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INTRODUCTION: Although rates of esophageal adenocarcinoma (EAC) in the United States continue to rise, many patients at risk of disease are not screened. EsoCheck (EC), a nonendoscopic esophageal balloon sampling device coupled with EsoGuard (EG), a DNA-based screening assay, is an US Food and Drug Administration-approved minimally invasive alternative to the traditional screening method of upper endoscopy. The objective of this study was to prospectively determine the diagnostic accuracy, tolerance, and acceptability of the EC/EG test in a screening population. METHODS: We recruited veterans who met the American College of Gastroenterology Guideline criteria for endoscopic Barrett's esophagus (BE) and EAC screening at the Louis Stokes Cleveland Veterans Affairs Medical Center. All study participants completed unsedated EC-guided distal esophageal sampling followed by a sedated esophagogastroduodenoscopy (EGD). Diagnostic yield of the EG assay and EGD was recorded and used in calculation of sensitivity and specificity of EC/EG in prospective screening. The abbreviated Spielberger State-Trait Anxiety Inventory questionnaire was administered before and after completion of EC. Overall tolerance of EC sampling was evaluated on a 10-point Likert scale. RESULTS: Esophageal cancer screening was accepted by 130 of 782 eligible veterans (16.6%), and we analyzed results of those who completed both screening tests (N = 124). Prevalence of BE/EAC among studied veterans was 12.9% (16/124), based on EGD. Sensitivity and specificity of EC/EG for EGD-detected BE/EAC were 92.9% (95% confidence interval [CI] 66.1-99.8) and 72.2% (95% CI 62.1-80.8), respectively. Positive and negative predictive values were 32.5% (95% CI 18.6-49.1) and 98.6% (95% CI 92.4-100), respectively. Baseline Spielberger State-Trait Anxiety Inventory-6 scores were reflective of notable levels of anxiety among veterans in the periprocedural setting. The mean postprocedure acceptability score for the EC test was 7.23 (SD 2.45). DISCUSSION: Our data suggest excellent sensitivity and negative predictive value of EC/EG in a screening population of veterans, making this modality a powerful screening tool for BE and EAC.
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BACKGROUND & AIMS: Mechanisms contributing to the onset and progression of Barrett's (BE)-associated esophageal adenocarcinoma (EAC) remain elusive. Here, we interrogated the major signaling pathways deregulated early in the development of Barrett's neoplasia. METHODS: Whole-transcriptome RNA sequencing analysis was performed in primary BE, EAC, normal esophageal squamous, and gastric biopsy tissues (n = 89). Select pathway components were confirmed by quantitative polymerase chain reaction in an independent cohort of premalignant and malignant biopsy tissues (n = 885). Functional impact of selected pathway was interrogated using transcriptomic, proteomic, and pharmacogenetic analyses in mammalian esophageal organotypic and patient-derived BE/EAC cell line models, in vitro and/or in vivo. RESULTS: The vast majority of primary BE/EAC tissues and cell line models showed hyperactivation of EphB2 signaling. Transcriptomic/proteomic analyses identified EphB2 as an endogenous binding partner of MYC binding protein 2, and an upstream regulator of c-MYC. Knockdown of EphB2 significantly impeded the viability/proliferation of EAC and BE cells in vitro/in vivo. Activation of EphB2 in normal esophageal squamous 3-dimensional organotypes disrupted epithelial maturation and promoted columnar differentiation programs, notably including MYC. EphB2 and MYC showed selective induction in esophageal submucosal glands with acinar ductal metaplasia, and in a porcine model of BE-like esophageal submucosal gland spheroids. Clinically approved inhibitors of MEK, a protein kinase that regulates MYC, effectively suppressed EAC tumor growth in vivo. CONCLUSIONS: The EphB2 signaling is frequently hyperactivated across the BE-EAC continuum. EphB2 is an upstream regulator of MYC, and activation of EphB2-MYC axis likely precedes BE development. Targeting EphB2/MYC could be a promising therapeutic strategy for this often refractory and aggressive cancer.
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Esôfago de Barrett , Carcinoma de Células Escamosas , Neoplasias Esofágicas , Suínos , Animais , Esôfago de Barrett/patologia , Efrina-B2/genética , Proteômica , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas/patologia , Proto-Oncogenes , Proteínas Tirosina Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mamíferos/genéticaRESUMO
The identification of hereditary cancer genes for esophageal adenocarcinoma (EAC) and its precursor, Barrett's esophagus (BE), may prove critical for the development of novel prevention and treatment strategies. Specifically, efforts for detecting BE and EAC susceptibility genes have focused on families with three or more affected members, since these individuals have an earlier age onset compared to non-familial individuals. Given that the use of BE may overestimate the likelihood of disease heritability, we evaluated the age of diagnosis in kindreds with a restricted definition including only confirmed high-grade dysplasia (HGD) or EAC. The Familial Barrett's Esophagus Consortium database was used to identify individuals with HGD and EAC. These individuals were subsequently split into three kindred groups: non-familial-a single affected family member, duplex-two affected family members, and multiplex-three or more affected family members. Age of cancer diagnosis and other risk factors were compared between individuals in these groups. The study included 441 non-familial, 46 duplex, and 13 multiplex individuals. There was a statistically significant difference for age of diagnosis for individuals in the multiplex families compared to the non-familial and duplex families (56.0 versus 64.3, 63.5; p = 0.049). There was no significant difference between demographic factors and other cancer risk factors between family types. The results of this study support a genetic basis for familial Barrett's associated neoplasia and evaluation of the genetic susceptibility to this disease should continue to focus on families with multiple (three or more) affected members.
