RESUMO
INTRODUCTION: Colorectal carcinoma (CRC) is among the most common carcinomas in women and men. In the advanced stage, patients are treated based on the RAS status. Recent studies indicate that in the future, in addition to KRAS and NRAS, alterations in other genes, such as PIK3CA or TP53, will be considered for therapy. Therefore, it is important to know the mutational landscape of routinely diagnosed CRC. METHOD: We report the molecular profile of 512 Swiss CRC patients analyzed by targeted next-generation sequencing as part of routine diagnostics at our institute. RESULTS: Pathogenic and likely pathogenic variants were found in 462 (90%) CRC patients. Variants were detected in TP53 (54.3%), KRAS (48.2%), PIK3CA (15.6%), BRAF (13.5%), SMAD4 (10.5%), FBXW7 (7.8%), NRAS (3.5%), PTEN (2.7%), ERBB2 (1.6%), AKT1 (1.5%), and CTNNB1 (0.9%). The remaining pathogenic alterations were found in the genes ATM(n= 1), MAP2K1(n= 1), and IDH2(n= 1). DISCUSSION/CONCLUSIONS: Our analysis revealed the prevalence of potential predictive markers in a large cohort of CRC patients obtained during routine diagnostic analysis. Furthermore, our study is the first of this size to uncover the molecular landscape of CRC in Switzerland.
Assuntos
Neoplasias Colorretais , Proteínas Proto-Oncogênicas p21(ras) , Masculino , Humanos , Feminino , Prevalência , Proteínas Proto-Oncogênicas p21(ras)/genética , Suíça/epidemiologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Mutação , Classe I de Fosfatidilinositol 3-Quinases/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
BACKGROUND: Nestin, a class VI intermediate filament protein of the cytoskeleton, and CD34, a transmembrane phosphoglycoprotein, are markers of progenitor cells. This study aimed to evaluate their expression and clinical significance in colorectal cancer. METHODS: A clinically annotated tissue microarray, including 599 patients with colorectal cancer, was analyzed by immunohistochemistry. Furthermore, nestin and CD34 correlations with HIF-1a and a panel of cytokines and chemokines were assessed using quantitative reverse transcription PCR and The Cancer Genome Atlas dataset. RESULTS: Expression of nestin and CD34 was observed only in the tumor stroma. Patients displaying high expression of nestin and CD34 demonstrated higher rates of T1 and T2 tumors (p = .020), lower vascular invasion (p < .001) and improved 5-year overall survival (65%; 95% CI = 55-73 vs 45%; 95% CI = 37-53) after adjusting for clinicopathological characteristics (HR: 0.67; 95% CI = 0.46-0.96). A moderate to strong correlation (r = 0.37-0.78, p < .03) of nestin and CD34 was demonstrated for the following markers; HIF-1α, CD4, CD8, FOXP3, IRF1, GATA3, CCL2, CCL3, CXCL12 and CCL21. CONCLUSIONS: Combined expression of nestin and CD34 expression is associated with better overall survival possibly by modulating a favorable immune response.
Assuntos
Neoplasias Colorretais , Neovascularização Patológica , Antígenos CD34 , Neoplasias Colorretais/genética , Humanos , Imuno-Histoquímica , Nestina/genéticaRESUMO
BACKGROUND: Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. METHODS: Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for which an outcome was available and correct. Additional data on the used test method were collected from the participants. Chi-squared tests on contingency tables and a biserial rank correlation were applied by IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). RESULTS: In 26 of the 1190 tests (2.2%) a technical failure occurred. For the remaining 1164 results, 1008 (86.6%) were correct, 151 (12.9%) were false-negative and 5 (0.4%) included incorrect mutations. Correct p.(Thr790Met) detection improved over time and for repeated scheme participations. In-house non-next-generation sequencing (NGS) techniques performed worse (81.1%, n = 293) compared to non-NGS commercial kits (85.2%, n = 656) and NGS (97.0%, n = 239). Over time there was an increase in the users of NGS. Resection specimens performed worse (82.6%, n = 610 tests) compared to cell line material (90.9%, n = 578 tests), except for NGS (96.3%, n = 344 for resections and 98.6%, n = 312 for cell lines). Samples with multiple mutations were more difficult compared to samples with the single p.(Thr790Met) variant. A change of the test method was shown beneficial to reduce errors but introduced additional analysis failures. CONCLUSIONS: A significant number of laboratories that offer p.(Thr790Met) testing did not detect this relevant mutation compared to the other EQA participants. However, correct identification of this variant is improving over time and was higher for NGS users. Revising the methodology might be useful to resolve errors, especially for resection specimens with low frequency or multiple variants. EQA providers should include challenging resections in the scheme.
