Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors.

Salivary gland cancers are rare but aggressive tumors that have poor prognosis and lack effective cure. Of those, parotid tumors constitute the majority. Functioning as metabolic machinery contributing to cellular redox balance, peroxisomes have emerged as crucial players in tumorigenesis. Studies on murine and human cells have examined the role of peroxisomes in carcinogenesis with conflicting results. These studies either examined the consequences of altered peroxisomal proliferators or compared their expression in healthy and neoplastic tissues. None, however, examined such differences exclusively in human parotid tissue or extended comparison to peroxisomal proteins and their associated gene expressions. Therefore, we examined differences in peroxisomal dynamics in parotid tumors of different morphologies. Using immunofluorescence and quantitative PCR, we compared the expression levels of key peroxisomal enzymes and proliferators in healthy and neoplastic parotid tissue samples. Three parotid tumor subtypes were examined: pleomorphic adenoma, mucoepidermoid carcinoma and acinic cell carcinoma. We observed higher expression of peroxisomal matrix proteins in neoplastic samples with exceptional down regulation of certain enzymes; however, the degree of expression varied between tumor subtypes. Our findings confirm previous experimental results on other organ tissues and suggest peroxisomes as possible therapeutic targets or markers in all or certain subtypes of parotid neoplasms.

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.

Sclerosing Polycystic Adenoma of Salivary Glands: A Novel Neoplasm Characterized by PI3K-AKT Pathway Alterations-New Insights Into a Challenging Entity.

Sclerosing polycystic adenoma (SPA) is a rare salivary gland neoplasm originally thought to represent a non-neoplastic lesion. Recently we have encountered an index case of apocrine intraductal carcinoma of parotid gland of 62-year-old man with invasive salivary duct carcinoma component arising from SPA, a combination of tumor entities that has never been published so far. Here, we further explore the nature of SPA by evaluating 36 cases that were identified from the authors' consultation files. The patients were 25 females and 11 males aged 11 to 79 years (mean, 47.8 y). All tumors originated from the parotid gland. Their size ranged from 11 to 70 mm (mean, 28 mm). Histologically, all cases revealed characteristic features of SPA, such as lobulated well-circumscribed growth, focal hyalinized sclerosis, presence of large acinar cells with abundant brightly eosinophilic intracytoplasmic granules, and ductal components with variable cytomorphologic characteristics, including foamy, vacuolated, apocrine, mucous, clear/ballooned, squamous, columnar and oncocyte-like cells. In all cases, there were foci of intraluminal solid and cribriform intercalated duct-like epithelial proliferations with variable dysplasia which were positive for S100 protein and SOX10, and fully enveloped by an intact layer of myoepithelial cells. In addition, 14/36 cases (39%) had focal intraductal cribriform and micropapillary apocrine-type dysplastic epithelial structures composed of cells positive for androgen receptors and negative for S100/SOX10. The intraductal proliferations of both types showed focal mild to severe dysplasia in 17 cases (17/36; 47%). Two cases showed overt malignant morphology ranging from high-grade intraductal carcinoma to invasive carcinoma with an apocrine ductal phenotype. Next generation sequencing using ArcherDX panel targeting RNA of 36 pan-cancer-related genes and/or a TruSight Oncology 170/500 Kit targeting a selection of DNA from 523 genes and RNA from 55 genes was performed. Tumor tissue was available for molecular analysis in 11 cases, and 9 (9/11; 82%) of them harbored genetic alterations in the PI3K pathway. Targeted sequencing revealed HRAS mutations c.37G>C, p.(Gly13Arg) (2 cases) and c.182A>G, p.(Gln61Arg) (2 cases), and PIK3CA mutations c.3140A>G, p.(His1047Arg) (3 cases), c.1633G>A, p.(Glu545Lys) (1 case), and c.1624G>A, p.(Glu542Lys) (1 case). Moreover, mutations in AKT1 c.49G>A, p.(Glu17Lys) and c.51dup, p.(Tyr18ValfsTer15); c.49_50delinsAG, p.(Glu17Arg) (as a double hit) were found (2 cases). In addition, germinal and somatic mutation of PTEN c.1003C>T, p.(Arg335Ter); c.445C>T, p.(Gln149Ter), respectively, were detected. Gene fusions were absent in all cases. These prevalent molecular alterations converging on one major cancer-related pathway support the notion that SPA is a true neoplasm with a significant potential to develop intraluminal epithelial proliferation with apocrine and/or intercalated duct-like phenotype. The name SPA more correctly reflects the true neoplastic nature of this enigmatic lesion.

