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.

The EGF signaling pathway influences cell migration and the secretion of metalloproteinases by myoepithelial cells in pleomorphic adenoma.

During tumor development, benign neoplastic cells are influenced by the expression of cytokines, growth factors, and proteases present in the tumor microenvironment. Epidermal growth factor (EGF) is the most studied growth factor and is considered important for cell proliferation and migration. Metalloproteinases (MMPs) are also involved in tumor progression. The present study aimed to analyze the proliferation, viability and migration index of pleomorphic adenoma myoepithelial cells, in addition to the secretion of MMPs with EGF supplementation. Benign myoepithelial cells were cultured with two different EGF doses (5 and 10 ng/ml), and the influence of EGF on cell proliferation and viability, using trypan blue and MTT assays, respectively, after 24, 48, and 72 h, was evaluated. To analyze cellular morphology, hematoxylin-eosin staining and indirect immunofluorescence using the anti-vimentin antibody, was performed. In vitro migration assays were performed in Transwell chambers with an 8-µm pore covered with Matrigel and supplemented with 5 or 10 ng/ml of EGF, after 96 h. After 4 days of cell culture, ELISA was performed to determine the MMP-2 and MMP-13 levels. One-way analysis of variance (ANOVA) with post hoc Tukey test was applied, with a significance level of 0.05. The results revealed that EGF influences myoepithelial cell morphology, without alteration of proliferation and viability. The migration assay showed that EGF increased the mean index from 16 % in the control group to 40 and 76 % for 5 and 10 ng/ml of EGF, respectively. ELISA revealed that when the cells were supplemented with either of the EGF doses, an increase in MMP-2 levels was observed when compared with the control group (C). This study concludes that EGF aids in the production of MMP-2, which favors the dissolution of the basement membrane, contributing to cell migration and tumor progression, hence permitting contact between the myoepithelial cells and stroma.

Kallikrein-related peptidase 10 expression in salivary gland tissues and tumours.

OBJECTIVES: Kallikrein-related peptidase 10 (KLK10) has been implicated in the development of several types of cancer. The purpose of this study was to analyze the expression of KLK10 in 3 types of salivary gland tumour and normal salivary glands. MATERIALS AND METHODS: A standard immunoperoxidase staining technique was used to assess the immunoexpression profile of KLK10 in normal salivary glands and 3 types of salivary gland tumour: pleomorphic adenoma, adenoid cystic carcinoma and mucoepidermoid carcinoma. RESULTS: Pleomorphic adenomas showed significantly lower KLK10 levels than control tissues. Neither of the malignant tumours (adenoid cystic carcinoma and mucoepidermoid carcinoma) showed a significant alteration in the immunoreactive scores of KLK10 in comparison with the normal salivary gland tissues. KLK10 immunoreactive scores were comparable in adenoid cystic carcinoma and mucoepidermoid carcinoma. Pleomorphic adenoma had significantly lower levels of KLK10 than mucoepidermoid carcinoma. CONCLUSIONS: The finding of lower KLK10 levels in pleomorphic adenoma suggests aberrant expression in a tumour that develops primarily from myoepithelial cells. A kallikrein cascade may play a role in the development and/or outcome of some salivary gland tumours.

Expression of gelatinases (MMP-2, MMP-9) and cyclooxygenases (COX-1, COX-2) in some benign salivary gland tumors.

Salivary gland tumors, most of which are rare benign tumors, represent a histologically heterogenous group with the greatest diversity of morphological and cellular features. The aim of this study is to analyse the expression and possible interactions between gelatinases (MMP-2, MMP-9) and cyclooxygenases (COX-1, COX-2) in some benign salivary gland tumors. We investigated the expression of gelatinases and cyclooxigenases in control salivary gland, Pleomorphic adenoma and Warthin's tumor through immunohistochemistry and Reverse Transcription - Polymerase Chain Reaction (PCR). We identified the expression of both classes of enzyme in normal samples and in the two types of pathological samples without any quantitative differences. From the present data no significant differences emerge in the expression of these enzymes among the different pathologies examined. Nevertheless, due to the small number of samples included in this study, general statements regarding correlation between the degree of severity of the tumoral pathology and the quantitative expression of these potential tumoral markers can not be made.