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Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Mutação , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Receptores ErbB/genética , Seguimentos , Testes Genéticos/métodos , Testes Genéticos/normas , Humanos , Estudos Longitudinais , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/enzimologia , Polimorfismo de Nucleotídeo Único , Controle de Qualidade , Células Tumorais CultivadasRESUMO
INTRODUCTION: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. It has distinct molecular features and primarily affects the KIT and PDGFRA genes. OBJECTIVE: We wanted to assess the molecular profile of 68 GIST patients who were sequenced consecutively between 2014 and 2019 at our institute of pathology. METHODS: Our cohort comprised 60 primary and 8 metastatic GIST patients; 43 and 57% of the cases, respectively, were analyzed by Sanger sequencing or next-generation sequencing (NGS). RESULTS: Of the 60 primary GIST patients, 47 (78%) showed a KIT mutation; 2 cases showed a double KIT mutation, and 1 of these was a therapy-naive GIST. Nine (15%) patients harbored a PDGFRA mutation, 2 (3%) had a BRAF mutation, 1 (2%) had a PIK3CA mutation, and 1 (2%) did not show any mutation. One BRAF and the PIK3CA mutation have not been described in GIST before. All metastatic GIST harbored exclusively KIT mutations. CONCLUSION: A retrospective analysis of GIST sequenced at our institute revealed incidences of KIT and PDGFRA mutations comparable to those in other cohorts from Europe. Interestingly, we found 2 previously undescribed mutations in the BRAF and PIK3CA genes as well as 1 treatment-naive case with a double KIT mutation in exon 11.
Assuntos
Análise Mutacional de DNA , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/genética , Mutação , Idoso , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Estudos Retrospectivos , SuíçaRESUMO
Hepatocellular carcinoma (HCC) represents the fifth and ninth cause of mortality among male and female cancer patients, respectively and typically arises on a background of a cirrhotic liver. HCC develops in a multi-step process, often encompassing chronic liver injury, steatosis and cirrhosis eventually leading to the malignant transformation of hepatocytes. Aberrant expression of the class I homeobox gene family (HOX), a group of genes crucial in embryogenesis, has been reported in a variety of malignancies including solid tumors. Among HOX genes, HOXA13 is most overexpressed in HCC and is known to be directly regulated by the long non-coding RNA HOTTIP. In this study, taking advantage of a tissue microarray containing 305 tissue specimens, we found that HOXA13 protein expression increased monotonically from normal liver to cirrhotic liver to HCC and that HOXA13-positive HCCs were preferentially poorly differentiated and had fewer E-cadherin-positive cells. In two independent cohorts, patients with HOXA13-positive HCC had worse overall survival than those with HOXA13-negative HCC. Using HOXA13 immunohistochemistry and HOTTIP RNA in situ hybridization on consecutive sections of 16 resected HCCs, we demonstrated that HOXA13 and HOTTIP were expressed in the same neoplastic hepatocyte populations. Stable overexpression of HOXA13 in liver cancer cell lines resulted in increased colony formation on soft agar and migration potential as well as reduced sensitivity to sorafenib in vitro. Our results provide compelling evidence of a role for HOXA13 in HCC development and highlight for the first time its ability to modulate response to sorafenib.
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Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Fígado/efeitos dos fármacos , Sorafenibe/farmacologia , Sítios de Ligação , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Diferenciação Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Estudos de Coortes , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Proteínas de Homeodomínio/genética , Humanos , Hibridização In Situ , Fígado/metabolismo , Fígado/patologia , Fígado/cirurgia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Análise de Sobrevida , Análise Serial de TecidosRESUMO
Accreditation is a procedure by which an authoritative body gives formal recognition that an organization is competent to carry out specific tasks according to certain standards. Accreditation of pathology laboratories according to ISO 15189 is now becoming more and more a matter of course in continental Europe. This review describes some practical experience aspects with our own pathology laboratory accreditation according to ISO 15189, and outlines the advantages, addresses critical points, and discusses certain caveats of this process.