Epidermal growth factor receptor and human epidermal growth receptor 2 expression in parotid mucoepidermoid carcinoma: possible implications for targeted therapy.

The expression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) was analyzed in immunohistochemical preparations from 46 primary parotid mucoepidermoid carcinomas (MEC). For the cases with lymph node metastases, the receptor expressions were investigated in parallel samples, primary tumour and metastasis, from each patient (n=11). The goal was to evaluate whether any of these receptors are suitable as a target for radionuclide-based imaging and therapy. The HercepTest scoring was used for the analysis of both HER2 and EGFR expression (0, 1+, 2+ or 3+). EGFR overexpression (2+/3+) was found in 67.4% (31/46) of the primary tumours. Out of the 11 cases with evaluated paired samples, EGFR overexpression was observed in 81.8% (9/11) of the primary tumours and 72.7% (8/11) of the corresponding lymph node metastases. There was only one patient who had EGFR overexpression in the primary tumours which changed to negative in the lymph node metastases but no changes occurred reciprocally. The HER2 overexpression was only found in 4.3% (2/46) of the primary mucoepidermoid carcinoma and none of the lymph node metastases (0/11). EGFR and HER2 stainings were mainly found in the cell membranes. It was concluded that the majority of parotid mucoepidermoid carcinomas express EGFR strongly in their cell membranes and that lymph node metastases generally express EGFR to approximately the same extent as in the primary tumours. The stability in the EGFR expression is encouraging in the effort to develop radionuclide-based EGFR imaging agents. It is also possible that EGFR targeting agents (e.g. Iressa, Tarceva, Erbitux or radiolabelled antibodies) can be applied for the therapy of mucoepidermoid carcinoma.

Expression of four histone lysine-methyltransferases in parotid gland tumors.

Methylation of histones is one of the important "epigenetic" mechanisms associated with the transcriptional silencing and/or activating of tumor suppressor genes. To assess whether epigenetic phenomena could be involved in salivary gland carcinogenesis, the expression levels of four histone lysine-methyltransferases (HMT) were investigated, in both pleomorphic adenoma and the adjacent normal tissue of the parotid glands. The expression levels of three HMTs, SETB1, Eu-HMTase and SET08, were higher in tumor tissues. On the contrary, DOTL1 presented a lower expression level in the tumor tissues than in the corresponding normal tissues. These data suggest that the HMTs may be involved in the differentiation of pleomorphic adenoma, probably through chromatin structural changes, and indicates that the study of the epigenetic mechanism which modulates the variation in the methylation profile of histones may be useful to obtain information concerning those genes involved in tumor transformation in human parotid glands.

Constitutive (HO-2) and inducible (HO-1) haem oxygenase in pleomorphic adenomas of the human parotid: an immunocytochemical study.

This study examines the expression HO-1 and HO-2 isozymes in human parotid pleomorphic adenomas. They are members of the heat shock protein family, and are thought to play a role in the regulation of tumoral blood flow. Immunocytochemistry using antibodies specific for HO-1 and HO-2 were undertaken in 12 pleomorphic adenoma specimens, all sections of which contained adjacent normal salivary tissue. Normal salivary gland acini and ducts displayed significantly stronger immunoreactivity for HO-2 compared to tumour cells (p < 0.001). Expression for HO-1 was minimal in both normal salivary gland acini and tumour cells with no difference (p = 1.000). However, positive staining for HO-1 was seen in normal salivary ducts and in pleomorphic adenomas showing ductal differentiation. In conclusion, this is the first study to examine the expression of HO-1 and HO-2 within normal salivary glands and pleomorphic adenomas. Our findings suggest that HO may be implicated in the pathogenesis of salivary pleomorphic adenomas.