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Adenocarcinoma , Esôfago de Barrett , Neoplasias Esofágicas , Síndromes Neoplásicas Hereditárias , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Esôfago de Barrett/diagnóstico , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Humanos , Fatores de RiscoRESUMO
BACKGROUND & AIMS: Esophageal adenocarcinoma (EAC) is resistant to standard chemoradiation treatments, and few targeted therapies are available. We used large-scale tissue profiling and pharmacogenetic analyses to identify deregulated signaling pathways in EAC tissues that might be targeted to slow tumor growth or progression. METHODS: We collected 397 biopsy specimens from patients with EAC and nonmalignant Barrett's esophagus (BE), with or without dysplasia. We performed RNA-sequencing analyses and used systems biology approaches to identify pathways that are differentially activated in EAC vs nonmalignant dysplastic tissues; pathway activities were confirmed with immunohistochemistry and quantitative real-time polymerase chain reaction analyses of signaling components in patient tissue samples. Human EAC (FLO-1 and EsoAd1), dysplastic BE (CP-B, CP-C, CP-D), and nondysplastic BE (CP-A) cells were incubated with pharmacologic inhibitors or transfected with small interfering RNAs. We measured effects on proliferation, colony formation, migration, and/or growth of xenograft tumors in nude mice. RESULTS: Comparisons of EAC vs nondysplastic BE tissues showed hyperactivation of transforming growth factor-ß (TGFB) and/or Jun N-terminal kinase (JNK) signaling pathways in more than 80% of EAC samples. Immunohistochemical analyses showed increased nuclear localization of phosphorylated JUN and SMAD proteins in EAC tumor tissues compared with nonmalignant tissues. Genes regulated by the TGFB and JNK pathway were overexpressed specifically in EAC and dysplastic BE. Pharmacologic inhibition or knockdown of TGFB or JNK signaling components in EAC cells (FLO-1 or EsoAd1) significantly reduced cell proliferation, colony formation, cell migration, and/or growth of xenograft tumors in mice in a SMAD4-independent manner. Inhibition of the TGFB pathway in BE cell lines reduced the proliferation of dysplastic, but not nondysplastic, cells. CONCLUSIONS: In a transcriptome analysis of EAC and nondysplastic BE tissues, we found the TGFB and JNK signaling pathways to be hyperactivated in EACs and the genes regulated by these pathways to be overexpressed in EAC and dysplastic BE. Inhibiting these pathways in EAC cells reduces their proliferation, migration, and formation of xenograft tumors. Strategies to block the TGFB and JNK signaling pathways might be developed for treatment of EAC.
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Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Sistema de Sinalização das MAP Quinases/genética , RNA Neoplásico/análise , Fator de Crescimento Transformador beta/metabolismo , Animais , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Dioxóis/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Testes Farmacogenômicos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Pirazóis/farmacologia , Quinolinas/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Proteínas Smad/genética , Proteínas Smad/metabolismo , Biologia de Sistemas , Transcriptoma , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética , Ensaio Tumoral de Célula-TroncoRESUMO
Esophageal adenocarcinoma is a deadly cancer with increasing incidence in the United States, but mechanisms underlying pathogenesis are still mostly elusive. In addressing this question, we assessed gene fusion landscapes by comprehensive RNA sequencing (RNAseq) of 55 pretreatment esophageal adenocarcinoma and 49 nonmalignant biopsy tissues from patients undergoing endoscopy for Barrett's esophagus. In this cohort, we identified 21 novel candidate esophageal adenocarcinoma-associated fusions occurring in 3.33% to 11.67% of esophageal adenocarcinomas. Two candidate fusions were selected for validation by PCR and Sanger sequencing in an independent set of pretreatment esophageal adenocarcinoma (N = 115) and nonmalignant (N = 183) biopsy tissues. In particular, we observed RPS6KB1-VMP1 gene fusion as a recurrent event occurring in approximately 10% of esophageal adenocarcinoma cases. Notably, esophageal adenocarcinoma cases harboring RPS6KB1-VMP1 fusions exhibited significantly poorer overall survival as compared with fusion-negative cases. Mechanistic investigations established that the RPS6KB1-VMP1 transcript coded for a fusion protein, which significantly enhanced the growth rate of nondysplastic Barrett's esophagus cells. Compared with the wild-type VMP1 protein, which mediates normal cellular autophagy, RPS6KB1-VMP1 fusion exhibited aberrant subcellular localization and was relatively ineffective in triggering autophagy. Overall, our findings identified RPS6KB1-VMP1 as a genetic fusion that promotes esophageal adenocarcinoma by modulating autophagy-related processes, offering new insights into the molecular pathogenesis of esophageal adenocarcinomas. Cancer Res; 76(19); 5628-33. ©2016 AACR.
