Gene expression profiling in sinonasal adenocarcinoma

Abstract

Background: Sinonasal adenocarcinomas are uncommon tumors which develop in the ethmoid sinus after exposure to wood dust. Although the etiology of these tumors is well defined, very little is known about their molecular basis and no diagnostic tool exists for their early detection in high-risk workers.

Methods: To identify genes involved in this disease, we performed gene expression profiling using cancer-dedicated microarrays, on nine matched samples of sinonasal adenocarcinomas and non-tumor sinusal tissue. Microarray results were validated by quantitative RT-PCR and immunohistochemistry on two additional sets of tumors.

Results: Among the genes with significant differential expression we selected LGALS4, ACS5, CLU, SRI and CCT5 for further exploration. The overexpression of LGALS4, ACS5, SRI, CCT5 and the downregulation of CLU were confirmed by quantitative RT-PCR. Immunohistochemistry was performed for LGALS4 (Galectin 4), ACS5 (Acyl-CoA synthetase) and CLU (Clusterin) proteins: LGALS4 was highly up-regulated, particularly in the most differentiated tumors, while CLU was lost in all tumors. The expression of ACS5, was more heterogeneous and no correlation was observed with the tumor type.

Conclusion: Within our microarray study in sinonasal adenocarcinoma we identified two proteins, LGALS4 and CLU, that were significantly differentially expressed in tumors compared to normal tissue. A further evaluation on a new set of tissues, including precancerous stages and low grade tumors, is necessary to evaluate the possibility of using them as diagnostic markers.

Figure 1

Heat map of the two-class comparison (A) and unsupervised (B) analysis. Expression levels are color coded with red, green, black and gray, corresponding to an increase, decrease or no change in gene expression, or missing data, respectively.

Figure 2

Relative expression levels of LGALS4, ACS5, CLU, in tumors versus matched normal sinusal tissue as determined by RT-qPCR. Fold change was calculated according to the equation described in the Materials and Methods with normalization against the average of three housekeeping genes, RPLPO, β2 microglobulin, and ubiquitin C. *tumor tissue versus average of all normal sinusal tissues (cf. RT-qPCR Results for details).

Figure 3

Representative cases of LGALS4, CLU and ACS5 expression in matched normal mucosa (×100), and tumor tissue (×25). A-B-C: Normal sinusal mucosa immunostaining. (A-C): Weak and focal cytoplasmic staining of serous cells in a few seromucinous glands with LGALS4 and CLU. (A):Weak staining of respiratory epithelium with LGALS4. (B): Strong and diffuse immunostaining of serous cells with ACS5. D-E-F: Tumor immunostaining. (D): Poorly-differentiated "solid-type" component showing no immunoreactivity for LGALS4 while the "colonic-type" component is positive in a mixed ITAC (patient 5). (E): Example of ACS5 expression in a "colonic-type" ITAC. (F): No immunoreactivity for CLU in tumor samples (×100) except in one non-ITAC (Insert * (×25), Patient 11).