Jantine W. van Baal

Comparison of Kinome Profiles of Barrett's Esophagus with Normal Squamous Esophagus and Normal Gastric Cardia

The precursor metaplastic mucosal lesion that predisposes for esophageal adenocarcinoma is Barretts esophagus. Because the signal transduction events that occur in Barretts esophagus are poorly understood, this study aimed at generating a comprehensive description of cellular kinase activity in Barretts esophagus, normal squamous esophagus, and gastric cardia to gain more insight into the pathogenesis of Barretts esophagus. Peptide arrays, exhibiting 1,176 specific consensus sequences for protein kinases, were used to produce a global analysis of cellular kinase activity in biopsies of Barretts esophagus, and results were compared with the neighboring cardia and squamous epithelia. Several differences in kinase activity using immunoblot analysis and enzyme activity assays were validated in biopsies of 27 Barretts esophagus patients. Three unique kinome profiles are described and compared. We identified cascades of activated kinases showing that mitogen-activated protein kinase and epidermal growth factor receptor activity are both significantly altered in Barretts esophagus compared with squamous and gastric cardia epithelia. Another novel finding is that the glycolysis pathway is significantly up-regulated in Barretts esophagus, which is illustrated by an up-regulated pyruvate kinase activity. Here, the unique kinome profile of Barretts esophagus is made available as a comprehensive database. Several signaling pathways are revealed as specifically expressed in Barretts esophagus when compared with the adjacent normal epithelia. These unique findings provide novel insight in the pathogenesis of Barretts esophagus that will ultimately help to resolve the increasing problem of Barretts esophagus and prevention of esophageal adenocarcinoma.

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

OBJECTIVES:Barretts 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.

Efficient automated assessment of genetic abnormalities detected by fluorescence in situ hybridization on brush cytology in a Barrett esophagus surveillance population

Automated assessment of genetic abnormalities detected by fluorescence in situ hybridization (FISH) in brush cytology specimens from patients with Barrett esophagus (BE) may enhance the clinical applicability of this methodology. The objectives of this study were to validate a novel, automated, proprietary system (CytoVison SPOT AX) for the assessment of FISH abnormalities in BE brush cytology and, subsequently, to use this automated method for screening of a BE surveillance cohort.

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

Objective Barretts 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.

Stepwise Radical Endoscopic Resection of the Complete Barrett's Esophagus With Early Neoplasia Successfully Eradicates Pre-Existing Genetic Abnormalities

OBJECTIVES: Malignant transformation of Barretts mucosa is associated with the accumulation of genetic alterations. Stepwise radical endoscopic resection of the Barretts segment with early neoplasia is a promising new treatment resulting in complete re-epithelialization of the esophagus with neosquamous epithelium. It is unknown whether radical resection also eradicates genetic abnormalities. The aim of this study was to prospectively evaluate whether genetic abnormalities as found in the Barretts segment before radical resection are effectively eradicated and absent in the neosquamous epithelium.METHODS: Nine patients with early neoplasia who successfully underwent radical resection were included. Immunohistochemistry (IHC) was performed to assess p53 protein overexpression. DNA fluorescent in-situ hybridization was (DNA-FISH) performed for evaluation of numerical abnormalities of chromosomes 1 and 9, and losses of p16 and p53. Immunohistochemistry and DNA-FISH were performed on endoscopic resection specimens of the neoplasia and on follow-up biopsies of the neosquamous epithelium.RESULTS: DNA-FISH and IHC showed alterations in the pretreatment samples of all patients. All showed aneusomy of chromosome 1 and 9. Loss of p16 and p53 were seen in 6 and 8 patients. IHC showed intense p53 nuclear staining in seven patients. Post-treatment biopsies showed neosquamous epithelium with a normal diploid signal count for all DNA-FISH probes and normal IHC stainings in all patients.CONCLUSIONS: Radical resection of Barretts esophagus with early neoplasia successfully eradicates pre-existing genetic abnormalities and results in neosquamous epithelium without these genetic abnormalities.