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Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Fusão Gênica , Proteínas de Membrana/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Análise de Sequência de RNA , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Autofagia , Linhagem Celular Tumoral , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , HumanosAssuntos
Histiocitoma Fibroso Benigno/patologia , Neoplasias Cutâneas/patologia , Adolescente , Biópsia por Agulha , Diagnóstico Diferencial , Feminino , Seguimentos , Histiocitoma Fibroso Benigno/diagnóstico , Humanos , Imuno-Histoquímica , Osteopecilose/diagnóstico , Osteopecilose/patologia , Índice de Gravidade de Doença , Dermatopatias Genéticas/diagnóstico , Dermatopatias Genéticas/patologia , Neoplasias Cutâneas/diagnósticoRESUMO
We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755-763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca(2+) mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.
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Trifosfato de Adenosina/metabolismo , Astrocitoma/metabolismo , Líquido Extracelular/metabolismo , Canais Iônicos/metabolismo , Receptor PAR-1/metabolismo , Trombina/metabolismo , Ânions , Sinalização do Cálcio , Carbenoxolona/farmacologia , Linhagem Celular Tumoral , Quelantes/farmacologia , Colforsina/análogos & derivados , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Ativação Enzimática , Humanos , Soluções Hipertônicas , Soluções Hipotônicas , Canais Iônicos/efeitos dos fármacos , Cinética , Concentração Osmolar , Probenecid/farmacologia , Receptor PAR-1/efeitos dos fármacos , Toxina Tetânica/farmacologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Previously, we reported that activation of G protein-coupled receptors (GPCR) in 1321N1 human astrocytoma cells elicits a rapid release of ATP that is partially dependent on a G(q)/phophospholipase C (PLC)/Ca(2+) mobilization signaling cascade. In this study we assessed the role of Rho-family GTPase signaling as an additional pathway for the regulation of ATP release in response to activation of protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca(2+) mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme, which inhibit Rho-GTPases, attenuated the thrombin- and LPA-stimulated ATP release but did not decrease carbachol-stimulated release. Thus the ability of certain G(q)-coupled receptors to additionally stimulate Rho-GTPases acts to strongly potentiate a Ca(2+)-activated ATP release pathway. However, pharmacological inhibition of Rho kinase I/II or myosin light chain kinase did not attenuate ATP release. PAR1-induced ATP release was also reduced twofold by brefeldin treatment suggesting the possible mobilization of Golgi-derived, ATP-containing secretory vesicles. ATP release was also markedly repressed by the gap junction channel inhibitor carbenoxolone in the absence of any obvious thrombin-induced change in membrane permeability indicative of hemichannel gating.
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Trifosfato de Adenosina/metabolismo , Astrócitos/fisiologia , Cálcio/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , ADP Ribose Transferases/farmacologia , Antígenos CD/metabolismo , Apirase/metabolismo , Astrócitos/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Toxinas Botulínicas/farmacologia , Brefeldina A/farmacologia , Carbacol/farmacologia , Carbenoxolona/farmacologia , Linhagem Celular , Permeabilidade da Membrana Celular , Humanos , Receptor Muscarínico M3/metabolismo , Receptor PAR-1/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Vesículas Secretórias/fisiologia , Trombina/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidoresRESUMO
The inherent electron-capture properties of triamcinolone acetonide (TAA) fatty acid conjugates were exploited for development of a GC-MS technique for quantitation of C21 long-chain fatty esters of TAA synthesized in BEAS-2B cells, an immortalized airway epithelium cell line. TAA esters extracted from BEAS-2B cells were purified and detected via selected ion monitoring of the molecular anions generated from the TAA esters under electron-capture negative-ion mass spectrometric conditions. Standard curves were linear over a range of 0.0 to >4.5 ng/mg protein with r(2) values = 1. Levels of TAA conjugates extracted from BEAS-2B treated with 10(-5)M TAA for 24h ranged from 0.024 to 0.301 ng/mg protein. Further evidence for confirmation of the identity of TAA fatty esters formed in BEAS-2B cells was obtained via selected reaction monitoring. The transition monitored was formation of the carboxy anion generated from each of the respective molecular anions of the TAA esters during collision-induced decomposition. These findings indicate that the GC-MS analysis is suitable for studies of the kinetics of the TAA fatty acid conjugates formation in vitro and may be directly applicable to determination of the kinetics of TAA fatty acid conjugation in vivo.