RESUMO
OBJECTIVE: Barrett's oesophagus is a premalignant condition that occurs in the context of gastro-oesophageal reflux. However, most Barrett's cases are undiagnosed because of reliance on endoscopy. We have developed a non-endoscopic tool: the Cytosponge, which when combined with trefoil factor 3 immunohistochemistry, can diagnose Barrett's oesophagus. We investigated whether a quantitative methylation test that is not reliant on histopathological analysis could be used to diagnose Barrett's oesophagus. DESIGN: Differentially methylated genes between Barrett's and normal squamous oesophageal biopsies were identified from whole methylome data and confirmed using MethyLight PCR in biopsy samples of squamous oesophagus, gastric cardia and Barrett's oesophagus. Selected genes were then tested on Cytosponge BEST2 trial samples comprising a pilot cohort (n=20 cases, n=10 controls) and a validation cohort (n=149 cases, n=129 controls). RESULTS: Eighteen genes were differentially methylated in patients with Barrett'soesophagus compared with squamous controls. Hypermethylation of TFPI2, TWIST1, ZNF345 and ZNF569 was confirmed in Barrett's biopsies compared with biopsies from squamous oesophagus and gastric cardia (p<0.05). When tested in Cytosponge samples, these four genes were hypermethylated in patients with Barrett's oesophagus compared with patients with reflux symptoms (p<0.001). The optimum biomarker to diagnose Barrett's oesophagus was TFPI2 with a sensitivity and specificity of 82.2% and 95.7%, respectively. CONCLUSION: TFPI2, TWIST1, ZNF345 and ZNF569 methylation have promise as diagnostic biomarkers for Barrett's oesophagus when used in combination with a simple and cost effective non-endoscopic cell collection device.
Assuntos
Esôfago de Barrett/diagnóstico , Biomarcadores/metabolismo , Metilação de DNA/genética , Esôfago/patologia , Adulto , Idoso , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Biópsia , Técnicas Citológicas/métodos , Esofagoscopia/métodos , Esôfago/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer and is one of the leading causes of cancer-related death. The risk factors for HCC include cirrhosis, chronic viral hepatitis, heavy alcohol intake and metabolic diseases such as obesity, type 2 diabetes and metabolic syndrome. Insulin resistance is a common denominator of all of these conditions and is tethered to hyperinsulinaemia. Here, we give an overview of the recent advances linking hyperinsulinaemia to HCC development and progression. In particular, we summarise the underlying causes of hyperinsulinaemia in the setting of chronic liver diseases. We present epidemiological evidence linking metabolic diseases to HCC risk and HCC-related mortality, as well as the pathogenic cellular and molecular mechanisms explaining this relation. A better understanding of the mechanisms by which insulin participates in HCC biology might ultimately provide novel opportunities for prevention and treatment.
Assuntos
Carcinoma Hepatocelular/metabolismo , Hiperinsulinismo/metabolismo , Resistência à Insulina , Neoplasias Hepáticas/metabolismo , Transdução de Sinais , Animais , Carcinoma Hepatocelular/patologia , Diabetes Mellitus Tipo 2/complicações , Modelos Animais de Doenças , Progressão da Doença , Hepatite C/complicações , Humanos , Hiperinsulinismo/patologia , Insulina/fisiologia , Cirrose Hepática/complicações , Neoplasias Hepáticas/patologia , Síndrome Metabólica/complicações , Camundongos , Hepatopatia Gordurosa não Alcoólica/complicações , Obesidade/complicações , Ratos , Fatores de RiscoRESUMO
BACKGROUND & AIMS: Sorafenib is the standard of care for the treatment of advanced hepatocellular carcinoma (HCC). However, primary and acquired resistance is observed in patients. We examined whether gefitinib, which inhibits both epidermal growth factor receptor (EGFR) and HER-3 phosphorylation, could improve HCC cell response to sorafenib. METHODS: Sorafenib and gefitinib were tested in HCC tumor xenografts and in sorafenib-sensitive and sorafenib-resistant HCC cell lines. Biomarkers relevant to the HER system were analyzed by Western blotting and ELISA. RNA interference was used to downregulate the HER system. Amphiregulin concentrations were measured by ELISA in sera from patients under sorafenib treatment. RESULTS: Sorafenib combined with gefitinib significantly inhibited tumor growth in mice and reduced cell viability in vitro compared to single agents. In cell lines cultured in 10% serum or treated with EGF, sorafenib alone inhibited phospho-STAT3 while it maintained or even increased phospho-ERK and/or phospho-AKT. The paradoxical effects of sorafenib were prevented by gefitinib or by downregulation of EGFR and HER-3 expression. In cells with acquired resistance to sorafenib, aberrant activation of EGFR/HER-3 receptors as well as overexpression of several EGFR ligands were observed. These enhanced autocrine/paracrine loops led to the constitutive activation of ERK and AKT and conferred increased sensitivity to gefitinib. Increased serum concentrations of amphiregulin were observed in 10 out of 14 patients under sorafenib treatment compared to baselines. CONCLUSIONS: Signaling pathways controlled by EGFR and HER-3 restrict sorafenib effects both in naive and sorafenib-resistant HCC cells. Consequently, gefitinib cooperates with sorafenib to increase antiproliferative response and to prevent resistance.
