Toll-like Receptor 2 Signalling and the Lysosomal Machinery in Barrett's Esophagus.

BACKGROUND AND AIMS: Inflammation plays an important role in the development of esophageal adenocarcinoma and its metaplastic precursor lesion, Barrett's esophagus. Toll-like receptor (TLR) 2 signalling and lysosomal function have been linked to inflammation-associated carcinogenesis. We examined the expression of TLR2 in the esophagus and the effect of long-term TLR2 activation on morphological changes and expression of factors involved in lysosomal function in a Barrett's esophagus epithelium cell line. METHODS: TLR2 expression in normal squamous esophagus, reflux esophagitis, Barrett's esophagus and esophageal adenocarcinoma biopsies was assessed with Q-RT-PCR, in situ hybridization and immunohistochemistry. Barrett's esophagus epithelium cells (BAR-T) were incubated with acid and bile salts in the presence or absence of the TLR2 agonist Pam3CSK4 for a period up to 4 weeks. Morphological changes were assessed with electron microscopy, while Q-RT-PCR was used to determine the expression of lysosomal enzymes (Cathepsin B and C) and factors involved in endocytosis (LAMP-1 and M6PR) and autophagy (LC3 and Rab7). RESULTS: TLR2 was expressed in normal squamous esophagus, reflux esophagitis, Barrett's esophagus but was most prominent in esophageal adenocarcinoma. Long-term TLR2 activation in acid and bile salts exposed BAR-T cells resulted in more and larger lysosomes, more mitochondria and increased expression of LAMP-1, M6PR, Cathepsin B and C when compared to BAR-T cells incubated with acid and bile salts but no TLR2 agonist. Factors associated with autophagy (LC3 and Rab7) expression remained largely unchanged. CONCLUSION: Activation of TLR2 in acid and bile salts exposed Barrett epithelium cells resulted in an increased number of mitochondria and lysosomes and increased expression of lysosomal enzymes and factors involved in endocytosis.

Cryospray ablation using pressurized CO2 for ablation of Barrett's esophagus with early neoplasia: early termination of a prospective series.

BACKGROUND: Cryotherapy is a relatively novel ablation modality for the endoscopic ablation of Barrett's esophagus (BE). Data on the use of pressurized carbon dioxide (CO2) gas for cryoablation are scarce. STUDY AIM: To determine the efficacy and safety of cryospray ablation using pressurized CO2 gas in the treatment of BE with early neoplasia. METHODS: In this prospective single center case series, we aimed to include 30 patients with BE and early neoplasia. Nodular neoplastic lesions were treated with endoscopic mucosal resection (EMR). Residual BE mucosa was treated with cryospray ablation every 4 weeks until the complete BE segment was eliminated or up to seven treatment sessions. If no reduction of the BE segment was observed after two subsequent treatment sessions, cryoablation was terminated. Patients were contacted at days 1 and 4 post-treatment to evaluate the level of discomfort. Endoscopic and histologic follow-up evaluations were performed up to 24 months post-treatment. RESULTS: After the inclusion of 10 patients, insufficient effect of cryoablation was observed, resulting in early termination of the study. In total, seven patients with intramucosal carcinoma (IMC) and three with high grade dysplasia (HGD) were included. Prior EMR was performed in nine patients. A median of 2.5 (IQR 2.0 - 4.0) cryoablation sessions were performed. At 6 months of follow-up, complete eradication of intestinal metaplasia was observed in 11 % (1 /9; one patient died, not treatment or disease related) of the patients and complete eradication of dysplasia in 44 % (4 /9). In three patients, HGD or IMC was detected during follow-up, and was endoscopically treated. Apart from a gastric perforation as a result of gastric distension caused by CO2 gas during the first treatment, cryospray treatments were well tolerated. CONCLUSION: After a short learning curve, cryoablation using CO2 gas was found to be a safe and well tolerated treatment modality. However, in our experience, the efficacy of CO2 cryoablation combined with EMR for nodular lesions is disappointing for the treatment of BE associated neoplasia.

Surveillance of Barrett's esophagus and mortality from esophageal adenocarcinoma: a population-based cohort study.