Amylase expression in human parotid neoplasms: evidence by in situ hybridization for lack of transcription of the amylase gene.

Salivary alpha-amylase (EC 3.2.1.1) is the major protein component of human parotid gland secretion. We studied amylase gene structure and expression in tissue from a series of normal and neoplastic parotid glands by Southern blot analysis, in situ hybridization, and immunohistochemistry. Thirty-two tumors were examined. Southern blot analysis of DNA extracted from a Warthin tumor, an adenoid cystic carcinoma, and a mucoepidermoid carcinoma showed no evidence of structural rearrangement of amylase genes. Eleven parotid Warthin tumors were negative for amylase protein and mRNA by immunocytochemistry and in situ hybridization. One pleomorphic adenoma in the group of 10 examined showed focal staining for amylase protein, although amylase mRNA could not be demonstrated in the same population of cells by in situ hybridization in serial tissue sections. Five mucoepidermoid carcinomas and three acinar cell carcinomas were devoid of amylase protein and mRNA. Normal parotid tissue obtained from all patients studied revealed abundant acinar cell amylase mRNA and protein. In situ hybridization, in conjunction with immunocytochemistry, allows precise cellular localization of mRNA and protein, thereby establishing the site of production of specific transcripts. We conclude that the interruption in amylase gene expression in parotid gland neoplasms occurs at the transcriptional level.

Immunohistochemical demonstration of ribonuclease and amylase in normal and neoplastic parotid glands.

Antibodies specific for bovine ribonuclease A (antiRNase A) were raised in rabbits, and immunologic cross-reactivity between bovine RNase A and human salivary gland RNase was demonstrated. The antiRNase A served as the primary antibody in the peroxidase-antiperoxidase immunohistochemical technique. Paraffin blocks of five normal human parotids and 20 parotid tumors were examined. In normal parotid and in cases of cystadenoma lymphomatosum, immunoreactive RNase was localized in the ductal epithelium, evidence of the ductal cell origin of these benign tumors. RNase immunoreactivity was noted in the adenomatous structures and in cells isolated in the myxoid matrix of pleomorphic adenomas, which supports recent evidence of an epithelial origin of these tumors. Malignant acinar cells of acinic cell carcinoma were strongly positive for immunoreactive RNase, while acinar cells of normal parotid were uniformly negative. This expression of the gene for RNase A probably represents a loss of differentiation (i.e., control) of the neoplastic acinar cells. Further evidence for this hypothesis was obtained by treating these tumors with an antihuman salivary amylase antibody, which is localized in normal acinar cells. No immunoreactive amylase was observed. The results support the idea that immunoreactivity need not accompany enzyme activity, as the presence of immunoreactive RNase was noted in all neoplastic tissues examined. Immunohistochemical localization of two antigens in the same tissue demonstrates the varied biochemical changes associated with parotid neoplasia.

Malignant fibrous histiocytomas of salivary glands.

The light microscopic, immunohistological and ultrastructural findings in two cases of malignant fibrous histiocytoma arising in salivary glands are presented and the features of seven previously reported cases are reviewed. This neoplasm is extremely rare in this site and may pose problems in diagnosis. It has to be distinguished from other spindled cell tumours, in particular from epithelial tumours of predominantly spindled cell pattern; immunohistological markers for histiocytic cells may be of value. The histogenesis of this neoplasm is controversial but our electron microscopic findings support an origin from mesenchymal cells which differentiate along a broad fibrohistiocytic spectrum.

Over-expression of glutathione S-transferases, DT-diaphorase and an apparently tumour-specific cytosolic class-3 aldehyde dehydrogenase by Warthin tumours and mucoepidermoid carcinomas of the human parotid gland.

