Computational protocol: Up-regulation of CLDN1 in gastric cancer is correlated with reduced survival

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Protocol publication

[…] R/BioConductor [,] with the package Beadarray [] were used for preprocessing of the microarray text data from BeadStudio. Spatial artifacts were removed using BASH [] before the expression data were log2-transformed and quantile normalized. The log2 fold change (FC) of each probe on the array within each tissue pair (tumor vs matched normal mucosa) was then calculated, and the data were loaded into the J-express software package []. Rank product testing [] was then performed to test whether the differential expression between tumor tissue and matched normal mucosa was significant. The differential expression was declared significant if the adjusted p-value, i.e. the FDR q-value, was less than 0.05. Hierarchical clustering was performed using average linkage and Euclidean distance measure. The analyses were performed using the J-express software package [].To produce a reasonably sized list of the most differentially expressed genes, lesser expressed genes were filtered out at a cutoff level of FC > 1.5, producing a list of the 130 most differentially expressed genes. This dataset was imported into Onto-Express and Pathway Express [,], part of the Onto-Tools software suite, for functional analysis, and grouped into Gene Ontology (GO) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) cellular signaling pathways []. Pathway Express calculates an Impact Factor (IF) which is used to rank the affected signaling pathways, based on the fold change, the number of the involved genes in the pathway, and the amount of perturbation of downstream genes [].The dataset was entered into PASW Statistics (SPSS version 18.0.2) to perform bivariate correlation analysis to select genes that associated with clinicopathological parameters. Both Pearson and Spearman correlation coefficients were employed to identify correlating genes. Among genes that correlated, we were particularly interested in those that showed a similar expression in our previously published study of H. pylori-exposed gastric epithelial cells []. The selected genes were then subjected to a Cox multivariate regression analysis to investigate whether any of the genes were independent predictors of post-operative survival in the GC patients, independent of histological type, tumor stage and size, nodal disease, and age at surgery. In the one predictor gene that was identified, different cut-off levels were applied to construct high and low expression level groups, before statistical significance between the groups was assessed using a log-rank (Mantel-Cox) test. A Kaplan-Meier survival plot was created to demonstrate the difference in survival between the high- and low-expression groups.The microarray data are available under the accession number E-MTAB-1440 in the ArrayExpress database []. […]

Pipeline specifications

Software tools BASH, J-Express, Onto-Express, Pathway-Express, Onto-Tools
Application aCGH data analysis
Organisms Helicobacter pylori
Diseases Gastritis, Infection, Neoplasms, Stomach Neoplasms, Helicobacter Infections