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Acreditação , Laboratórios/normas , Patologia Clínica , HumanosRESUMO
Notch signaling pathways have recently been implicated in the pathogenesis of metabolic diseases. However, the role of hepatic Notch signaling in glucose and lipid metabolism remains unclear and needs further investigation as it might be a candidate therapeutic target in metabolic diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). We used hepatocyte-specific Notch1 knockout (KO) mice and liver biopsies from NASH and NAFLD patients to analyze the role of Notch1 in glucose and lipid metabolism. Hepatocyte-specific Notch1 KO mice were fed with a high fat diet (HFD) or a regular diet (RD). We assessed the metabolic phenotype, glucose and insulin tolerance tests, and liver histology. Hepatic mRNA expression was profiled by Affymetrix Mouse Gene arrays and validated by quantitative reverse transcription PCR (qPCR). Akt phosphorylation was visualized by immunoblotting. Gene expression was analyzed in liver biopsies from NASH, NAFLD, and control patients by qPCR. We found that Notch1 KO mice had elevated fasting glucose. Gene expression analysis showed an upregulation of glucose-6-phosphatase, involved in the final step of gluconeogenesis and glucose release from glycogenolysis, and perilipin-5, a regulator of hepatic lipid accumulation. When fed with an HFD KO mice developed overt diabetes and hepatic steatosis. Akt was highly phosphorylated in KO animals and the Foxo1 target gene expression was altered. Accordingly, a reduction in Notch1 and increase in glucose-6-phosphatase and perilipin-5 expression was observed in liver biopsies from NAFLD/NASH compared with controls. Notch1 is a regulator of hepatic glucose and lipid homeostasis. Hepatic impairment of Notch1 expression may be involved in the pathogenesis of human NAFLD/NASH.
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Diabetes Mellitus/genética , Fígado Gorduroso/genética , Predisposição Genética para Doença/genética , Glucose-6-Fosfatase/genética , Perilipina-1/genética , Receptor Notch1/genética , Animais , Diabetes Mellitus/etiologia , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/etiologia , Perfilação da Expressão Gênica/métodos , Hepatócitos/metabolismo , Humanos , Immunoblotting , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
UNLABELLED: The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature. CONCLUSIONS: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/patologia , Proteínas de Ligação a DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Neovascularização Patológica/etiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Apoptose , Neoplasias dos Ductos Biliares/irrigação sanguínea , Neoplasias dos Ductos Biliares/tratamento farmacológico , Proteínas de Ciclo Celular , Proliferação de Células , Colangiocarcinoma/irrigação sanguínea , Colangiocarcinoma/tratamento farmacológico , Proteínas Contráteis/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Oncogenes , Fatores de Transcrição de Domínio TEARESUMO
OBJECTIVES: Analysis of a large local autopsy collective to gather epidemiological and histopathological data on hepatocellular carcinoma (HCC). METHODS: We examined a large dataset of 44,104 autopsies performed at the Institute of Pathology, Basel, Switzerland, including 2 autopsy collectives (1969-1983 and 1988-2012) to gather current data on HCC in the advanced stage. A total of 398 HCC were diagnosed, accounting for around 1% of all autopsies. RESULTS: As expected, most patients developing HCC had advanced stages of liver fibrosis or cirrhosis (F3/F4). However, in the more recent autopsy collective (1988-2012), our data also show an increase of HCC arising in livers without or with only mild to moderate fibrosis (F0-F2). Extrahepatic metastasis was found in 156 of 398 HCC (39.1%), with lung metastasis (74.5%) being the most common, followed by the bones (24.8%) and adrenal glands (19.1%). CONCLUSIONS: Our data therefore seem to suggest that, in the last 2 decades, despite the introduction of new therapeutic modalities for HCC, no significant changes have been observed regarding the metastatic pattern of advanced HCC.