Assuntos
Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Receptores ErbB/metabolismo , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Piridinas/farmacologia , Receptor ErbB-3/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Anfirregulina , Animais , Antineoplásicos/uso terapêutico , Benzenossulfonatos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Família de Proteínas EGF , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe , Glicoproteínas/metabolismo , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Niacinamida/análogos & derivados , Compostos de Fenilureia , Piridinas/uso terapêutico , Quinazolinas/farmacologia , Sorafenibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Radiofrequency ablation (RFA) is an effective treatment for dysplastic Barrett's esophagus (BE), but recurrence can occur after initial response. Currently there is uncertainty about how to best define histological remission. A DNA methylation panel on esophageal samples was previously shown to have high diagnostic accuracy for BE. We aimed to investigate this biomarker panel in the assessment of response to RFA treatment. METHODS: We retrospectively analyzed esophageal and gastroesophageal junction (GEJ) biopsies from patients with BE before and after RFA treatment. We quantified the extent of intestinal metaplasia (IM) based on number of glands with goblet cells (IM-Score) and expression of the intestinal factor trefoil factor-3 (TFF3-Score). Promoter methylation of 3 genes (ZNF345, TFP12, ZNF569) was measured by methylight (Meth-Score) throughout the RFA treatment pathway. FINDINGS: We included 45 patients (11 non-dysplastic BE, 14 low-grade dysplasia, 20 high-grade dysplasia/intramucosal cancer). Meth-Scores were significantly higher in BE with and without dysplasia and GEJ with IM compared to GEJ without IM (P<·001). Meth-scores significantly correlated with the extent of IM at the GEJ measured both with IM-Scores (rho=66·0%, P<·001), and TFF3-Scores (rho=75·6%, P<·001). In patients with residual IM at the GEJ, RFA re-treatment brought about a 7·6-fold reduction in the methylation levels. The Meth-score had an area under the ROC curve of 95·1% (95%CI 91·1% - 99·1%) differentiating BE from normal GEJ. INTERPRETATION: A DNA methylation panel can discriminate between the extent of histological IM in esophageal and junctional biopsies and could be used to objectively quantify residual disease following RFA.
Assuntos
Esôfago de Barrett/patologia , Esôfago de Barrett/terapia , Biomarcadores Tumorais/genética , Metilação de DNA , Ablação por Radiofrequência/métodos , Idoso , Esôfago de Barrett/genética , Biópsia , Estudos de Casos e Controles , Progressão da Doença , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Repressoras/genética , Estudos Retrospectivos , Resultado do Tratamento , Fator Trefoil-3/genéticaRESUMO
BACKGROUND: Barrett's oesophagus predisposes to adenocarcinoma. However, most patients with Barrett's oesophagus will not progress and endoscopic surveillance is invasive, expensive, and fraught by issues of sampling bias and the subjective assessment of dysplasia. We investigated whether a non-endoscopic device, the Cytosponge, could be coupled with clinical and molecular biomarkers to identify a group of patients with low risk of progression suitable for non-endoscopic follow-up. METHODS: In this multicentre cohort study (BEST2), patients with Barrett's oesophagus underwent the Cytosponge test before their surveillance endoscopy. We collected clinical and demographic data and tested Cytosponge samples for a molecular biomarker panel including three protein biomarkers (P53, c-Myc, and Aurora kinase A), two methylation markers (MYOD1 and RUNX3), glandular atypia, and TP53 mutation status. We used a multivariable logistic regression model to compute the conditional probability of dysplasia status. We selected a simple model with high classification accuracy and applied it to an independent validation cohort. The BEST2 study is registered with ISRCTN, number 12730505. FINDINGS: The discovery cohort consisted of 468 patients with Barrett's oesophagus and intestinal metaplasia. Of these, 376 had no dysplasia and 22 had high-grade dysplasia or intramucosal adenocarcinoma. In the discovery cohort, a model with high classification accuracy consisted of glandular atypia, P53 abnormality, and Aurora kinase A positivity, and the interaction of age, waist-to-hip ratio, and length of the Barrett's oesophagus segment. 162 (35%) of 468 of patients fell into the low-risk category and the probability of being a true non-dysplastic patient was 100% (99% CI 96-100) and the probability of having high-grade dysplasia or intramucosal adenocarcinoma was 0% (0-4). 238 (51%) of participants were classified as of moderate risk; the probability of having high-grade dysplasia was 14% (9-21). 