A Novel Gene List Identifies Tumors with a Stromal-Mesenchymal Phenotype and Worse Prognosis in Gastric Cancer

Abstract

Background: Molecular biomarkers that predict disease progression can help identify tumor subtypes and shape treatment plans. In this study, we aimed to identify robust biomarkers of prognosis in gastric cancer based on transcriptomic data obtained from primary gastric tumors.

Methods: Microarray, RNA sequencing, and single-cell RNA sequencing-based gene expression data from gastric tumors were obtained from public databases. Freshly frozen gastric tumors (n = 42) and matched FFPE (formalin-fixed, paraffin-embedded) (n = 40) tissues from a Turkish gastric cancer cohort were used for quantitative real-time PCR and immunohistochemistry-based assessments of gene expression, respectively.

Results: A novel list of 20 prognostic genes was identified and used for the classification of gastric tumors into two major tumor subgroups with differential stromal gene expression ("Stromal-UP" (SU) and "Stromal-DOWN" (SD)). The SU group had a more mesenchymal profile with an enrichment of extracellular matrix-related gene sets and a poor prognosis compared to the SD group. Expression of the genes within the signature correlated with the expression of mesenchymal markers ex vivo. A higher stromal content in FFPE tissues was associated with shorter overall survival.

Conclusions: A stroma-rich, mesenchymal subgroup among gastric tumors identifies an unfavorable clinical outcome in all cohorts tested.

Keywords: biomarker; gastric cancer; prognosis; stroma.

Figure 1

Hierarchical clustering analysis of gastric cancer tumors with prognostic genes. Twenty probesets that were significantly associated with survival data were used for hierarchical clustering in discovery cohorts: (a) GSE62254 (n = 300) and (b) GSE15459 (n = 192). Red, black and green colors indicate high, intermediate and low expression, respectively. Two groups with discernible high and low expressions for all 20 probesets were labeled as “SU” and “SD” groups, respectively. Kaplan–Meier plots are shown for patients with the SU/SD label in (c) GSE62254 and (d) GSE15459. Log-rank p-values are indicated.

Figure 2

Hierarchical clustering analysis and Kaplan–Meier graphs of gastric cancer tumors with prognostic genes in validation cohorts. (a) Out of 20, 13 prognostic probesets that were available on the HGU133A platform were used for hierarchical clustering in GSE29272, and (c) Eighteen Illumina probesets of prognostic genes were used for GSE84437. Red, black and green colors indicate high, intermediate and low expression, respectively. Two major branches with low and high overall expression were assigned to the SU and SD groups. Kaplan–Meier graphs and log-rank p-values are shown for (b) GSE29272 and (d) GSE84437.

Figure 3

Kaplan–Meier plots for SU–SD groups within intestinal and diffuse type tumors in GSE62254 and GSE15459. Log-rank p-values are shown.

Figure 4

IHC-based staining of protein levels in tissue (representative low and high areas, ×40 magnification) and protein expression patterns of the proteins regarding the five genes (×200 magnification). ACTA2, TAGLN, and CALD1: cytoplasmic staining in spindle-shaped stromal cells, mostly regarding fibroblasts (asterisk) and negative in neoplastic epithelial glands (arrowhead). Staining intensity was stronger in ACTA2, while TAGLN and CALD1 showed weaker staining. TMP2: Moderate to strong cytoplasmic and membranous staining in neoplastic epithelial glands (arrowhead). Additionally, stromal cells showed positivity (an asterisk) in various degrees. HEYL: weak to moderate cytoplasmic and nuclear staining in neoplastic epithelial glands (arrowhead) with moderate expression in stromal cells, including inflammatory cells (asterisk).