Assuntos
Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/secundário , Cirrose Hepática/complicações , Neoplasias Hepáticas/complicações , Neoplasias Pulmonares/secundário , Neoplasias das Glândulas Suprarrenais/secundário , Idoso , Idoso de 80 Anos ou mais , Autopsia , Neoplasias Ósseas/secundário , Carcinoma Hepatocelular/epidemiologia , Feminino , Humanos , Fígado/patologia , Cirrose Hepática/epidemiologia , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/epidemiologia , Masculino , Pessoa de Meia-Idade , Suíça/epidemiologiaRESUMO
UNLABELLED: Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related death. Despite the advances in diagnosis and management of HCC, the biology of this tumor remains poorly understood. Recent evidence highlighted long noncoding RNAs (lncRNAs) as crucial determinants of HCC development. In this study we report the lncRNA HOXA transcript at the distal tip (HOTTIP) as significantly up-regulated in HCC specimens. The HOTTIP gene is located in physical contiguity with HOXA13 and directly controls the HOXA locus gene expression by way of interaction with the WDR5/MLL complex. HOX genes encode transcription factors regulating embryonic development and cell fate. We previously described HOX genes deregulation to be involved in hepatocarcinogenesis. Indeed, we observed the marked up-regulation of HOXA13 in HCC. Here, by correlating clinicopathological and expression data, we demonstrate that the levels of HOTTIP and HOXA13 are associated with HCC patients' clinical progression and predict disease outcome. In contrast to the majority of similar studies, our data were obtained from snap-frozen needle HCC biopsies (n=52) matched with their nonneoplastic counterparts collected from patients who had not yet received any HCC-tailored therapeutic treatments at the time of biopsy. In addition, taking advantage of gain and loss of function experiments in liver cancer-derived cell lines (HuH-6 and HuH-7), we uncover a novel bidirectional regulatory loop between HOTTIP/HOXA13. CONCLUSION: Our study highlights the key role of HOTTIP and HOXA13 in HCC development by associating their expression with metastasis and survival in HCC patients, provides novel insights on the function of lncRNA-driven hepatocarcinogenesis, and paves the way for further investigation about the possible role of HOTTIP as a predictive biomarker of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Homeodomínio/genética , Neoplasias Hepáticas/genética , RNA Longo não Codificante/genética , Idoso , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/secundário , Linhagem Celular Transformada , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Gradação de Tumores , Valor Preditivo dos Testes , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: Angiogenesis plays a pivotal role in neoplastic growth and metastasis formation. Vascular endothelial growth factor A (VEGFA) is a major player in physiological and tumour-induced angiogenesis and numerous human tumours have been show to overexpress VEGFA. Moreover increased VEGFA gene expression has been found frequently to correlate with tumour progression, recurrences and survival. Interestingly, several studies have demonstrated that gene amplification may result in protein overexpression and that amplification of the therapeutics' target gene can serve as an excellent predictive marker (i.e. HER2 and trastuzumab). However the impact of VEGFA gene amplification has been only recently assessed for some cancer types such as osteosarcoma, colorectal, breast and liver cancer. AIMS: This study aimed to assess VEGFA gene amplification status using fluorescent in situ hybridization (FISH) in a large cohort of different tumour entities. Thus, we investigated the incidence of VEGFA amplification using a multi-tumour tissue microarray (TMA) containing 2,837 evaluable specimens from 80 different tumour entities and 31 normal tissue types. Moreover, we validated FISH analysis as reference method to evaluate VEGFA gene status by comparing it to comparative genomic hybridization (CGH). RESULTS: We observed that VEGFA locus amplification and/or polysomy represented a small but regularly detected population in several tumour entities while was not present in normal tissues. VEGFA gene alterations were predominantly observed in hepatocarcinomas, adenocarcinomas of the pancreas and intestine, large cell carcinoma of the lung and in endometrium serous carcinoma. Furthermore our data demonstrated that VEGFA detection by FISH provided highly comparable results to those generated by CGH. CONCLUSION: Albeit with low percentage, VEGFA amplification is commonly observed across several tumour entities. Furthermore, our results demonstrated that FISH test could be used as a reliable diagnostic tool to evaluate VEGFA gene status in human specimens.
