Table 1 summarizes our results. The vast majority of the tumors expressed SCF ( Figure 1B and Supplemental Figure 1B and F); it was largely found in the duct-type epithelial component ( Figure 1C) where c-Kit was predominantly elevated ( Figure 1B). We used antibody-based IHC to detect active forms of ERK1/2 on tumor sections (Figure 1E). The Ras-Raf-MEK1/2-ERK1/2 cascade is a major downstream effector-signaling pathway of RTKs, including c-Kit. Thus, SCF-induced Regorafenib research buy activation of c-Kit would accompany

active ERK1/2 expression in the inner duct-type epithelial component of the tumor cells where c-Kit was elevated. Table summarizes our results. In 17 of 27 ACCs, active ERK1/2 protein was substantially increased in more than 20% of tumor cells. Interestingly, other types of non-cancerous cells adjacent to tumors

within salivary glands were positive for SCF. They included stromal fibroblasts (Figure 2A and Supplemental Figure 2A and B), neutrophils ( Figure 2B and Supplemental Figure 2C and D), peripheral nerve ( Figure 2, C–E and Supplemental Figure 2E), skeletal muscle ( Figure 2F and Supplemental Figure 2F), vascular endothelial cells ( Figure 2G), and mucous acinar cells and intercalated ducts GDC-0449 price ( Figure 2H). Strong immunoreactivity to the SCF antibody was found in neutrophils and peripheral nerve ( Figure 2, B and D). In addition, Figure 2E shows that staining for SCF highlights a peripheral nerve with a tumor wrapping around the nerve bundle,

creating a targetoid pattern. We investigated whether mRNA expression of c-Kit and SCF was also elevated in ACC (Figure 3, A and B), and also included EGFR because it has been implicated in the development of ACC ( [17] and [18]; Figure 3C). mRNA was isolated from FFPE sections as described above, and quantitative PCR performed. Figure 3A shows that c-Kit mRNA expression was elevated others in ACC, with the relative expression increased by 1.88 (P < .05) over the average of normal samples. The top quartile of mRNA expression of c-Kit particularly distinguished ACCs from normal salivary tissues. In contrast, the expression levels of SCF and EGFR mRNA showed a broad range, which overlapped with those in normal tissue ( Figure 3, B and C) and showed no significant difference (P > .05) from ACC samples. Given that SCF-mediated c-Kit activity is important for local invasion and metastasis, we determined the strength of correlation between SCF and c-Kit mRNA expression in the presence (cases 1, 11, 15, 16, 21, 23, 25 and 26; Table) or absence of perineural invasion (PNI). We generated scatter plots with trend lines to show correlations (Figure 4, A–C). Trend line equations and R-squared values were calculated with Microsoft Excel and are displayed atop each chart.