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Salivary gland cancers comprise a heterogeneous group of neoplasms whose biological and clinical characteristics differ considerably from those of mucosal squamous cell carcinomas of the head and neck. One of the most common subtypes, adenoid cystic carcinoma (ACC), is notable for its myoepithelial differentiation, proclivity for hematogenous spread, and slow but progressive clinical course. The molecular alterations that underlie its development and progression are poorly characterized. Here we used oligonucleotide microarray analysis to survey the expression of 8920 different human genes in 15 ACCs, one ACC cell line, and five normal major salivary glands. We observed expression of genes indicative of myoepithelial differentiation, as expected, including those whose protein products are components of basement membranes and extracellular matrix. Other genes that were highly ranked for their expression in ACC were those encoding the transcription factors SOX4 and AP-2␥, the latter of which also was overexpressed in ACC relative to 175 other carcinomas from 10 anatomical sites that we had previously profiled. Additional genes, which were highly expressed in ACC compared to the other carcinomas, included casein kinase 1, epsilon and frizzled-7, both members of the Wnt/␤-catenin signaling pathway. Our study documents for the first time the diverse spectrum of genes overexpressed in ACC and highlights gene products and pathways that in the future might be exploited as therapeutic targets for this cancer, which up until Unlike mucosal squamous cell carcinomas of the head and neck, carcinomas of the salivary glands consist of a diverse histopathological spectrum of neoplasms. Adenoid cystic carcinoma (ACC) is one of the most common subtypes of salivary gland cancer, and in several series is the most frequent malignant tumor of the submandibular and minor salivary glands. 1,2 Histopathologically, ACC often forms both true lumens as well as pseudolumens in varying proportions. It characteristically shows myoepithelial differentiation and produces in large amounts specific proteins that comprise basement membranes and extracellular matrix. 3 The neoplasm has a proclivity for invading nerves, but it infrequently spreads via the lymphatic system. ACC has a protracted clinical course with local recurrences, hematogenous metastases, and poor response to classical chemotherapeutic approaches. After surgery and radiation therapy for patients with ACC, the disease-specific survival at 15 years is ϳ40%. 4 The constellation of genes that are critical for the development and progression of ACC are not known. Furthermore, genes whose expression in ACC is altered relative to those in normal salivary glands and other carcinomas have not yet been compiled. In this study, we used large-scale microarray analysis in an effort to characterize the expression profiles in this salivary gland malignancy, and to specifically identify those genes differentially expressed between ACC and normal salivary gland epithelium. Importantly, we have als...

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We surveyed the expression of 557 cancer-related genes in 15 cases of well-differentiated OSCC by cDNA microarray analysis. To identify potential biomarkers for lymph node metastasis, all microarray data were compared by the MannWhitney test and the significance analysis of microarrays between OSCCs with and those without lymph node metastasis. The tissues of OSCCs with lymph node metastasis exhibited increased expression levels of MMP-1, MMP-3, uPA, integrin-␣3, paxillin, tenascin C and IL-6 transcripts. All of these genes were included in common clusters on the Cluster/TreeView analysis, implying that functional gene groups of proteolytic enzymes and integrin-related molecules are involved in cervical lymph node metastasis. The results of RTQ-PCR for differentially expressed genes were in accord with those of cDNA microarray analyses, suggesting that the data obtained by microarray gene expression analyses were valid. Consistent with cooperative expression patterns, immunohistochemical analyses demonstrated that products of MMP-1, MMP-3 and uPA were colocalized to components of the neoplastic stroma, particularly mononuclear inflammatory cells with well-developed eosinophilic cytoplasm. Our results suggest that expression levels of molecules involved in tissue remodeling and cell-ECM adhesion, especially MMP-1 and integrin-␣3, can provide an accurate biomarker system for predicting the risk of cervical lymph node metastasis in OSCC.

To make reproducible diagnoses for oral carcinoma in situ (CIS), combined immunohistochemistry directed at the positioning of squamous cell proliferation (Ki-67) and differentiation (keratin (K) 13 and K19) was used, both of which support histological evaluations by providing biological evidence. Normal/hyperplastic epithelia was defined by K19+ cells only in the first basal layer, K13+ cells in the third basal and upper layers, and sporadic Ki-67+ cells in the second basal layer. These profiles indicated that a proliferating center of the oral epithelium is located in the parabasal cell layer, and K19 and K13 can be regarded as markers for basal and prickle cells, respectively. Epithelial dysplasia was characterized by irregular stratification of Ki-67+ cells and the absence of K19/K13 in proliferating cells. Irregular emerging of K19+ and K13+ cells in proliferating foci with unique stratification of atypical Ki-67+ cells indicated CIS. When the definition was applied, surgical margins in 172 recurrent cases were shown to contain CIS (39.4%) and squamous cell carcinoma (55.8%), indicating that the new diagnostic criteria for CIS reflected clinical behaviors of the cases. The results indicate that oral CIS contain more histological variations, especially those with definite keratinization, than what had been previously defined.

Tissue samples from 30 patients with adenoid cystic carcinoma and 20 with adenocarcinoma of salivary gland origin were studied by immunohistochemical staining with specific antibodies to the four macromolecules that are present in normal basement membranes: type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. In the adenoid cystic carcinoma samples, the four proteins were localized in different types of extracellular matrices in the tumor, namely pseudocystic spaces, hyaline stroma, and around tumor cell nests. The staining intensity was enhanced by pretreatment with hyaluronidase. The tumor cells of adenoid cystic carcinoma showed a tendency to proliferate with individual cells in contact with the basement membrane and to infiltrate through basement membrane‐rich tissues, such as peripheral nerves, blood vessels, and skeletal muscles. In contrast, only circumferential staining of tumor cell nests was obtained in adenocarcinoma samples. The results suggest that adenoid cystic carcinoma is a tumor with affinity for basement membranes, and this basic feature is reflected in its histology and presumably in its biologic behavior. Immunostaining with antibodies to basement membrane proteins appears to be useful for differential diagnosis of some types of these two carcinomas.

The results indicate that the ECM remodeling steps in OSF are similar to each phase of usual granulation tissue formation. Restricted mouth opening may be a result of loss of variety of ECM molecules including elastin into the homogeneity of collagen type I replacing muscle fibers.

The biosynthesis of basement membrane molecules and fibronectin was studied in vitro in the two different human cell systems (ACC2 and ACC3) established from adenoid cystic carcinomas (ACC) of the salivary gland using immunofluorescence and confocal microscopy. When cells were attached and spread on dishes, fine granular immunofluorescence for type IV collagen, laminin, heparan sulphate proteoglycan, entactin, and fibronectin first appeared diffusely in the cytoplasm, and then changed in aggregation of coarse granules in the perinuclear area. With formation of colonies, these signals were present in the extracellular space, initially in the basal aspect of attached cells and consequently in the lateral intercellular space. After the cells formed a confluent monolayer, extracellular signals started to decrease in inverse proportion to the reappearance of intracellular ones. The results indicate that the parenchymal cells of ACC synthesize these five extracellular matrix molecules, secrete them into the extracellular milieu and remodel the extracellular deposits. It is suggested that the characteristic stromal architecture of ACC, represented by stromal pseudocysts, results from their own secretion of the basement membrane molecules and fibronectin.