58 (12%) of participants were classified as high-risk; the probability of having non-dysplastic endoscopic biopsies was 13% (5-27), whereas the probability of having high-grade dysplasia or intramucosal adenocarcinoma was 87% (73-95). In the validation cohort (65 patients), 51 were non-dysplastic and 14 had high-grade dysplasia. In this cohort, 25 (38%) of 65 patients were classified as being low-risk, and the probability of being non-dysplastic was 96·0% (99% CI 73·80-99·99). The moderate-risk group comprised 27 non-dysplastic and eight high-grade dysplasia cases, whereas the high-risk group (8% of the cohort) had no non-dysplastic cases and five patients with high-grade dysplasia. INTERPRETATION: A combination of biomarker assays from a single Cytosponge sample can be used to determine a group of patients at low risk of progression, for whom endoscopy could be avoided. This strategy could help to avoid overdiagnosis and overtreatment in patients with Barrett's oesophagus. FUNDING: Cancer Research UK.
Assuntos
Esôfago de Barrett/diagnóstico , Citodiagnóstico/métodos , Medição de Risco/métodos , Adenocarcinoma/patologia , Idoso , Esôfago de Barrett/patologia , Biomarcadores/análise , Estudos de Casos e Controles , Progressão da Doença , Neoplasias Esofágicas/patologia , Esofagoscopia , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
BACKGROUND: The heregulin-1ß/HER3-driven pathway is implicated in several epithelial malignancies and its blockade is currently undergoing clinical investigation. Paradoxically, the status and the regulation of this pathway is poorly known in hepatocellular carcinoma (HCC). METHODS: Using 85 HCC obtained after tumour resection, heregulin-1ß and HER3 expression was evaluated by real-time RT-PCR, ELISA and/or immunohistochemistry. Statistics were performed to analyze associations between gene expression and clinicopathological parameters. The effects of insulin on the heregulin-1ß/HER3 pathway was investigated in four HCC cell lines. RESULTS: HER3 mRNA was upregulated in 52 % of tumours, while heregulin-1ß mRNA was downregulated in 82 %. Hepatitis B and C viral infections were respectively associated with high and low HER3 mRNA expression. No association was seen between neither HER3 or heregulin-1ß mRNA and prognostic factors, survival or recurrence. Immunohistochemistry showed predominant cytoplasmic staining of HER3 in tumours but the staining was nonreproducible. HER3 mRNA and protein levels were not correlated in liver tissues. In HCC cells, insulin promoted HER3 proteasomal degradation and inhibited heregulin-1ß stimulation of cell migration. HER3 and insulin receptor co-immunoprecipitated in these cells. The loss of insulin receptor expression by RNA interference sensitized cells to heregulin-1ß-induced AKT phosphorylation. CONCLUSIONS: Autocrine heregulin-1ß loop is uncommon in HCC and HER3 mRNA expression is differentially influenced by hepatitis viruses. Insulin is a negative regulator of HER3 protein expression and function in HCC cells. Altogether these data may explain why HER3 and heregulin-1ß expression have no prognostic value and suggest that HCC patients are unlikely to derive benefit from HER3-targeted monotherapies.
Assuntos
Carcinoma Hepatocelular/metabolismo , Hepatite B/complicações , Hepatite C/complicações , Neoplasias Hepáticas/metabolismo , Neuregulina-1/genética , Receptor ErbB-3/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Comunicação Autócrina , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Hepatite B/genética , Hepatite B/metabolismo , Hepatite C/genética , Hepatite C/metabolismo , Humanos , Insulina/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Neuregulina-1/metabolismo , Prognóstico , Receptor ErbB-3/metabolismo , Transdução de Sinais , Análise de Sobrevida , Adulto JovemRESUMO
Esophageal adenocarcinoma (EAC) has a poor outcome, and targeted therapy trials have thus far been disappointing owing to a lack of robust stratification methods. Whole-genome sequencing (WGS) analysis of 129 cases demonstrated that this is a heterogeneous cancer dominated by copy number alterations with frequent large-scale rearrangements. Co-amplification of receptor tyrosine kinases (RTKs) and/or downstream mitogenic activation is almost ubiquitous; thus tailored combination RTK inhibitor (RTKi) therapy might be required, as we demonstrate in vitro. However, mutational signatures showed three distinct molecular subtypes with potential therapeutic relevance, which we verified in an independent cohort (n = 87): (i) enrichment for BRCA signature with prevalent defects in the homologous recombination pathway; (ii) dominant T>G mutational pattern associated with a high mutational load and neoantigen burden; and (iii) C>A/T mutational pattern with evidence of an aging imprint. These subtypes could be ascertained using a clinically applicable sequencing strategy (low coverage) as a basis for therapy selection.