A comparative analysis by SAGE of gene expression profiles of esophageal adenocarcinoma and esophageal squamous cell carcinoma

Esophageal adenocarcinoma (EA) and esophageal squamous cell carcinoma (ESCC) are the two main types of esophageal cancer. Despite extensive research the exact molecular basis of these cancers is unclear. Therefore we evaluated the transcriptome of EA in comparison to non-dysplastic Barrett’s esophagus (BE), the metaplastic epithelium that predisposes for EA, and compared the transcriptome of ESCC to normal esophageal squamous epithelium. For obtaining the transcriptomes tissue biopsies were used and serial analysis of gene expression (SAGE) was applied. Validation of results by RT-PCR and immunoblotting was performed using tissues of an additional 23 EA and ESCC patients. Over 58,000 tags were sequenced. Between EA and BE 1013, and between ESCC and normal squamous epithelium 1235 tags were significantly differentially expressed (p < 0.05). The most up-regulated genes in EA compared to BE were SRY-box 4 and Lipocalin2, whereas the most down-regulated genes in EA were Trefoil factors and Annexin A10. The most up-regulated genes in ESCC compared to normal squamous epithelium were BMP4, E-Cadherin and TFF3. The results could suggest that the BE expression profile is closer related to normal squamous esophagus then to EA. In addition, several uniquely expressed genes are identified.

The effect of socioeconomic status on staging and treatment decisions in esophageal cancer.

Background: Optimal treatment choice for patients with esophageal cancer (EC) is complex and largely determined by tumor characteristics, comorbidity, and age. Goals: This study describes the role of patient characteristics, among which is socioeconomic status (SES), in EC treatment. Study: Patients diagnosed with primary EC between 1990 and 2008 in the southern part of the Netherlands were identified using the Eindhoven Cancer Registry. Multivariable logistic and proportional hazard regression analyses were used to identify determinants of treatment and survival. Results: We included 1914 patients, and 37% of them underwent intentionally curative treatment. Low-SES patients were diagnosed at older age (16% vs. 9%, age more than or equal to 80) and with more advanced tumor stages (13% vs. 10%, stage T4) than high-SES patients. Age less than 60 compared with 70 to 79 years [adjusted odds ratio, 4.51; 95% confidence interval (CI), 2.98-6.84] and high SES compared with low SES (adjusted odds ratio 1.59; 95% CI, 1.07-2.37) were independent predictors for curative treatment. Probability of death for high-SES patients undergoing palliative treatment was decreased compared with low-SES patients (hazard ratio, 0.84; 95% CI, 0.71-0.99). Conclusions: SES is an important factor in treatment choice of EC. As health care is equally accessible to the whole population in the Netherlands, this suggests that both patient-related and physician-related factors are involved in this phenomenon.

Cytokeratin and CDX-2 expression in Barrett's esophagus

Objective. Barretts esophagus (BE) is a premalignant condition of the distal esophagus. For diagnostic purposes it is important to find biomarkers that can specifically identify BE, for instance to differentiate BE epithelial cells from gastric cardia epithelial cells in brush cytology specimens. The objective of this study was to determine the specificity of CDX-2 and a set of cytokeratins (CKs) as specific markers for BE as compared with normal squamous esophageal and gastric cardia tissue. Material andmethods. Immunohistochemistry (IHC) with specific antibodies against CDX-2, and a set of CKs was performed on fresh frozen consecutive tissue sections of normal squamous, gastric cardia and non-dysplastic BE of 80 patients. Results. IHC results showed CK8, CK18 and CK20 expression in both BE and gastric cardia, while CK7 was seen in all BE but also in 26% of gastric cardia biopsies. CK10/13 was only expressed in normal squamous epithelium. CDX-2 nuclear staining was found in 87.5% of the BE biopsies, whereas normal squamous esophagus and cardia biopsies were negative. Conclusions. CDX-2 in combination with a set of CKs can be used as biomarkers to distinguish between BE and normal squamous esophagus. In order to distinguish BE from cardia tissue, a combination of CDX-2 and CK7 is most informative.

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

BackgroundBarrett’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.MethodsTLR4 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.ResultsTLR4 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.ConclusionTLR4 activation in BE results in a strong increase in COX-2 and may contribute to malignant transformation.

Familial Clustering of Barrett's Esophagus and Esophageal Adenocarcinoma in a European Cohort

BACKGROUND & AIMS Up to 7% of cases of Barretts 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.