Sustained response of carcinoma ex pleomorphic adenoma treated with trastuzumab and capecitabine.

BACKGROUND: Carcinoma ex pleomorphic adenoma is a rare histologic subtype of salivary gland cancer with an overall poor prognosis. Limited histopathologic analyses have shown that some such tumors exhibit significant HER2/neu immunoreactivity, suggesting a potential role for HER2-based therapy. We report here a case of a 58-year old man with metastatic carcinoma ex pleomorphic adenoma who achieved a sustained long term response to combination therapy with trastuzumab and capecitabine. CASE PRESENTATION: A 58 year old man presented with T1N2bM0 carcinoma ex pleomorphic adenoma and underwent surgery followed by adjuvant radiation therapy. Multiple metastases to bone were documented one year later. Since the original tumor was strongly HER2/neu positive by immunohistochemistry, the patient was treated with trastuzumab, capecitabine, and zoledronic acid. He experienced total resolution of symptoms and repeat FDG-PET scan after three cycles revealed interval disease resolution. Continued treatment has resulted in maintenance of disease control for over 2 years. CONCLUSION: This case illustrates the successful long term treatment of carcinoma ex pleomorphic adenoma with targeted therapy with trastuzumab in combination with chemotherapy. In the absence of definitive clinical trials which are unlikely to be performed due to the rarity of this tumor, case reports such as this one suggest potential utility for trastuzumab in combination with chemotherapy in the treatment of HER2/neu-overexpressing carcinoma ex pleomorphic adenoma.

Human kallikrein 14 (KLK14) expression in salivary gland tumors.

OBJECTIVE: To analyze the expression of human kallikrein 14 (KLK14) in salivary gland tumors. METHODS: A standard immunoperoxidase staining technique was used to assess the expression profile of KLK14 in normal salivary glands and tumors including pleomorphic adenoma (PA; n=17), adenoid cystic carcinoma (ACC; n=13) and mucoepidermoid carcinoma (MEC; n=9). Tumor stage, grade, patient age and gender, and site of occurrence were recorded. These clinical parameters were correlated with KLK14 levels in malignant tumors. The expression profiles for KLK3, 5, 6, 8 and 13 were also retrieved. RESULTS: Normal salivary glands, PA, ACC and MEC showed strong expression of KLK14 in ductal and non-ductal cells. Both PA and ACC showed higher KLK14 levels than normal glands and MEC tissues. There were no statistically significant associations between levels of KLK14 and clinical parameters. CONCLUSIONS: The differences in the levels of KLK14 suggest that KLKs may aid in the differential diagnosis of salivary gland tumors. The coexpression of KLKs suggests their possible involvement in an enzymatic pathway activated in salivary gland. KLK14 may be a promising new biomarker in salivary gland tumors.

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.

Isoenzymes of N-acetyl-beta-hexosaminidase in human pleomorphic adenoma and healthy salivary glands: a preliminary study.

BACKGROUND: Pleomorphic adenoma (PA) is a benign tumour of the salivary gland with a tendency to malignancy which creates many diagnostic problems. N-acetyl-beta-hexosaminidase (HEX) is a lysosomal exoglycosidase involved in degradation of oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans, known as a potential tumour marker. In the majority of tissues and body fluids, HEX exists as two major isoenzymes: HEX A and HEX B. The aim of our study was to evaluate HEX A and HEX B activity in healthy and PA human salivary glands using colorimetric and isoelectrofocusing methods. METHODS: PA (n=8) and macroscopically unchanged salivary glands (n=8), served as controls, were used for the study. After preliminary preparation, isoenzymes of HEX were determined by colorimetric and isoelectrofocusing methods. RESULTS: Total activity of HEX, as well as HEX A and HEX B, in PA specimens determined by a colorimetric method was significantly higher compared with normal human salivary gland specimens. After isoelectrofocusing, in normal human salivary and PA glands, two sets of HEX isoforms were found corresponding to HEX A and HEX B. There was no significant difference in the amount of HEX A and HEX B isoforms. In PA tissue, activities of HEX isoforms in the pI ranges 1, 3b, 6 and 8 were significantly lower, and in ranges 5 and 8 significantly higher than in normal tissue. The observed significant shifts were localised mostly in HEX B activity area. CONCLUSIONS: The present data indicate that HEX activity and activity of its isoenzymes in tumour specimens is significantly and consistently elevated, and thus suggest the need for further studies on the degradation of glycoconjugates, both in healthy salivary glands and PA. It appears that HEX may be considered as a new tumour marker in these salivary gland diseases.

