Akt Signaling Pathway Is Activated in the Minor Salivary Glands of Patients with Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is an autoimmune exocrinopathy of mainly the salivary and lacrimal glands associated with high prevalence of lymphoma. Akt is a phosphoinositide-dependent serine/threonine kinase, controlling numerous pathological processes, including oncogenesis and autoimmunity. Herein, we sought to examine its implication in pSS pathogenesis and related lymphomagenesis. The expression of the entire and activated forms of Akt (partially and fully activated: phosphorylated at threonine-308 (T308) and serine-473 (S473), respectively), and two of its substrates, the proline-rich Akt-substrate of 40 kDa (PRAS40) and FoxO1 transcription factor has been immunohistochemically examined in minor salivary glands (MSG) of pSS patients (n = 29; including 9 with pSS-associated lymphoma) and sicca-complaining controls (sicca-controls; n = 10). The entire and phosphorylated Akt, PRAS40, and FoxO1 molecules were strongly, uniformly expressed in the MSG epithelia and infiltrating mononuclear cells of pSS patients, but not sicca-controls. Morphometric analysis revealed that the staining intensity of the fully activated phospho-Akt-S473 in pSS patients (with or without lymphoma) was significantly higher than sicca-controls. Akt pathway activation was independent from the extent or proximity of infiltrates, as well as other disease features, including lymphoma. Our findings support that the Akt pathway is specifically activated in MSGs of pSS patients, revealing novel therapeutic targets.

Matrix metalloproteinase-7, -8, -9, -15, and -25 in minor salivary gland adenoid cystic carcinoma.

Knowledge on the role of matrix metalloproteinases (MMPs) in adenoid cystic carcinoma (ACC) is limited. MMPs are capable of degrading almost all extracellular and pericellular components to promote invasion and metastasis. This study aimed to evaluate the immunohistochemical expression of MMP-7, -8, -9, -15, and -25 in ACC and to relate the results with clinicopathological factors and survival. The study included 68 patients with minor salivary gland ACC treated at the Helsinki University Hospital (Helsinki, Finland) in 1974-2012. Samples from 52 patients were available, consisting of 44 primary tumours and eight recurrent tumours. We scored immunostaining of MMP-7, -8, -9, -15, and -25 and analysed the immunoscore against clinical and pathological parameters using statistical correlation test. MMP-9 immunoexpression in pseudocysts of ACC and in peritumoural inflammatory cells associated with better survival and fewer treatment failures. High tumoural MMP-7 and -25 associated with better survival. High tumoural MMP-15 associated with poorer survival and high tumoural MMP-9 with advanced stage and regional recurrences. Tumour cells did not show MMP-8 immunopositivity. These results suggest that MMP-9 may contribute to ACC carcinogenesis in different roles. MMP-7, -8, and -9 can stimulate signalling pathways that may promote tissue modulation and metastatic potential. MMP-15 and -25 may reflect prognosis.

Intraductal Papillary Mucinous Neoplasms of Minor Salivary Glands With AKT1 p.Glu17Lys Mutation.

The spectrum of low-grade intraductal papillary proliferations of the salivary glands is heterogenous, and reproducible morphologic diagnostic criteria have not yet been established. Recognized types are sialadenoma papilliferum, inverted ductal papilloma, and intraductal papilloma, but some lesions have been possibly included in the morphologic spectrum of cystadenoma or low-grade intraductal carcinomas. We herein present detailed morphologic, immunophenotypic, and genotypic features of 3 minor salivary gland neoplasms affecting 2 men (aged 65 and 71 y) and 1 woman (aged 78 y). They ranged in size from 1 to 2.5 cm. All tumors showed atypical papillary intraductal growth that presented either as uninodular/unicystic lesions (intraductal papilloma-like; n=2) or as a discontinuous growth along the ductal system in a manner similar to pancreatic intraductal papillary mucinous neoplasm (n=1). Variable cytologic and architectural atypia was observed, ranging from bland intraductal papilloma-like features, to areas mimicking atypical ductal hyperplasia and low-grade ductal carcinoma in situ of the breast. Amplicon-based massive parallel sequencing revealed an identical AKT1 p.Glu17Lys mutation in all 3 cases, but absence of concurring mutations in other genes of the RAS or PI3K pathway. This small series represents the first genetic study on salivary intraductal papillary neoplasms. Our cases showed significant variation in the degree of cytologic and architectural atypia, which overlaps with intraductal papillomas at the one end and with low-grade intraductal carcinoma at the other end of the spectrum, suggesting a disease continuum. As the full biological and morphologic characteristics of these ductal papillary lesions remain to be defined, the noncommitted term "intraductal papillary neoplasms" might be more appropriate. Our novel genetic findings mirror similar activating mutations of AKT1 and other PI3K pathway members in intraductal papillary lesions of the breast and anogenital glands.

