Expression profiling of stem cell-related genes in neoadjuvant-treated gastric cancer: a NOTCH2, GSK3B and β-catenin gene signature predicts survival

Abstract

Cancer stem cell (CSC) based gene expression signatures are associated with prognosis in various tumour types and CSCs are suggested to be particularly drug resistant. The aim of our study was first, to determine the prognostic significance of CSC-related gene expression in residual tumour cells of neoadjuvant-treated gastric cancer (GC) patients. Second, we wished to examine, whether expression alterations between pre- and post-therapeutic tumour samples exist, consistent with an enrichment of drug resistant tumour cells. The expression of 44 genes was analysed in 63 formalin-fixed, paraffin embedded tumour specimens with partial tumour regression (10-50% residual tumour) after neoadjuvant chemotherapy by quantitative real time PCR low-density arrays. A signature of combined GSK3B(high), β-catenin (CTNNB1)(high) and NOTCH2(low) expression was strongly correlated with better patient survival (p<0.001). A prognostic relevance of these genes was also found analysing publically available gene expression data. The expression of 9 genes was compared between pre-therapeutic biopsies and post-therapeutic resected specimens. A significant post-therapeutic increase in NOTCH2, LGR5 and POU5F1 expression was found in tumours with different tumour regression grades. No significant alterations were observed for GSK3B and CTNNB1. Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of NOTCH2 staining, but not in the percentage of NOTCH2. Taken together, the GSK3B, CTNNB1 and NOTCH2 expression signature is a novel, promising prognostic parameter for GC. The results of the differential expression analysis indicate a prominent role for NOTCH2 and chemotherapy resistance in GC, which seems to be related to an effect of the drugs on NOTCH2 expression rather than to an enrichment of NOTCH2 expressing tumour cells.

Figure 1. Expression of GSK3B, CTNNB1 and NOTCH2 and association with survival. A)

Clustering of tumours based on expression of GSK3B, CTNNB1 and NOTCH2. B) The Kaplan-Meier curves of the patient clusters show better survival of patients in cluster 2 (median OS not reached) compared to cluster 1 (median OS 36.7 mo, 95% CI 24.4–49.1) or cluster 3 (median OS 55.9 mo, 95% CI 16.7–95.0). C) Kaplan-Meier curves of patients based on the categorisation of tumours according to the optimal cut-off values for the three genes (GSK3B ^high CTNNB1 ^high NOTCH2 ^low: median OS not reached; GSK3B ^low CTNNB1 ^low NOTCH2 ^high: median OS 18.0 mo, 95% CI 0–39.5; Others: median OS 42.1 mo, 95% CI 28.3–55.9). D) Analysis of publically available array data of gastric cancer . Kaplan-Meier curves of patients categorised according to the combined expression of GSK3B, CTNNB1 and NOTCH2 in the tumours using optimal cut-off values are shown (GSK3B ^high CTNNB1 ^high NOTCH2 ^low: median OS not reached; Others: median OS 14.6 mo, 95% CI 8.6–19.3). P-values were determined by the log-rank test.

Figure 2. Alterations in the immunohistochemical staining for NOTCH2 between pre-therapeutic biopsies and their corresponding post-therapeutic tumours.

Alterations of cytoplasmic staining intensities are shown. Each line indicates the alteration of the immunohistochemical staining score between the pre-therapeutic biopsy (Pre) and the corresponding post-therapeutic tumour specimen (Post) for each case. P-values were determined by the Wilcoxon signed rank test (exact).