c-Kit Expression is Rate-Limiting for Stem Cell Factor-Mediated Disease Progression in Adenoid Cystic Carcinoma of the Salivary Glands

Abstract

Adenoid cystic carcinoma (ACC) is an aggressive malignant neoplasm of the salivary glands in which c-Kit is overexpressed and activated, although the mechanism for this is as yet unclear. We analyzed 27 sporadic ACC tumor specimens to examine the biologic and clinical significance of c-Kit activation. Mutational analysis revealed expression of wild-type c-Kit in all, eliminating gene mutation as a cause of activation. Because stem cell factor (SCF) is c-Kit's sole ligand, we analyzed its expression in the tumor cells and their environment. Immunohistochemistry revealed its presence in c-Kit-positive tumor cells, suggesting an activation of autocrine signaling. We observed a significant induction of ERK1/2 in the cells. SCF staining was also found in other types of non-cancerous cells adjacent to tumors within salivary glands, including stromal fibroblasts, neutrophils, peripheral nerve, skeletal muscle, vascular endothelial cells, mucous acinar cells, and intercalated ducts. Quantitative PCR showed that the top quartile of c-Kit mRNA expression distinguished ACCs from normal salivary tissues and was cross-correlated with short-term poor prognosis. Expression levels of SCF and c-Kit were highly correlated in the cases with perineural invasion. These observations suggest that c-Kit is potentially activated by receptor dimerization upon stimulation by SCF in ACC, and that the highest quartile of c-Kit mRNA expression could be a predictor of poor prognosis. Our findings may support an avenue for c-Kit-targeted therapy to improve disease control in ACC patients harboring the top quartile of c-Kit mRNA expression.

Figure 1

Expression of c-Kit, SCF, and P-ERK1/2 in ACC of the salivary glands. (A) H&E staining. (B)–(F). Immunohistochemistry with antibodies to c-Kit (B), SCF (C and D), phospho-p44/42 ERK1/2 (E); antibody isotype control (F). SCF staining was largely in the duct-type epithelial component in the tumors (C and D; indicated by black arrows) where c-Kit was predominantly elevated (B). We observed a significant induction of ERK1/2 activity in the cells (E; black arrows). The staining intensity scales are as follows; c-Kit (B: 2-3 +), SCF (C and D: 1 +), and P-ERK1/2 (E: 2 +).

Figure 2

Immunohistochemical staining with antibodies to SCF in ACC cells and non-cancerous cells in the salivary glands (A-H). (A) Stromal fibroblasts (black arrows). (B) Neutrophils around tumor (black arrows). (C) Nerve twigs (black arrows) and lymphocytes (red arrows). (D) Tumor (black arrows) invading nerve (red arrows). (E) Targetoid pattern of tumor cells (black arrows) in perineural (red arrows) invasion. (F) Skeletal muscle (black arrows) and tumor (red arrows). (G) Tumor (black arrows) and endothelial cells (red arrows) in blood vessels. (H) Mucous acinar cells (black arrows) and intercalated ducts (red arrows).

Figure 3

Box plot analyses of relative gene expression of c-Kit (A), SCF (B), and EGFR (C) in ACC tumor samples and normal tissue. Each box represents the quartile distribution range (25–75%) with the median shown by a black horizontal line. The range and individual cases are displayed as a black vertical line and scatter dots, respectively. The y-axis indicates the fold-change relative to the average of normal samples' gene expression. Statistical comparisons between data sets were performed with two-tailed Student's t tests, with P < .05 considered significant.

Figure 4

Expression levels of SCF and c-Kit in cases with perineural invasion. (A)–(C) Scatter plot analysis for the strength of a correlation between SCF and c-Kit mRNA expression in the presence or absence of perineural invasion. (A) All cases. (B) Cases with perineural invasion. (C) Cases without perineural invasion. Trend lines are included. Trend line equations and R-squared values were calculated separately.

Figure 5

Kaplan-Meier curves showing overall survival rates of ACC patients according to mRNA expression of c-Kit (A and D), SCF (B and E), or EGFR (C and F). The ACC cohort was divided into groups according to relative gene expression. Panels A to C compare patients with expression in the top half overall (blue line) and the lower half (green line) for each gene. Panels D to F compare expression in the higher quartile (blue line) and lower three quartiles (green line). Circles identify censored data. Statistical comparisons between data sets were made with Wilcoxon tests and each P value is displayed, with P < .05 considered significant.