Abundant expression of mTOR kinase in salivary gland tumors - potentials as therapy target?

BACKGROUND: Salivary gland tumors constitute 3-6% of all head and neck neoplasms in adults. Because of limited advances made in the treatment of metastatic disease, the more important is the role of new therapeutic strategies, including molecular therapy. The mammalian target of rapamycin (mTOR) has recently been established as a therapeutic target for several drugs. MATERIAL: Evaluation of phospho-mTOR as a possible therapy target by patients with salivary gland tumors. Immunohistochemical semi-quantitative analyses of the expression of phospho-mTOR(Ser2448) were processed on a tissue microarray containing samples from more than 900 patients. For statistical analysis, contingency table and chi-squared test (likelihood) were used. RESULTS: We observed at least weak phospho-mTOR expression in 25.6-41.2% of all 4 histological adenoma and in 36.8-61.6% of all 11 histological carcinoma subtypes analyzed. No association was seen between phospho-mTOR expression and tumor grade in mucoepidermoid carcinomas. CONCLUSION: In conjunction with literature data providing the evidence for a functional role of mTOR in salivary gland tumors, we conclude that treatment with mTOR-antagonists might potentially also be efficient in wide variety of salivary gland carcinomas.

Expression of matrix metalloproteinases MMP-2, MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 in the epithelium and stroma of salivary gland pleomorphic adenomas.

AIMS: The balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) is involved in the morphogenesis of normal salivary gland as well as in the mechanisms of tumour invasion and metastasis. The role of MMPs and TIMPs in pleomorphic adenoma has not been elucidated sufficiently. Our aim was to analyse the mRNA and protein expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the epithelium and stroma of pleomorphic adenoma and to evaluate their roles. METHODS AND RESULTS: In each sample from six patients, cells from the epithelium and stroma were obtained by laser microdissection. The mRNA expression of MMPs and TIMPs was determined by real-time quantitative reverse transcriptase-polymerase chain reaction and protein expression was confirmed by immunohistochemistry. Results showed that mRNA expression of MMPs and TIMPs was significantly higher in stroma than in epithelium in most patients. MMPs and TIMPs were immunoreactive mainly in epithelium rather than in stroma. CONCLUSIONS: Our results provide preliminary evidence that stromal myoepithelium may be the primary source of MMPs and that the stroma has the potential to play a more important role than ductal epithelium in biological behaviour of pleomorphic adenomas. These findings seem worthy of further investigation.

Expression of two drug-metabolizing cytochrome P450-enzymes in human salivary glands.

OBJECTIVE: The oral cavity is constantly lubricated by saliva and even small amounts of xenobiotics and / or their metabolites in the saliva may affect the oral mucosa. Our aim was therefore to clarify if xenobiotic metabolizing enzymes CYP1A2 and CYP3A4 are expressed in salivary glands. METHODS: Formalin-fixed paraffin-embedded specimens from parotid (10), submandibular (7) and labial (10) salivary glands were examined immunohistochemically and by in situ hybridization for expression of CYP1A2 and CYP3A4 protein and mRNA. RESULTS: CYP1A2 and CYP3A4 protein and mRNA were detected in ductal and seromucous / serous acinar cells in all gland types although to a varying degree and intensity. Mucous acinar cells were positive to a lesser extent. CONCLUSION: The results indicate a xenobiotic metabolizing capability of salivary glands. This may have implications for development of oral mucosal disease as a result of mucosal exposure to metabolites originating from internal sources (blood) as well as from saliva.

Expression of metalloproteinase-2 (gelatinase A) in labial salivary glands of patients with Sjögren's syndrome with HTLV-I infection.

