Molecular classification of gastric cancer: a new paradigm

Abstract

Purpose: Gastric cancer may be subdivided into 3 distinct subtypes--proximal, diffuse, and distal gastric cancer--based on histopathologic and anatomic criteria. Each subtype is associated with unique epidemiology. Our aim is to test the hypothesis that these distinct gastric cancer subtypes may also be distinguished by gene expression analysis.

Experimental design: Patients with localized gastric adenocarcinoma being screened for a phase II preoperative clinical trial (National Cancer Institute, NCI #5917) underwent endoscopic biopsy for fresh tumor procurement. Four to 6 targeted biopsies of the primary tumor were obtained. Macrodissection was carried out to ensure more than 80% carcinoma in the sample. HG-U133A GeneChip (Affymetrix) was used for cDNA expression analysis, and all arrays were processed and analyzed using the Bioconductor R-package.

Results: Between November 2003 and January 2006, 57 patients were screened to identify 36 patients with localized gastric cancer who had adequate RNA for expression analysis. Using supervised analysis, we built a classifier to distinguish the 3 gastric cancer subtypes, successfully classifying each into tightly grouped clusters. Leave-one-out cross-validation error was 0.14, suggesting that more than 85% of samples were classified correctly. Gene set analysis with the false discovery rate set at 0.25 identified several pathways that were differentially regulated when comparing each gastric cancer subtype to adjacent normal stomach.

Conclusions: Subtypes of gastric cancer that have epidemiologic and histologic distinctions are also distinguished by gene expression data. These preliminary data suggest a new classification of gastric cancer with implications for improving our understanding of disease biology and identification of unique molecular drivers for each gastric cancer subtype.

Figure 1

Venn Diagram demonstrating the distribution of probes that are significantly different between gastric cancer subtypes and normal stomach. Although there is a large overlap between differentially expressed genes in each subgroup comparison versus normal, a large number of genes uniquely differentiate each individual gastric cancer subtype and normal stomach. Analysis is done using limma (with fold-change cut-off 2, and FDR=0.01; values for multiple probe values for the same gene were averaged). Type 1 = proximal non diffuse, type 2 = diffuse, and type 3 = distal non-diffuse gastric cancer.

Figure 2

Discriminant Plot for Gene Signatures showing that the RNA expression signatures of samples from different gastric cancers are quite well separated and very tightly grouped into 3 types of gastric cancer (supplemental table 1): Proximal non-diffuse (type 1), Diffuse (type 2), and Distal non-diffuse (type 3). This sample classification was done using pdmclass R-function, using ridge regression. The leave-one-out cross validation error is 0.14, which implies that >85% samples are classified correctly. The output from pdmClass cross-validation confusion matrix is provided to the right of the discriminant plot.