Cytosolic class-3 aldehyde dehydrogenase (ALDH-3) may help to protect organisms from certain environmental aldehydes by catalysing their detoxification. Consistent with this notion are the reports that relatively high levels of this enzyme are present in tissues, e.g. stomach mucosa and lung, that are so-called ports of entry for such agents. Further, it is found in human saliva. The present investigation revealed that small amounts of this enzyme are also present in human salivary glands; mean values for ALDH-3 activities (NADP-dependent enzyme-catalysed oxidation of benzaldehyde) in cytosolic fractions prepared from submandibular and parotid glands were 52 (range: 29-92) and 44 (range: 13-73) mIU/g tissue, respectively. Essentially identical or slightly lower levels of this enzyme activity were found in pleomorphic adenomas, an undifferentiated carcinoma, and an adenocystic carcinomas, of the parotid gland. On the other hand, Warthin tumours, and mucoepidermoid carcinomas of the parotid gland exhibited relatively elevated levels of ALDH-3 activity; mean values were 1200 (range: 780-1880) and 810 (range: 580-1200) mIU/g tissue, respectively. The ALDH-3 found in normal salivary glands was, as judged by physical, immunological and kinetic criteria, identical to human stomach mucosa ALDH-3 whereas the ALDH-3 present in Warthin tumours, and mucoepidermoid carcinomas, of the parotid gland appeared to be a subtle variant thereof. Qualitatively paralleling the relatively elevated ALDH-3 levels in mucoepidermoid carcinomas and Warthin tumours were relatively elevated levels of glutathione S-transferase (alpha and pi) and DT-diaphorase. As was the case with ALDH-3 levels, glutathione S-transferase (alpha and pi) and DT-diaphorase levels were not elevated in pleomorphic adenomas. Glutathione S-transferase mu was not detected in the two normal parotid gland samples, or in the single pleomorphic adenoma sample, tested. It was found in the single mucoepidermoid carcinoma sample, and in one of the two Warthin tumour samples tested. Cellular levels of ALDH-3, glutathione S-transferases and/or DT-diaphorase could be useful criteria when the decision to be made is whether a salivary gland tumour is a mucoepidermoid carcinoma. ALDH-3 and glutathione S-transferases are known to catalyse the detoxification of two agents that are used to treat salivary gland tumours, viz. cyclophosphamide and cisplatin, respectively. Thus, elevated levels of these enzymes in the mucoepidermoid carcinomas must account for, or at least contribute to, the relative ineffectiveness of these agents when used to treat this tumour.

Phosphatase enzymes. Cytochemical study of pleomorphic adenoma and normal human salivary glands.

Human parotid glands, submandibular glands, and pleomorphic adenomas were examined by electron microscopic histochemistry. All epithelial cells of the normal salivary glands showed plasma membrane adenosine triphosphatase (ATPase) and inosine diphosphatase (IDPase) activity. However, myoepithelial cells reacted most intensely. Pleomorphic adenomas showed epithelial cells within solid and ductal portions of the tumors that were variably reactive for both ATPase and IDPase. Histochemical examination of the epithelial cells in the myxoid portions of the tumors did not provide conclusive evidence as to the nature of their progenitor cells. Surface-associated phosphatases (alkaline phosphatase, ATPase, and IDPase) cannot be reliably used as histochemical markers of salivary gland myoepithelial cells. Therefore, morphological and phosphatase histochemical studies that intend to examine the role of myoepithelial cells in salivary gland neoplasms must be interpreted with care.

Collagenase activity in squamous cell carcinoma of the human parotid gland.

A collagenase obtained from a single squamous cell carcinoma of human parotid gland was purified to homogeneity by procedures including precipitation with ammonium sulfate, gel filtration, ion-exchange chromatography with DEAE-cellulose, and extraction from polyacrylamide gel after electrophoresis. The parotid tumor collagenase had a molecular weight of approximately 68,000 and appeared similar to other mammalian collagenases in many respects such as action on collagen and response to common collagenase inhibitors. Analysis of specimens of various human parotid gland tumors revealed that the levels of collagenase activity were higher in squamous cell carcinoma than in nonsquamous tumors. A high level of collagenase activity from squamous cell carcinoma suggests that collagenase may promote local connective tissue destruction associated with tumor invasion and lymph node metastasis.