OBJECTIVE: To determine whether gelatinase A (MMP-2) plays a significant role in the pathogenesis of Sjögren's syndrome (SS) with or without HTLV-I infection. METHODS: We examined 24 patients with SS (14 HTLV-I-seropositive and 8 HTLV-I-seronegative). Labial salivary gland tissue samples were analysed immunohistochemically using anti-MMP-2 monoclonal antibodies. RESULTS: In normal salivary glands, MMP-2 expression was not detected. All biopsy samples of 8 SS patients with HTLV-I-associated myelopathy (HAM) and 3 of 6 HTLV-I-seropositive SS patients without manifestation of HAM stained positively for MMP-2. However, the other samples were negative for MMP-2. CONCLUSION: Our study showed the MMP-2 expression in labial salivary glands of HTLV-I seropositive SS patients, especially in all SS patients with HAM. The presence of MMP-2 in the salivary glands of these patients suggests that it may play a role in cellular infiltration and destruction in salivary glands of SS.

Secretion from minor salivary glands following ablation of the major salivary glands in rats.

Since minor salivary glands are tiny and dispersed, ductal cannulation cannot be used when studying their function. The present study was devised to develop a method of measuring minor salivary gland function by excision of the major glands. Female rats (230-280 g) were anaesthetized with sodium pentobarbital. Ablation of the submandibular, sublingual and parotid glands was performed through a sagittal neck incision. Sham-operated rats served as controls. Groups of sialadenectomized animals were investigated immediately and after 1 week, 2 weeks and 3 months. To study secretory function, the mouth was rinsed with 250 microl water in every 5 min and protein and amylase concentrations were measured. After an initial 50 min of basal secretion pilocarpine (1 mg/kg, i.p.) was given. Bilateral ablation of both submandibular, sublingual and parotid glands led to a moderate loss of body weight and a considerable increase in water intake. No other obvious abnormality was observed for periods up to 90 days following surgery. We deduce that the minor glands secrete approximately 14 % of protein and 1% of amylase in whole saliva Secretion is maintained even after 90 days following removal of the major glands. Surgical removal of the major salivary glands allows the secretory function of the minor glands in rats to be studied in vivo.

Immunolocalization of aromatase in human minor salivary glands of the lower lip with primary Sjögren's syndrome.

The enzyme aromatase is involved in the conversion of androgens to estrogens and in the modulation of various androgenic and estrogenic actions. Abnormalities of estrogen metabolism have been postulated to play roles in the development and/or pathophysiology of Sjögren's syndrome. In the present study, aromatase was immunolocalized in 75 cases of inflammatory disorders of human minor salivary glands of the lower lip. These included cases of primary Sjögren's syndrome (19 cases), of chronic sialadenitis (34 cases) and of mucous extravasation cysts (22 cases), in order to clarify the possible involvement of in situ estrogen production in primary Sjögren's syndrome. Aromatase immunoreactivity was detected in myoepithelial cells of acini and in interstitial cells adjacent to acini and ducts in 13/19 (68%) cases of primary Sjögren's syndrome. In contrast, aromatase expression was detected in only six of 34 (18%) cases of chronic sialadenitis and in seven of 22 (32%) cases of mucous extravasation cyst. These results suggest that increased aromatase expression in minor salivary glands with primary Sjögren's syndrome in premenopausal women may be involved in the biological features of primary Sjögren's syndrome through the production of estrogens in situ and possibly through the aggravation of the inflammatory reaction.

Expression of the histo-blood group ABO gene defined glycosyltransferases in epithelial tissues.

The histo-blood group ABO carbohydrate antigens are differentially expressed in epithelia in close correlation with cellular differentiation. In order to gain insight into the biosynthetic regulation of these carbohydrate antigens, we correlated the expression of A carbohydrate antigens with that of the A gene defined glycosyl-transferase by immunohistology of human oral epithelia using monoclonal antibodies. In glandular epithelium the A transferase was found in mucous cells similar to that of the A carbohydrate antigens. In stratified non-keratinized squamous epithelium the A transferase was expressed only in spinous cell layers, which is in accordance with the appearance of the A carbohydrate antigens in these more mature cell layers. This simultaneous acquisition of the primary and secondary gene product of a glycosyltransferase gene, provides evidence that the well-defined sequential expression of histo-blood group carbohydrate antigens in stratified squamous epithelium may be directly regulated at the transcriptional level of the glycosyltransferase. Future studies will address the mechanism behind loss of A antigens in premalignant lesions and carcinomas.

