Quantitative Expressionsanalyse multipler Gene zur molekularen Charakterisierung des Plattenepithels des Ösophagus von gesunden Patienten und Patienten mit Barrett-Ösophagus oder Barrett-Carcinom

Abstract

Background: In order to identify genes or combination of genes that have the power to descriminate between patients with premalignant Barrett’s esophagus and Barrett’s-associated adenocarcinoma based on the molecular profile of the corresponding normal squamous esophagus tissues, we analyzed a panel of 23 genes using quantitative real-time RT-PCR (qRT-PCR, Taqman®) and bioinformatic tools. Methods: 48 histological normal squamous esophageal tissues collected from 19 patients with Barrett’s esophagus (BE), 19 patients with Barrett’s - associated esophageal adenocarcinoma (EA), and a healthy control group of 10 patients (CG) were studied. A quantitative real time RT-PCR method (TaqMan®) was used to measure, relative to the internal standard beta actin, the mRNA expression levels of the following 23 genes: c-Myb, ODC, CDX2, DNMT1, DNMT3a, DNMT3b, RXRalpha, RXRbeta, RXRgamma, RARalpha, RARgamma, BFT, GSTPI, COX1, COX2, DPD, SPARC, BCL2, TP. BAX, DAPK, TM4SF3, TSPAN. Triplicate analysis for the full 23 gene of interest panel was performed for all samples for a total of more than 3528 single PCR reactions. Results: Median mRNA gene expression levels were significantly different in normal squamous esophagus tissues from patients with Barrett’s esophagus, Barrett’s-associated adenocarcinoma and the healthy control group for the following genes: Bax (P = 0.002), BFT (P = 0.001), CDX2 (P = 0.005), COX2 (P = 0.042), DAPK (P = 0.2), DNMT1 (P< 0.001), GSTPI (P = 0.003), RARalpha (P = 0.042), RARgamma (P< 0.001), RXRalpha (P = 0.002), RXRbeta (P = 0.008), SPARC (P = 0.039), TSPAN (P = 0.041), VEGF (P = 0.006). A blinded linear discriminant analysis was able to distinguish three genetically different groups of normal squamous esophagus tissues. The three groups consisted solely of normal tissues from patients with EA, BE or CG. A cross-validation study revealed that molecular separation for the three groups could be achieved with an error-rate of 0.1. Conclusion: This study provides the first non-array parallel mRNA quantitation analysis of a panel of genes in normal squamous esophagus tissues in Barrett’s esophagus disease. It shows substantial differences in gene expression levels in normal esophagus tissues of patients with EA, BE and CG. Our results suggest that mRNA expression quantitation of a panel of genes in combination with a linear discriminant analysis can discriminate between healthy patients, premalignant Barrett’s patients, and malignant Barrett’s cancer based on the molecular profile of corresponding histological normal squamous esophagus tissues. Further studies are warranted to determine the potential clinical value for the treatment of patients with this disease.