Extra-cellular signal-regulated ERK-1/ERK-2 pathway activation in human salivary gland mucoepidermoid carcinoma: association to aggressive tumor behavior and tumor cell proliferation

Abstract

Information on oncogenetic events accompanying salivary gland mucoepidermoid carcinoma is so far limited. Activation of extracellular signal-regulated kinases ERK-1 and ERK-2 is strongly correlated to cancer. Using an antibody specific for phosphorylated (active) ERK-1/ERK-2, we examined human salivary gland mucoepidermoid carcinoma samples by immunohistochemistry. The comparison in paired tumor and normal tissue samples showed that phosphorylated ERK-1/ERK-2 immunoreactivity was higher in tumor cells as compared to surrounding normal salivary parenchyma. ERK-1/ERK-2 phosphorylation was observed in approximately 39% of mucoepidermoid carcinomas. Those tumors where the ERK-1/ERK-2 pathway was activated had a more aggressive tumor behavior as compared to the group where this pathway was inactive. The association of ERK-1/ERK-2 phosphorylation to a worse prognosis was independent of histological grade. ERK-1/ERK-2 phosphorylation was associated with increased Ki67 and cyclin A indexes, which indicated that ERK-1/ERK-2 pathway activation increased tumor cell proliferation. There was no relationship between ERK-1/ERK-2 phosphorylation and HER-2/neu or p16/INK4a protein expression. In conclusion, ERK-1/ERK-2 pathway is active in salivary gland mucoepidermoid carcinoma and this activation is associated to a more aggressive tumor behavior and a higher proliferative activity. These data suggest that deregulation of ERK-1/ERK-2 pathway contributes to mucoepidermoid carcinoma phenotype and, possibly, represents a target for new anticancer drugs.

Figure 1.

Representative examples of salivary gland mucoepidermoid carcinoma and surrounding tissue samples analyzed by immunohistochemistry for phosphorylated ERK-1/ERK-2, total ERK-1/ERK-2, P16, Ki67, and cyclin A. A: Immunoperoxydase visualization of phosphorylated ERK-1/ERK-2. a: The nodular tumor tissue (arrowheads) is strongly reactive, while the normal adjacent parenchyma (asterisk) is mainly unreactive. b: Same sample as in A. Reactive endothelial nuclei (arrowheads) and normal salivary parenchyma with few reactive ductal cells (arrow) and unreactive acini (asterisk). c: Low-grade mucoepidermoid carcinoma. The basal myoepithelial cells (arrowheads) are consistently reactive, while the mucous cells (arrows) are unreactive. d: High-grade mucoepidermoid carcinoma. Intermediate cells (asterisk) with nuclear anaplasia are diffusely and strongly reactive. Note a few unreactive mucous cells (arrowhead). e: Intermediate-grade mucoepidermoid carcinoma. Reactive clear cells. f: A majority of intermediate cells are reactive (asterisk). Some glandular nonspecific cells are also reactive (arrowheads). Original magnification, ×25 (a); ×100 (b); ×400 (c); ×200 (d); ×200 (e); ×200 (f). B: Total ERK-1/ERK-2, Ki67, cyclin A, and P16 expression analyzed with phosphatase alkaline staining (red). a: Total ERK-1/ERK-2 expression in tumor (arrowheads) and surrounding salivary gland tissue (asterisk). b: Ki67 expression in one intermediate-grade phospho-ERK-positive mucoepidermoid carcinoma (6.6 positive nuclei per 1000 nuclei). Three positive nuclei (arrows) are present among 77 total tumor nuclei in this field. c: Cyclin A expression in one low-grade phospho-ERK-positive mucoepidermoid carcinoma (5.9 positive nuclei per 1000 nuclei). Four positive nuclei (arrows) are present among 191 total tumor nuclei in this field. d: Diffuse immunostaining for P16 in a phospho-ERK-positive mucoepidermoid carcinoma. Original magnification, ×100 (a); ×400 (b); ×400 (c); ×100 (d).

Figure 2.

Kaplan-Meier curves showing progression-free survival according to histological grade (A) or phosphorylated ERK-1/ERK-2 immunoreactivity (B). P values were determined using the two-sided Cox-Mantel log-rank test. Events (open circles) account for local recurrence, metastasis, and cancer-related death.

Figure 3.

Box plots of Ki67 (A) and cyclin A (B) index values according to phosphorylated ERK-1/ERK-2 immunoreactivity. Lower and upper limits of boxes indicate the 25th and 75th percentiles; squares within boxes indicate the median values; the lines extending from the boxes indicate the range of non-outlying values. Outliers were not plotted, but included in the statistical analysis.