Krishnadath, K.K.

Reproducibility of protein identification of selected cell types in barrett's esophagus analyzed by combining laser-capture microdissection and mass spectrometry (Article)

2011-01-07T00:00:00Z

Barrett's esophagus (BE) is associated with increased risk of esophageal adenocarcinoma (EAC) and characterized by replacement of normal esophageal squamous epithelium by columnar epithelium. These alterations are also reflected in changes in the protein-expression profiles of the cell types involved. To separately investigate the proteomes of selected cell-types we combined laser-capture microdissection (LCM) and liquid chromatography-mass spectrometry (LC-MS). Aims were to determine the sensitivity, specificity, and technical reproducibility of the sampling method, and the biological variability within and between biopsies and patients. Frozen biopsies were cryo-sectioned, samples of around 2000 epithelial or stroma cells microdissected, digested and measured by Orbitrap LC-MS. Proteins were then identified by MS/MS database search and quantified by label-free analysis. An average of 366 protein-groups were identified per sample, and more protein-groups were found in epithelial samples than in stromal samples (442 vs 301, p < 0.0001). Altogether, 1254 distinct proteingroups were found, 289 and 88 of them significantly more often in epithelial and stroma samples, respectively. We assessed five different types of reproducibilities (run-to-run, intrabiopsy, biopsy-tobiopsy, experiment-to-experiment, and patient-to-patient) for protein identification and protein quantification. Reproducibility of protein identification ranged from 78 to 57%, and standard deviation of protein quantification was on patient-to-patient level four times higher than for run-to-run. We conclude that sampling around 2000 cells requires groups of 32 samples to detect significant, over 10-fold differences in protein abundances and thus creates a successful compromise between throughput and quality of results. We therefore believe that this method is suitable for investigating protein-expression profiles during carcinogenesis.

COX-2 CA-haplotype is a risk factor for the development of esophageal adenocarcinoma (Article)

2007-11-01T00:00:00Z

BACKGROUND: Neoplastic progression of BE towards EAC is associated with increased expression of COX-2. Increased COX-2 expression and enzyme activity is linked to the COX-2 CA haplotype, which consists of two gene polymorphisms in the COX-2 promoter. AIM: To study the impact of COX-2 haplotypes on the risk of developing EAC in patients with different forms of gastroesophageal reflux disease including BE. METHODS: DNA was obtained from a total of 635 Dutch white patients comprised of 140 patients with EAC, 255 with BE, and 240 with reflux esophagitis. COX-2 haplotypes were based on the gene polymorphisms at -765C/G and -1195A/G, as determined by PCR-RFLP. RESULTS: The tested population contained 170 (14%) CA- (-765C and -1195A) haplotypes, 829 (65%) GA and 271 (21%) GG-haplotypes, and no GC-haplotypes. The haplotype distribution in patients with reflux esophagitis and BE was similar (CA 12%, GA 68%, GG 21%), but differed significantly from that in patients with EAC (CA 21%, GA 58%, GG 20%). Particularly, the CA-haplotype was more common (P < 0.001) in EAC patients. CA-carriership was associated with EAC (OR 2.8, 95% CI 1.3-6.2, P = 0.008), with homozygosity for the CA-allele being statistically most significantly associated (OR 6.1, 95% CI 1.6-24.2, P = 0.01). CONCLUSION: The COX-2 CA-haplotype is more frequently observed in patients with EAC than in patients with BE and reflux esophagitis. These data suggest a direct link between COX-2 activity and neoplastic progression in patients with BE and reflux esophagitis.

From Barrett's esophagus to adenocarcinoma and metastasis (Doctoral Thesis)

1997-06-26T00:00:00Z

The first description of islets of ectopic gastric mucosa in the esophagus was by Schmidt in 1805. One century later, in 1906, Tileston described peptic ulcerations in columnar epithelium lining the distal esophagus. In 1950 Norman Barrett gave a detailed description of the columnar lined esophagus. He regarded the distal columnar lined esophagus a mediastinal extension of the stomach, as a result of a congenitally short esophagus. Barrett based his theory on the nature of the mucosa and mucosal secretions of the columnar lining, whereas absence of the musculature and peritoneal covering of the normal stomach were ignored. In that period Lortat-Jacob described the same condition, which he named endobrachyesophagus, a term still used in French literature. Barrett's observation was that of gastric mucosa, extending as a continuous sheet into the mediastinum. He observed that the columnar mucosa could extend for a varying distance and could reach as far as the aortic arch. Allison and Johnstone in 1953 showed that anatomically and functionally the segment of digestive tract described by Barrett is part of the esophagus. These authors suggested that the so-called Barrett's esophagus might be an acquired rather than a congenital condition. This implies that as a consequence of gastroesophageal reflux, oesophageal squamous epithelium is converted to columnar epithelium through metaplasia. In several studies, authors noted "upward migration" of the squamo-columnar junction during follow-up of patients with gastro-esophageal reflux. Animal experiments proved that columnar epithelium in the esophagus is generated in the presence of gastro-oesophageal reflux. Through these observations it had become apparent that Barrett's esophagus is an acquired rather than a congenital condition. Around 1980 most authors appear to favor the view of the acquired origin of Barrett's esophagus However, congenital islands of ectopic gastric mucosa do occur. They are found in up to 10% of individuals undergoing endoscopy. These so called "inlet patches" occur principally in the cervical esophagus and are mostly surrounded by normal squamous epithelium.