OBJECTIVES: Barrett's esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma (EAC). Patients with a known diagnosis of BE are usually advised to participate in an endoscopic surveillance program, but its clinical value is unproven. Our objective was to compare patients participating in a surveillance program for BE before EAC diagnosis with those not participating in such a program, and to determine predictive factors for mortality from EAC. METHODS: All patients diagnosed with EAC between 1999 and 2009 were identified in the nationwide Netherlands Cancer Registry. These data were linked to Pathologisch-Anatomisch Landelijk Geautomatiseerd Archief, the Dutch Pathology Registry. Prior surveillance was evaluated, and multivariable Cox proportional hazards regression analysis was performed to identify predictors for all-cause mortality at 2-year and 5-year follow-up. RESULTS: In total, 9,780 EAC patients were included. Of these, 791 (8%) patients were known with a prior diagnosis of BE, of which 452 (57%) patients participated in an adequate endoscopic surveillance program, 120 (15%) patients in an inadequate program, and 219 (28%) patients had a prior BE diagnosis without participating. Two-year (and five-year) mortality rates were lower in patients undergoing adequate surveillance (adjusted hazard ratio (HR)=0.79, 95% confidence interval (CI)=0.64-0.92) when compared with patients with a prior BE diagnosis who were not participating. Other factors associated with lower mortality from EAC were lower tumor stage (stage I vs. IV, HR=0.19, 95% CI=0.16-0.23) and combining surgery with neoadjuvant chemo/radiotherapy (HR=0.66, 95% CI=0.58-0.76). CONCLUSIONS: Participation in a surveillance program for BE, but only if adequately performed, reduces mortality from EAC. Nevertheless, it remains to be determined whether such a program is cost-effective, as more than 90% of all EAC patients were not known to have BE before diagnosis.

Familial clustering of Barrett's esophagus and esophageal adenocarcinoma in a European cohort.

BACKGROUND & AIMS: Up to 7% of cases of Barrett's esophagus (BE) or esophageal adenocarcinoma (EAC) in the United States occur in family clusters. We identified first-degree and second-degree relatives of patients with BE and EAC to determine the extent of familial clustering in a European cohort and studied differences between familial and nonfamilial cases. METHODS: A questionnaire was sent to all patients diagnosed with BE or EAC from 2000-2011 at 3 hospitals in the Netherlands (n = 838). Diagnoses of affected relatives were confirmed by using the Dutch Pathology Registry. Familial statuses of BE were defined as definitive (≥1 first-degree or second-degree relative with BE or EAC), possible (≥1 reported relative with BE or esophageal cancer without histologic confirmation), unlikely (no family history), or unknown. RESULTS: A total of 603 patients with BE or EAC (71%) responded and were included in the analysis. Familial BE was definitive for 7% of cases (n = 39, 10% of first-degree relatives affected), possible for 6% (n = 36), unlikely for 49% (n = 297), and unknown for 38% (n = 231). Definitive cases of familial BE were younger at onset of heartburn and EAC diagnosis; their first-degree relatives more frequently had reflux symptoms and a prior upper endoscopy, compared with unlikely cases of familial BE. CONCLUSIONS: In a database analysis of patients diagnosed with BE or EAC in the Netherlands, 7% of cases of BE and EAC were familial. These cases have a younger average age of onset of reflux symptoms and diagnosis of EAC than unlikely familial cases. These findings may indicate that genetic factors contribute to BE susceptibility, with a possible central role of gastroesophageal reflux.

Consistency of a high-grade dysplasia diagnosis in Barrett's oesophagus: a Dutch nationwide cohort study.

BACKGROUND: Consistency of high-grade dysplasia in Barrett's oesophagus is incompletely known and the clinical course may vary between patients. AIMS: To evaluate the consistency of high-grade dysplasia diagnosis in a Dutch nationwide cohort and to identify predictors for (re-)detecting high-grade dysplasia or oesophageal adenocarcinoma when ≥ 1 follow-up evaluations after an initial high-grade dysplasia diagnosis were scored with a lower histological grade. METHODS: In this retrospective cohort study, all patients diagnosed with high-grade dysplasia in Barrett's oesophagus between 1999 and 2008 in the Netherlands were selected using the nationwide histopathology registry. Multivariate analysis was performed to identify predictors for (re-)detecting high-grade dysplasia or oesophageal adenocarcinoma in patients with ≥ 1 follow-up evaluations scored with a lower grade. RESULTS: In total, 512 high-grade dysplasia patients were included, of whom 53% had ≥ 1 follow-up evaluations scored with a lower grade. The (re-)detection risk was increased when follow-up was performed in a university hospital and when endoscopic/surgical resection was performed and decreased with an increasing number of follow-up evaluations scored with a lower grade. CONCLUSION: High-grade dysplasia diagnosis was inconsistent in more than half of patients. (Endoscopic) resection in an expert centre is recommended to (re-)detect high-grade dysplasia or oesophageal adenocarcinoma when an endoscopic follow-up protocol with biopsies repeatedly shows a lower histological grade.

Toll-like receptor 4 activation in Barrett's esophagus results in a strong increase in COX-2 expression.