Assuntos
Amplificação de Genes , Loci Gênicos , Neoplasias/classificação , Neoplasias/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Cromossomos Humanos Par 6/genética , Hibridização Genômica Comparativa , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Nus , Microvasos/patologia , Neoplasias/patologia , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC) are the most common liver tumors and a leading cause for cancer-related death in men. Notch2 regulates cellular differentiation in the developing and adult liver. Although aberrant Notch signaling is implicated in various cancers, it is still unclear whether Notch2 regulates proliferation and differentiation in liver carcinogenesis and thereby contributes to HCC and CCC formation. Here, we investigated the oncogenic potential of constitutive Notch2 signaling in the liver. We show that liver-specific expression of the intracellular domain of Notch2 (N2ICD) in mice is sufficient to induce HCC formation and biliary hyperplasia. Specifically, constitutive N2ICD signaling in the liver leads to up-regulation of pro-proliferative genes and proliferation of hepatocytes and biliary epithelial cells (BECs). Using the diethylnitrosamine (DEN) HCC carcinogenesis model, we further show that constitutive Notch2 signaling accelerates DEN-induced HCC formation. DEN-induced HCCs with constitutive Notch2 signaling (DEN(N2ICD) HCCs) exhibit a marked increase in size, proliferation, and expression of pro-proliferative genes when compared with HCCs from DEN-induced control mice (DEN(ctrl) HCCs). Moreover, DEN(N2ICD) HCCs exhibit increased Sox9 messenger RNA (mRNA) levels and reduced Albumin and Alpha-fetoprotein mRNA levels, indicating that they are less differentiated than DEN(ctrl) HCCs. Additionally, DEN(N2ICD) mice develop large hepatic cysts, dysplasia of the biliary epithelium, and eventually CCC. CCC formation in patients and DEN(N2ICD) mice is accompanied by re-expression of hepatocyte nuclear factor 4α(HNF4α), possibly indicating dedifferentiation of BECs. CONCLUSION: Our data establish an oncogenic role for constitutive Notch2 signaling in liver cancer development.
Assuntos
Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/fisiopatologia , Dietilnitrosamina/efeitos adversos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/fisiopatologia , Receptor Notch2/fisiologia , Transdução de Sinais/fisiologia , Animais , Neoplasias dos Ductos Biliares/fisiopatologia , Ductos Biliares Intra-Hepáticos , Carcinoma Hepatocelular/metabolismo , Diferenciação Celular , Proliferação de Células , Colangiocarcinoma/fisiopatologia , Modelos Animais de Doenças , Feminino , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Receptor Notch2/genéticaRESUMO
BACKGROUND & AIMS: Nodular regenerative hyperplasia (NRH) is a rare liver disease characterized by small regenerative nodules without fibrosis and can cause portal hypertension. Aetiology and pathogenesis of NRH remain unclear. We have recently shown that Notch1 knockout induces NRH with portal hypertension through vascular remodelling in mice. The aim of this study was to analyse histological and clinical data of NRH patients and to explore if the endothelial pathways identified in our NRH mouse model are also regulated in human NRH. METHODS: Patients were identified retrospectively from the pathology database. Clinical and laboratory patient data were retrieved. mRNA expression was measured in liver biopsies from a subset of NRH patients. RESULTS: Diagnosis of NRH was confirmed in needle biopsies of 51 patients, including 31 patients with grade 1, 12 patients with grade 2 and 8 patients with grade 3 NRH. Grade 3 nodularity significantly correlated with the presence of portal hypertension: 50% of the patients with grade 3 NRH vs. 6.5% with grade 1 (P = 0.0105). mRNA expression analysis in liver biopsies from 14 NRH patients and in primary human sinusoidal endothelial cells revealed downregulation of identical genes as in the murine NRH model, which are implicated in vascular differentiation: Notch1, delta-like 4 (Dll4) and ephrinB2. CONCLUSIONS: In this large NRH needle biopsy cohort, we demonstrated that advanced nodularity correlates with presence of portal hypertension. Downregulation of the endothelial signalling pathways Dll4/Notch1 and ephrinB2/EphB4 supports the hypothesis that human NRH is caused by a sinusoidal injury providing first insights into the molecular pathogenesis of this liver condition.
