381 (R = 61.73%). In contrast, SCF and c-Kit expression correlated poorly in the absence of PNI ( Figure 4C; R-squared values 0.0099 [R = 9.94%]). To determine the biologic and prognostic significance of c-Kit, SCF, and EGFR mRNA expression, we performed overall survival analysis

by generating Kaplan-Meier plots with Wilcoxon testing (Figure 5, A–D). We divided our ACC cohort into 2 groups according to gene expression scores (group 1: above the median; group 2: below it; Figure 5, A, C, and E) and also created groups whose expression values were in the highest or lowest quartiles ( Figure 5, B, D and F). c-Kit expression correlated with survival AZD4547 clinical trial ( Figure 5, A and D). Specifically, the subset with the highest c-Kit gene expression (top quartile), which did not overlap with the gene expression in normal tissue ( Figure 3A), EPZ015666 molecular weight had the poorest survival (P = .008). To determine whether our sample size in the analysis provided significance to this result, we performed a statistical power analysis as described in Methods [19]. The total number of 27 cases corresponded to a power of 0.87, providing confidence to this result, where a power of ≥ 0.80 (equivalent

to ≥ 22 total cases) is sufficient to detect a large difference between two groups. In contrast, we did not find a significant correlation between survival and expression of SCF and EGFR (Figure 5, B, C, E, and F). c-Kit is overexpressed and phosphorylated in sporadic ACCs without CYTH4 gene mutations [5]. The presence of SCF mRNA in tumor and normal salivary tissue has been reported as a potential mechanism for c-Kit activation in ACC [9]. However, it is not clear how SCF is expressed or to what extent it contributes to c-Kit activation. The goal of this study was to characterize the pattern of SCF protein expression in ACC tumor cells, and/or in the tumor environment, and to examine the clinical and

biologic significance of c-Kit activation in ACC patients. c-Kit is an oncogene [6]. Gain-of-function mutations in it occur in a range of human cancers and are advantageous for tumor growth, survival, and disease progression. For example, c-Kit mutations are often found in mast cell leukemia and gastrointestinal stromal tumors (GIST; 5, 6). However, gene mutations were not a cause of c-Kit activation in our cohort of 27 ACCs studied here. Our results confirmed studies from other laboratories [7] and [11]. We investigated whether ACC cells expressed c-Kit’s ligand, SCF. SCF was present not only in the tumor cells, which could mediate autocrine signaling, but also in other types of cells adjacent to the salivary glands. These cells might facilitate paracrine signaling [20]. In particular, SCF expression was highest in nerve cells in the tumor microenvironment. Peripheral nerves appear to release SCF into the neural space, where it could act as a chemo-attractant and growth factor critical for ACC.