An immunohistochemical analysis of anti-amylase antibody reactivity in acinic cell adenocarcinoma.

In many salivary acinic cell adenocarcinomas, well-differentiated serous acinar-type cells may be few and inconspicuous. In these cases it may be difficult to distinguish acinic cell adenocarcinoma from other types of salivary gland neoplasms such as cystadenocarcinoma. The usefulness of antisalivary amylase antibody immunohistochemical staining as a diagnostic aid was assessed on paraffin-embedded tissue sections from 27 typical acinic cell adenocarcinomas. Only 4 of 27 tumors showed reactivity in tumor cells. We conclude that anti-amylase antibody is of limited value in the recognition of acinic cell adenocarcinoma when light morphologic features are insufficient for diagnosis.

Lymphoepithelial cyst with crystalloid formation. Cytologic features of two cases.

BACKGROUND: The presence of amylase crystalloids (AC) in cystic lesions of the parotid gland is a rare occurrence and has been diagnosed to date as sialadenitis. We report the first two cases of parotid lymphoepithelial cyst (LC) containing this type of crystalloid. CASES: Case 1, a 56-year-old male, presented with a 3-cm parotid cyst. Fine needle aspiration (FNA) was performed on the mass. Smears showed numerous crystalloids identical to those described as crystallized amylase. Case 2, a 36-year-old female, had a 2-cm parotid mass. FNA smears exhibited the same features as did case 1. The two patients were treated with superficial parotidectomy, and an LC containing AC was diagnosed in both cases. CONCLUSION: When the above findings are present on FNA of parotid gland, the diagnosis of LC must be considered.

Characteristics of the epithelial component of parotid adenolymphoma.

Parotid adenolymphoma is composed of two histologic components, epithelial and lymphoid. Although some theories regarding the histogenesis of this tumor have long been disputed, there have been no definite conclusions. The purpose of this study was to clarify the origin of the epithelial components of this tumor using histochemical and immunopathological techniques, electron microscopy and a survey of HE-stained tumor sections. The results obtained indicated that the functions of the epithelial components were similar to those of the striated duct of the normal parotid gland, and morphological studies showed that the origin of the epithelial components may arise from parotid ductal inclusion in the lymphnodes in or around the parotid gland.

Expression of nitric oxide synthase in pleomorphic adenomas of the parotid.

The actions of nitric oxide (NO) in the pathology of solid tumours are complicated and many are poorly understood because NO has both inhibitory and tumour-promoting activities. In the current study we aimed to find out immunohistochemically whether the expression of both the inducible (iNOS) and endothelial (eNOS) forms of the enzyme nitric oxide synthase (NOS) were changed in pleomorphic adenomas of the parotid compared with normal salivary tissue. There was a significant difference in staining for iNOS between the tumour and normal salivary tissue, with tumour epithelial cells being stained in 29 cases of the 30 cases studied (P< 0.0001). The luminal cells of the salivary ducts also stained, but not the normal salivary tissue. Immunohistochemistry for the eNOS isoenzyme showed moderate staining of the tumour epithelium in only three specimens. There was also mild staining in the salivary duct cells of the normal glandular tissue and in endothelium of blood vessels in both tumour and normal glandular tissue in the same 29 cases.

α-amylase crystalloid granuloma of the parotid gland: case report and review of the literature.

We report a case of α-amylase crystalloid granuloma of the parotid gland in a 65-year-old Japanese woman. Histopathologically, the lesion comprised cystlike dilatation of the ducts and foreign body granulomas, with deposits of numerous crystalloid structures. The crystalloids were eosinophilic and varied in size and shape. Immunohistochemically, the crystalloids were positive for α-amylase. Immunoelectron microscopy showed the crystalloids to be cuboidal or rectangular in shape with irregularly shaped central spaces. We discuss this rare condition and review the literature on α-amylase crystalloids.