Assuntos
Regulação para Baixo/fisiologia , Efrina-B2/metabolismo , Hiperplasia Nodular Focal do Fígado/genética , Hipertensão Portal/etiologia , Fígado/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/fisiologia , Biópsia , Estudos de Coortes , Células Endoteliais/metabolismo , Hiperplasia Nodular Focal do Fígado/complicações , Hiperplasia Nodular Focal do Fígado/metabolismo , Humanos , Fígado/patologia , Estudos RetrospectivosRESUMO
BACKGROUND & AIMS: Approximately 50% of patients with chronic hepatitis C (CHC) have a sustained virologic response to treatment with pegylated interferon (pegIFN)-α and ribavirin. Nonresponse to treatment is associated with constitutively increased expression of IFN-stimulated genes (ISGs) in the liver. Treatment of patients with acute hepatitis C (AHC) is more effective, with sustained virologic response rates greater than 90%. We investigated mechanisms of the different responses of patients with CHC and AHC to pegIFN-α therapy. METHODS: We analyzed IFN signaling and ISG expression in liver samples from patients with AHC, patients with CHC, and individuals without hepatitis C (controls) using microarray, immunohistochemical, and protein analyses. Findings were compared with those from primary human hepatocytes stimulated with IFN-α or IFN-γ, as reference sets. RESULTS: Expression levels of hundreds of genes, primarily those regulated by IFN-γ, were altered in liver samples from patients with AHC compared with controls. Expression of IFN-γ-stimulated genes was induced in liver samples from patients with AHC, whereas expression of IFN-α-stimulated genes was induced in samples from patients with CHC. In an expression analysis of negative regulators of IFN-α signaling, we did not observe differences in expression of suppresor of cytokine signaling 1 or SOCS3 between liver samples from patients with AHC and those with CHC. However, USP18 (another negative regulator of IFN-α signaling), was up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in AHC. CONCLUSIONS: Differences in expression of ISGs might account for the greater response of patients with AHC, compared with those with CHC, to treatment with pegIFN-α and ribavirin. Specifically, USP18 is up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in patients with AHC.
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Antivirais/uso terapêutico , Endopeptidases/genética , Hepatite C Crônica/tratamento farmacológico , Hepatite C/tratamento farmacológico , Interferon-alfa/uso terapêutico , Interferon gama/metabolismo , Fígado/efeitos dos fármacos , Polietilenoglicóis/uso terapêutico , Ribavirina/uso terapêutico , Doença Aguda , Adolescente , Adulto , Biópsia , Western Blotting , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Farmacorresistência Viral/genética , Quimioterapia Combinada , Endopeptidases/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Hepacivirus/patogenicidade , Hepatite C/diagnóstico , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/genética , Hepatite C Crônica/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteínas Recombinantes/uso terapêutico , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Suíça , Fatores de Tempo , Transcrição Gênica , Resultado do Tratamento , Ubiquitina TiolesteraseRESUMO
BACKGROUND & AIMS: Notch signaling mediates embryonic vascular development and normal vascular remodeling; Notch1 knockout mice develop nodular regenerative hyperplasia (NRH). The pathogenesis of NRH is unclear, but has been associated with vascular injury in the liver sinusoids in clinical studies. We investigated the role of Notch1 signaling in liver sinusoidal endothelial cells (LSECs). METHODS: We studied MxCre Notch1(lox/lox) mice (conditional knockout mice without tissue-specific disruption of Notch1); mice with hepatocyte-specific knockout were created by crossing Notch1(lox/lox) with AlbCre(+/-) mice. Portal vein pressure was measured; morphology of the hepatic vasculature was assessed by histologic and scanning electron microscopy analyses. We performed functional and expression analyses of isolated liver cells. RESULTS: MxCre-induced knockout of Notch1 led to NRH, in the absence of fibrosis, with a persistent increase in proliferation of LSECs. Notch1 deletion led to de-differentiation, vascular remodeling of the hepatic sinusoidal microvasculature, intussusceptive angiogenesis, and dysregulation of ephrinB2/EphB4 and endothelial tyrosine kinase. Time-course experiments revealed that vascular changes preceded node transformation. MxCre Notch1(lox/lox) mice had reduced endothelial fenestrae and developed portal hypertension and hepatic angiosarcoma over time. In contrast, mice with hepatocyte-specific disruption of Notch1 had a normal phenotype. CONCLUSIONS: Notch1 signaling is required for vascular homeostasis of hepatic sinusoids; it maintains quiescence and differentiation of LSECs in adult mice. Disruption of Notch1 signaling in LSECs leads to spontaneous formation of angiosarcoma, indicating its role as a tumor suppressor in the liver endothelium.