BACKGROUND: Barrett's esophagus (BE) is known to progress to esophageal adenocarcinoma in a setting of chronic inflammation. Toll-like receptor (TLR) 4 has been linked to inflammation-associated carcinogenesis. We aimed to determine the expression and functional activity of TLR4 in the esophagus and whether TLR4 activation in BE could promote carcinogenesis by inducing COX-2 expression. METHODS: TLR4 expression in esophageal adenocarcinoma, BE, duodenum, reflux esophagitis and normal squamous esophagus biopsies was assessed using real-time PCR and validated by in situ hybridization and immunohistochemistry. Ex vivo cultures of BE, duodenum and normal squamous esophagus biopsies and a BE cell line (BAR-T) were stimulated with the TLR4 agonist lipopolysaccharide (LPS). To evaluate the effect of TLR4 activation, NF-κB activation, IL8 secretion and expression and COX-2 expression were determined. RESULTS: TLR4 expression was significantly increased in esophageal adenocarcinoma, BE, duodenum and reflux esophagitis compared to normal squamous esophagus. LPS stimulation resulted in NF-κB activation and a dose-dependent increase of IL8 secretion and mRNA expression. The induction of IL8 was more evident in BE compared to normal squamous esophagus. Upon LPS stimulation, COX-2 expression increased significantly in ex vivo cultured BE biopsies, which was observed in both epithelium and lamina propria cells. However, no effect was found in duodenum and normal squamous esophagus biopsies. CONCLUSION: TLR4 activation in BE results in a strong increase in COX-2 and may contribute to malignant transformation.

microRNA-145 in Barrett's oesophagus: regulating BMP4 signalling via GATA6.

OBJECTIVE: Barrett's oesophagus (BE) is a metaplastic condition of the distal oesophagus which predisposes to oesophageal adenocarcinoma (EAC). It has been suggested that microRNAs (miRNAs) are involved in the process of development of BE and EAC; however, few functional miRNA data are available. The aim of the study was to perform a tissue-specific miRNA profile and, based on this, to examine the function of miRNA-145 in the oesophagus. DESIGN: miRNA expression profiling using microarray analysis in EAC, BE and normal squamous epithelium of the oesophagus (SQ) was performed and validated using real-time PCR in samples from 15 patients and in situ hybridisation in samples from 10 patients. The proliferative effect of miRNA-145 precursor transfection in the SQ (HET-1A) and BE cell line (BAR-T) was measured. Downstream targets of miRNA-145 were determined by analysing mRNA and protein expression from miRNA-145 transfected cells. RESULTS: Three unique miRNA expression profiles were found in tissue from EAC, BE and SQ, which showed that miRNA-145 was upregulated in BE compared with EAC and SQ. Overexpression of miRNA-145 in HET-1A and BAR-T cells reduced cell proliferation and inhibited GATA6, BMP4 and SOX9 mRNA expression. Furthermore, altered BMP4 signalling was observed in vitro on miRNA-145 overexpression. These effects were blocked when cells were co-transfected with a miRNA-145 specific inhibitor. Additionally, BMP4 incubation of HET-1A cells altered miRNA-145 and GATA6 expression over time. CONCLUSION: These results imply that miRNA-145 indirectly targets BMP4 via GATA6 and is potentially involved in the development of BE.

Surveillance and follow-up strategies in patients with high-grade dysplasia in Barrett's esophagus: a Dutch population-based study.

OBJECTIVES: In patients with high-grade dysplasia (HGD) in Barrett's esophagus (BE), it is incompletely known which factors are associated with developing esophageal adenocarcinoma (EAC). We analyzed prior biopsy and follow-up strategies in a large nationwide population-based cohort of patients with HGD in BE, and identified predictors of EAC progression. METHODS: Prior biopsy records and follow-up evaluations were studied in patients with HGD in BE diagnosed between 1999 and 2008, using PALGA, a nationwide network and registry of histopathology and cytopathology in the Netherlands. Multivariate Cox proportional hazards regression analysis was performed to identify predictors for prevalent (≤ 6 months) and incident (> 6 months) EAC. RESULTS: In total, 827 patients with HGD in BE were included. Follow-up data after HGD diagnosis were available in 699 (85%) patients. In 249 (36%) of these patients, an EAC was detected (14.1 EACs per 100 person-years). The risk of prevalent EAC (n=177) was lower with previous surveillance (hazards ratio 0.7; 95% confidence interval 0.5-0.9), unifocal HGD (0.3;0.2-0.6), diagnosis in a university hospital (0.5;0.3-0.9), endoscopic resection (0.5;0.3-0.7), or ablation (0.0;0.0-0.3); and higher when patients were 65-75 years (1.5;1.04-2.04). After exclusion of prevalent EACs, the progression rate was 4.2 EACs per 100 person-years. The risk of progression to incident EAC (n = 72) was lower with previous surveillance (0.6;0.3-0.9) and ablation (0.2;0.0-0.8), and higher when > 75 years (3.8;2.0-7.2) or with an interval > 6 months between HGD diagnosis and first follow-up (e.g., 7-12 months 2.9;1.3-6.3). CONCLUSIONS: In this cohort of patients with HGD in BE, the EAC detection rate was 14.1 per 100 person-years and 4.2 per 100 person-years after excluding prevalent cases. The risk of both prevalent and incident EAC was reduced with previous surveillance and endoscopic treatment, while it was increased with older age.