Assuntos
Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Mutação , Adenocarcinoma/classificação , Adenocarcinoma/imunologia , Adenocarcinoma/terapia , Idoso , Antineoplásicos/uso terapêutico , Antígenos CD8/imunologia , Linhagem Celular Tumoral , Estudos de Coortes , Dano ao DNA , DNA de Neoplasias , Neoplasias Esofágicas/classificação , Neoplasias Esofágicas/imunologia , Neoplasias Esofágicas/terapia , Feminino , Heterogeneidade Genética , Genoma Humano , Humanos , Masculino , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Análise de Sequência de DNARESUMO
The development and progression of liver cancer are characterized by increased levels of reactive oxygen species (ROS). ROS-induced oxidative stress impairs cell proliferation and ultimately leads to cell death. Although liver cancer cells are especially resistant to oxidative stress, mechanisms of such resistance remain understudied. We identified the MAPK-activated protein kinase 2 (MK2)/heat shock protein 27 (Hsp27) signaling pathway mediating defenses against oxidative stress. In addition to MK2 and Hsp27 overexpression in primary liver tumors compared to adjacent nontumorous tissues, the MK2/Hsp27 pathway is activated by hydrogen peroxide-induced oxidative stress in hepatobiliary cancer cells. MK2 inactivation or inhibition of MK2 or Hsp27 expression increases caspase-3 and PARP cleavage and DNA breaks and therefore cell death. Interestingly, MK2/Hsp27 inhibition decreases antioxidant defenses such as heme oxygenase 1 through downregulation of the transcription factor nuclear factor erythroid-derived 2-like 2. Moreover, MK2/Hsp27 inhibition decreases both phosphorylation of epidermal growth factor receptor (EGFR) and expression of its ligand, heparin-binding EGF-like growth factor. A new identified partner of MK2, the scaffold PDZ protein EBP50, could facilitate these effects through MK2/Hsp27 pathway regulation. These findings demonstrate that the MK2/Hsp27 pathway actively participates in resistance to oxidative stress and may contribute to liver cancer progression.
Assuntos
Neoplasias do Sistema Biliar/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Idoso , Apoptose/efeitos dos fármacos , Neoplasias do Sistema Biliar/tratamento farmacológico , Neoplasias do Sistema Biliar/genética , Neoplasias do Sistema Biliar/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Oxidantes/farmacologia , Fosforilação/efeitos dos fármacos , Prognóstico , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-HíbridoRESUMO
Insulin receptor (IR) exists as two isoforms resulting from the alternative splicing of IR pre-mRNA. IR-B promotes the metabolic effects of insulin, whereas IR-A rather signals proliferative effects. IR-B is predominantly expressed in the adult liver. Here, we show that the alternative splicing of IR pre-mRNA is dysregulated in a panel of 85 human hepatocellular carcinoma (HCC) while being normal in adjacent nontumor liver tissue. An IR-B to IR-A switch is frequently observed in HCC tumors regardless of tumor etiology. Using pharmacologic and siRNA approaches, we show that the autocrine or paracrine activation of the EGF receptor (EGFR)/mitogen-activated protein/extracellular signal-regulated kinase pathway increases the IR-A:IR-B ratio in HCC cell lines, but not in normal hepatocytes, by upregulating the expression of the splicing factors CUGBP1, hnRNPH, hnRNPA1, hnRNPA2B1, and SF2/ASF. In HCC tumors, there is a significant correlation between the expression of IR-A and that of splicing factors. Dysregulation of IR pre-mRNA splicing was confirmed in a chemically induced model of HCC in rat but not in regenerating livers after partial hepatectomy. This study identifies a mechanism responsible for the generation of mitogenic IR-A and provides a novel interplay between IR and EGFR pathways in HCC. Increased expression of IR-A during neoplastic transformation of hepatocytes could mediate some of the adverse effects of hyperinsulinemia on HCC.