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células Endoteliais/metabolismo , Hiperplasia Nodular Focal do Fígado/metabolismo , Hemangiossarcoma/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/irrigação sanguínea , Neovascularização Patológica/metabolismo , Receptor Notch1/metabolismo , Animais , Desdiferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Células Endoteliais/patologia , Efrina-B2/metabolismo , Hiperplasia Nodular Focal do Fígado/genética , Hiperplasia Nodular Focal do Fígado/patologia , Genótipo , Hemangiossarcoma/genética , Hemangiossarcoma/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Hipertensão Portal/genética , Hipertensão Portal/metabolismo , Hipertensão Portal/fisiopatologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Fenótipo , Pressão na Veia Porta , Receptor EphB4/metabolismo , Receptor Notch1/deficiência , Receptor Notch1/genética , Transdução de Sinais , Fatores de TempoRESUMO
Lymph node involvement is prognostically the most determinant clinical factor for patients with head and neck squamous cell carcinomas (HNSCCs). Ultrasound of the neck and fine-needle aspiration (FNA) cytology is one of the first diagnostic procedures and the most accurate diagnostic staging tool for the neck. Patients with HPV-positive oropharyngeal carcinomas (OPSCC) show a significantly better prognosis when compared with HPV-negative OPSCC. P16 overexpression is accepted as surrogate marker for HPV-positive in OPSCC. These HPV/p16-positive OPSCC are localized either in the palatal tonsils or the base of tongue and frequently present with lymph node metastases. We analyzed the correlation and reliability of p16 expression of the FNA of the lymph node metastasis with the immunohistochemical expression of p16 of the same lymph node metastasis and its corresponding primary tumor, as it could be of importance for determining the localization and different prognosis of the primary tumor. 54 HNSCC patients were evaluated, p16 expression of the primary tumors and their lymph node metastases correlated precisely. In 25 of the 54 HNSCC patients, a FNA of the lymph node metastases was taken before the treatment. The positive cytological and immunohistochemical p16 staining correlated exactly. Of the 17 histologically p16-negative lymph node metastases 15 FNA were p16-negative, whereas two samples were p16-positive. In our view, a cytological p16 analysis of cervical lymph node metastasis can facilitate the correct localization of the primary tumor and discriminate reliably HPV-positive OPSCC from HPV-negative HNSCC with their significantly diverse prognosis.
Assuntos
Biópsia por Agulha Fina , Carcinoma de Células Escamosas/virologia , Papillomavirus Humano 16/isolamento & purificação , Linfonodos/virologia , Neoplasias Orofaríngeas/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Pescoço , Neoplasias Orofaríngeas/patologiaRESUMO
MAGE-A10 is a highly immunogenic member of the MAGE-A family of cancer/testis tumor-associated antigens (C/T TAAs). Studies performed with broadly reactive antibodies have helped to initially characterize this TAA. However, no specific reagents have been developed so far, thus preventing a thorough analysis of its expression in healthy and tumoral tissues. We have produced MAGE-A10 gene product in soluble recombinant form, and we have used it to generate specific monoclonal antibodies (mAbs). One of these reagents, recognizing an epitope located at the COOH terminus of the MAGE-A10 gene product, was used to stain a multitumor tissue microarray comprising more than 2,500 paraffin-embedded specimens including healthy tissues, benign tumors and malignancies of different histological origin. MAGE-A10 protein was identified as an intranuclear protein of an apparent molecular weight of 70 kDa, expressed in normal spermatogonia and spermatocytes but in no other healthy tissue. Most importantly, this C/T TAA appears to be expressed in high (>50%) percentages of cancer cells from a number of malignancies, including lung, skin and urothelial tumors. Unexpectedly, high expression of MAGE-A10 TAA at the protein level was also detectable in gynecological malignancies and stomach and gall bladder cancers. The characterization of MAGE-A10-specific reagents might set the stage for the development of targeted active immunotherapy by clarifying potential indications and by allowing the selection of patients eligible for treatment and the monitoring of its effectiveness.
