High NOTCH1 mRNA Expression Is Associated with Better Survival in HNSCC

Abstract

The clinical impact of the expression of NOTCH1 signaling components in squamous cell carcinoma of the pharynx and larynx has only been evaluated in subgroups. The aim of this study was therefore to evaluate NOTCH1 expression in head and neck squamous cell cancer (HNSCC) patient tissue and cell lines. We analyzed tissue from 195 HNSCCs and tissue from 30 normal patients for mRNA expression of NOTCH1, NOTCH3, HES1, HEY1, and JAG1 using quantitative real-time PCR. Association of expression results and clinical orpathological factors was examined with multivariate Cox regression. NOTCH1 expression was determined in three Human Papilloma Virus (HPV)-positive and nine HPV-negative HNSCC cell lines. High expression of NOTCH1 was associated with better overall survival (p = 0.013) and disease-free survival (p = 0.040). Multivariate Cox regression confirmed the significant influence of NOTCH1 expression on overall survival (p = 0.033) and disease-free survival (p = 0.029). A significant correlation was found between p16 staining and NOTCH1 mRNA expression (correlation coefficient 0.28; p = 0.01). NOTCH1 was expressed at higher levels in HPV-positive HNSCC cell lines compared with HPV-negative cell lines, which was not statistically significant (p = 0.068). We conclude that NOTCH1 expression is associated with overall survival, and that inhibition of NOTCH1 therefore seems less promising.

Keywords: HNSCC; NOTCH signaling; overall survival.

Figure 1

Depiction of median of relative mRNA expression in tumor tissue and normal tissue. Data were normalized to GAPDH per sample.

Figure 2

Association of relative NOTCH1 mRNA expression with overall (OS) and disease-free (DFS) survival was analyzed using the Kaplan-Meier method as well as the log-rank test. Patients were stratified into a relative high expression (mean mRNA expression + 1 standard deviation (SD)) group and an intermediate–low mRNA expression group (remainder). Significantly longer overall survival (a) and disease-free survival (b) were found for patients with relatively high mRNA expression of NOTCH1 compared with those with intermediate or low expression (p = 0.013 (OS) and p = 0.040 (DFS)).

Figure 3

Depiction of median of relative mRNA expression of NOTCH1 in p16-positive and p16-negative tumor tissue. Expression of NOTCH1 was significantly higher in p16-positive tumor tissue (p ≤ 0.001, Mann–Whitney U Test). * represents extreme outliers.

Figure 4

Association of relative NOTCH1 mRNA expression and overall survival in p16-positive (a) and -negative patients (b) was analyzed by the Kaplan-Meier method followed by log-rank test. A longer overall survival was found for p16-positive patients with relative high mRNA expression of NOTCH1 (a) (n = 50, p = 0.139) but not for p16-negative patients (b) (n = 118, p = 0.967).

Figure 5

In Western blot analyses, NOTCH1 (NTM) could be detected in all cell lines except for HN (top). Quantification revealed higher NOTCH1 protein expression in HPV-positive cell lines compared with in HPV-negative cell lines (p = 0.068) (bottom). Error bars represent one standard deviation.