expressed in normal and tumor tissues. Biochemical evidence supports specific miR-191-dependent regulation of the MDM4-C, but not MDM4-A, variant. Consistently, the A-allele was associated with statistically significant increased expression of MDM4 mRNA and protein levels in ovarian carcinomas. Importantly, the wild-type genotype (A/A) is more frequent (57.8% vs. 42.2% for A/C and C/C, respectively) in patients with high-grade carcinomas than in patients with low-grade carcinomas (47.2% vs. 52.5% for A/A and A/C + C/C, respectively). Moreover, A/A patients who do not express the estrogen receptor had a 4.2-fold [95% confidence interval (CI) = 1.2-13.5; P = 0.02] increased risk of recurrence and 5.5-fold (95% CI = 1.5-20.5; P = 0.01) increased risk of tumor-related death. Unexpectedly, the frequency of p53 mutations was not significantly lower in A/A patients. We conclude that acquisition of an illegitimate miR-191 target site causes downregulation of MDM4 expression, thereby significantly delaying ovarian carcinoma progression and tumor-related death. Importantly, these effects appear to be, at least partly, independent of p53.