Assuntos
Antígenos de Neoplasias/metabolismo , Núcleo Celular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Urológicas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Núcleo Celular/genética , Feminino , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Análise Serial de Tecidos , Células Tumorais Cultivadas , Neoplasias Urológicas/genética , Neoplasias Urológicas/imunologiaRESUMO
Liver organogenesis and cancerogenesis share common mechanisms. HOX genes control normal development, primary cellular processes and are characterized by a unique genomic network organization. Less is known about the involvement of HOX genes with liver cancerogenesis. The comparison of the HOX gene network expression between nontumorous livers and hepatocellular carcinomas (HCCs) highlights significant differences in the locus A HOX genes, located on chromosome 7, with a consistent overexpression of HOXA13 mRNA thus validating this gene deregulation as a feature of HCC. HOXA13 is a determinant of gut primordia and posterior body structures. Transcriptome analysis of HCC/nontumorous liver mRNAs, selected on the basis of HOXA13 overexpression, recognizes a set of deregulated genes. The matching of these genes with previously reported HCC transcriptome analysis identifies cell-cycle and nuclear pore-related HCC phenotype displaying poor prognosis. HOXA13 and HOXA7 homeoproteins share a consensus sequence that physically links eIF4E nuclear bodies acting on the export of specific mRNAs (c-myc, FGF-2, vascular endothelial growth factor (VEGF), ornithine decarboxylase (ODC) and cyclin D1). We report the protein-protein interaction between HOXA13 and eIF4E in liver cancer cells and the deregulation of eIF4E mRNA and protein in cell cycle/nuclear pore HCC group phenotype and in T4 stage HCCs, respectively. Thus, transcriptional and post-transcriptional HOXA13 deregulation is involved in HCC possibly through the mRNA nuclear export of eIF4E-dependent transcripts.
Assuntos
Carcinoma Hepatocelular/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Neoplasias Hepáticas/genética , Sequência de Aminoácidos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Núcleo Celular/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos , Transcrição GênicaRESUMO
Sec62 is part of the protein translocation apparatus in the membrane of the endoplasmic reticulum (ER). In yeast, Sec62 participates in the post-translational translocation of proteins into the ER, but its function in mammals remains elusive. Previously we described the amplification and over-expression of the SEC62 gene in prostate cancer cell lines and the protein has been described as a potential target gene in prostate cancer. In the current study we show that in the tumor tissue of prostate cancer patients Sec62 protein levels are elevated compared with tumor-free tissue derived from the same patients or from prostates of control group patients and that the higher Sec62 protein content correlates with an increasing de-differentiation of the cells. Therefore, up-regulation of Sec62 protein content indeed is a phenomenon associated with prostate cancer progression. Analysis of a multi-tissue tumor array showed that in addition to prostate cancer, overproduction of Sec62 is observed in various other tumors, most significantly in tumors of the lung and the thyroid. To examine the tumor-related functions of Sec62, we silenced the SEC62 gene in the prostate cancer cell-line PC3 as well as in a set of other tumor cell-lines with two different siRNAs. In general, after silencing of SEC62 the cell migration and the invasive potential of the cells was blocked or at least dramatically reduced while cell viability was hardly affected. Thus, the SEC62 gene may indeed be considered as a target gene in the therapy of various tumors.
Assuntos
Inativação Gênica , Proteínas de Membrana Transportadoras/genética , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Humanos , Reação em Cadeia da Polimerase , RNA Interferente PequenoRESUMO
The prognostic significance of macrophage and natural killer (NK) cell infiltration in colorectal carcinoma (CRC) microenvironment is unclear. We investigated the CRC innate inflammatory infiltrate in over 1,600 CRC using two independent tissue microarrays and immunohistochemistry. Survival time was assessed using the Kaplan-Meier method and Cox proportional hazards regression analysis in a multivariable setting. Spearman's rank correlation tested the association between macrophage and lymphocyte infiltration. The Basel study included over 1,400 CRCs. The level of CD16+ cell infiltration correlated with that of CD3+ and CD8+ lymphocytes but not with NK cell infiltration. Patients with high CD16+ cell infiltration (score 2) survived longer than patients with low (score 1) infiltration (p = 0.008), while no survival difference between patients with score 1 or 2 for CD56+ (p = 0.264) or CD57+ cell (p = 0.583) infiltration was detected. CD16+ infiltrate was associated with improved survival even after adjusting for known prognostic factors including pT, pN, grade, vascular invasion, tumor growth and age [(p = 0.001: HR (95% CI) = 0.71 (0.6-0.9)]. These effects were independent from CD8+ lymphocyte infiltration [(p = 0.036: HR (95% CI) = 0.81 (0.7-0.9)] and presence of metastases [(p = 0.002: HR (95% CI) = 0.43 (0.3-0.7)]. Phenotypic studies identified CD16+ as CD45+CD33+CD11b+CD11c+ but CD64- HLA-DR-myeloid cells. Beneficial effects of CD16+ cell infiltration were independently validated by a study carried out at the University of Athens confirming that patients with CD16 score 2 survived longer than patients with score 1 CRCs (p = 0.011). Thus, CD16+ cell infiltration represents a novel favorable prognostic factor in CRC.