Transcriptional oncogenomic hot spots in Barrett's adenocarcinomas: serial analysis of gene expression

Abstract

Serial analysis of gene expression (SAGE) provides quantitative and comprehensive expression profiling in a given cell population. In our efforts to define gene expression alterations in Barrett's-related adenocarcinomas (BA), we produced eight SAGE libraries and obtained a total of 457,894 expressed tags with 32,035 (6.9%) accounting for singleton tags. The tumor samples produced an average of 71,804 tags per library, whereas normal samples produced an average of 42,669 tags per library. Our libraries contained 67,200 unique tags representing 16,040 known gene symbols. Five hundred and sixty-eight unique tags were differentially expressed between BAs and normal tissue samples (at least twofold; P<or=0.05), 395 of these matched to known genes. Interestingly, the distribution of altered genes was not uniform across the human genome. Overexpressed genes tended to cluster in well-defined hot spots located in certain chromosomes. For example, chromosome 19 had 26 overexpressed genes, of which 18 mapped to 19q13. Using the gene ontology approach for functional classification of genes, we identified several groups that are relevant to carcinogenesis. We validated the SAGE results of five representative genes (ANPEP, ECGF1, PP1201, EIF5A1, and GKN1) using quantitative real-time reverse-transcription PCR on 31 BA samples and 26 normal samples. In addition, we performed an immunohistochemistry analysis for ANPEP, which demonstrated overexpression of ANPEP in 6/7 (86%) Barrett's dysplasias and 35/65 (54%) BAs. ANPEP is a secreted protein that may have diagnostic and/or prognostic significance for Barrett's progression. The use of genomic approaches in this study provided useful information about the molecular pathobiology of BAs.

Figure 1

Chromosomal localization of deregulated genes. Chromosomal regions that contain up‐regulated genes are shown in red, whereas those that contain down‐regulated genes are displayed in green. Regions which contain both up‐ and down‐regulated genes are colored in yellow. The distribution of these genes did not follow a random distribution pattern and several genomic regions contain clusters of deregulated genes. Some of the more significant “hot spots” can be seen here on chromosomes 1 (P = 0.01), 3 (P = 0.02), 12 (P = 0.01), 15 (P = 0.01), and 19 (P = 0.01).

Figure 2

Quantitative real‐time reverse‐transcription PCR showing fold expression changes at the mRNA level of five representative genes. qRT‐PCR analysis was performed using iCycler on 31 lower esophageal and GEJ adenocarcinoma samples (Tu) and 6 Barrett's esophagus (BE) samples in comparison with 26 normal glandular mucosa samples (N). The horizontal axis shows sample numbers, whereas the fold expression in tumor samples compared with that in normal samples is shown on the vertical axis. The fold expression was calculated according to the formula: as detailed in the “Materials and Methods” section. Each bar represents one sample. The displayed mean fold expression for each sample is calculated in comparison with the expression average of the 26 normal samples. The expression of each gene was normalized to the expression of HPRT1, which showed minimal variation in all normal and neoplastic samples tested. GKN1 shows downregulation (≤0.4‐fold expression) whereas ANPEP, PP1201, EIF5A1, and ECGF1 demonstrate overexpression (≥2.5 fold expression) in primary tumors as compared to normal tissue samples.

Figure 3

Visualization of RT‐PCR products on gel electrophoresis. Five matched tumor and normal samples that were analyzed using qRT‐PCR were subjected to 1.2% agarose gel electrophoresis and ethidium bromide staining. The intensity of bands confirms the PCR results, indicating higher mRNA expression levels of ANPEP, PP1201, EIF5A1, and ECGF, as well as lower expression of GKN1 in most of the tumor samples as compared with their matched normal control samples. HPRT1 was used as a control to show similar levels in each matched normal and tumor samples.

Figure 4

Immunohistochemical staining for ANPEP. (A, B) Normal gastric tissue glands (A) and normal esophageal squamous tissues (B) are negative for ANPEP immunostaining (Score 0). (C) Barrett's dysplastic tissue demonstrates immunostaining for ANPEP that is secreted in the lumen (Score +2). (D) Barrett's metaplasia tissue shows glandular staining (Score +2). (E) Diffuse‐type esophageal adenocarcinoma tissue shows staining for ANPEP in the cell cytoplasm with significant localization along the cell membranes (Score +3). (F) Intestinal‐type esophageal adenocarcinoma tissue showing high levels of ANPEP along the cell membranes as well as luminal secretion (Score +3). All photos (insets at upper‐right quadrant) are taken at 200× and 400× magnification.