Immunohistochemical characterization of functional markers in human minor salivary gland tumors.

The distribution of carcinoembryonic antigen (CEA), secretory component (SC), immunoglobulin A (IgA), immunoglobulin M (IgM), J-chain, and lysozyme in tumors of minor salivary glands was investigated using an immunoperoxidase method. Although CEA was demonstrated in both benign and malignant tumors, its distribution was relatively more common and with increased staining intensity in malignant tissues. In pleomorphic adenomas, the distribution of SC was similar to that of IgA and J-chain, suggesting the presence of secretory IgA in the epithelial cells. However, some neoplastic epithelial cells contained SC but not IgA and J-chain. No IgM was detected in such cells. Lysozyme could be demonstrated only in pleomorphic adenomas. Mucoepidermoid tumors and adenoidcystic carcinomas were negative for lysozyme. These findings suggest that some neoplastic ductal epithelial cells of pleomorphic adenomas retain functional characteristics of normal epithelial cells.

Immunohistochemical distribution of immunoglobulins, lactoferrin, and lysozyme in human minor salivary glands.

The immunofluorescence technique was used to examine the distribution of immunoglobulin A and its subclasses, secretory component (SC), J chain, lactoferrin and lysozyme in labial and lingual (von Ebner's) glands. IgA-containing plasma cells were found in the connective tissue around intercalated or intralobular ducts and a few were noted around acini of both glands. IgA was detected in the apical cytoplasm of intercalated and intralobular duct cells and in acini of von Ebner's glands and in demilunes of labial glands. Most IgA-containing cells also stained for J chain. The ratio of IgA1:IgA2-containing cells was approximately equal in von Ebner's and labial glands. Cytoplasmic and surface membrane-related staining for SC was detected in epithelial cells of the intercalated and intralobular ducts in both glands, in the serous acini of von Ebner's gland, and in the demilunes of labial glands. Lactoferrin was found in serous acini, demilunes, intercalated and intralobular ducts. Lysozyme was found in acinar and intercalated ducts, but was rarely seen in intralobular ducts. These results disclose the presence of cells (plasma cells and epithelial cells) and their products (IgA and secretory component) that indicate the local production of secretory IgA in minor salivary glands.

[Mucoepidermoid tumors of minor salivary glands. Clinical and pathologic correlations. Histoenzymologic and ultrastructural studies (author's transl)]. / Les tumeurs mucoépidermoïdes des glandes salivaires accessoires. Dénombrement. Etude clinico-pathologique, histoenzymologique et ultrastructurale.

In a series of 331 minor salivary gland tumors (malignant in about 55,3% of cases), mucoepidermoid tumors, after cystic adenoïd carcinomas, are the most frequent malignant tumors (21,5% of cases). They are much more common in women than in men. The average age of patients at presentation (52,2 years) is higher than that of pleomorphic adenomas. They occur more frequently in buccal floor, tongue and gums. By a half-quantitative study of 71 mucoepidermoid tumors, these neoplasms are ranged in 3 main groups : differentiated epidermoid or glandular tumors, intermediate cell tumors with predominant oncocytic, clear glycogenic or basophilic cells and rare adenosquamous carcinomas. These 3 groups are well demonstrated by histoenzymological investigations, which show high level of oxydative enzymes activity in oncocytic cells and high level of ATPase and alkaline phosphatase activities around basophilic sheets. Besides, an ultrastructural study shows, in addition to well differentiated glandular or epidermoid cells, 3 forms of intermediate cells : young basophilic ribosome-rich cells and more differentiated oncocytic or glycogenic cells. The various structural features of mucoepidermoid tumors are positively correlated with clinical course and behaviour, after long term follow-up studies. Differentiated forms and intermediate clear or oxyphilic cell tumors are of low grade malignancy. Intermediate basophilic cell tumors grow rapidly or metastasize and a lethal course is often noted in these cases.