Overexpression of cyclooxygenase-2 correlates with cytoplasmic HuR expression in salivary mucoepidermoid carcinoma but not in pleomorphic adenoma.

BACKGROUND: The overexpression of cyclooxygenase (COX)-2 in several human carcinomas suggests that COX-2 is related to carcinogenesis. Although COX-2 expression has been shown to be up-regulated in carcinomas of the salivary gland, its mechanisms are not completely understood. HuR is an mRNA-binding protein that controls the stability of certain transcripts including COX-2. METHODS: The expression of COX-2 and HuR was determined by immunohistochemistry in 28 cases of salivary pleomorphic adenoma and 18 cases of salivary mucoepidermoid carcinoma. RESULTS: 28.6% and 72.2% of the pleomorphic adenomas and mucoepidermoid carcinomas showed high COX-2 expression respectively. 35.7% of pleomorphic adenomas and 72.2% of mucoepidermoid carcinomas were tested positive for HuR in the cytoplasm of tumor cells. There was a correlation between a high COX-2 immunoreactivity and cytoplasmic HuR expression in mucoepidermoid carcinomas but not in pleomorphic adenomas. CONCLUSION: This study suggests that cytoplasmic HuR is correlated with COX-2 expression in salivary mucoepidermoid carcinomas. In addition, the immunoreactivity of COX-2 and cytoplasmic HuR might be used to evaluate the nature of a borderline malignancy in the salivary glands.

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.

Thallium-201 chloride (Tl-201) accumulation and Na+/K+-ATPase expression in tumours of the head and neck.

OBJECTIVES: The purpose of this report was to evaluate the relationship between the tumour retention index of thallium-201 chloride (Tl-201) scintigraphy and the Na+/K+-ATPase expression in tumours of the head and neck. METHODS: Tl-201 scintigraphy was performed in 146 patients (129 with malignant tumours, ten with benign tumours and seven with inflammation). The tumour retention index was obtained from the early and delayed dynamic Tl-201 scans. The Na+/K+-ATPase expression was evaluated immunohistochemically in 61 of 129 patients with malignant tumour. Furthermore, another 22 patients with benign tumour were evaluated immunohistochemically as a benign control. Comparison of the correlations between the grade of histopathological differentiation of tumour, the tumour retention index of Tl-201 scintigraphy and the Na+/K+-ATPase expression was performed. RESULTS: The grade of histopathological differentiation of tumour, the tumour retention index of Tl-201 scintigraphy and the expression of Na+/K+-ATPase showed a good correlation indicating that Na+/K+-ATPase plays an important role in transportation for Tl-201 to go through the tumour cell membrane. CONCLUSIONS: Na+/K+-ATPase is one of the most important factors for Tl-201 accumulation in tumour.

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.

Maspin expression in normal and neoplastic salivary gland.

BACKGROUND: Maspin inhibits cell motility, invasion and metastasis. Loss or reduction in maspin expression has been associated with tumoral progression. METHODS: The presence of maspin was studied immunohistochemically in salivary gland tumours presenting cells with myoepithelial differentiation in their composition, and in normal salivary gland. RESULTS: Pleomorphic adenoma (PA) presented high expression of maspin, except in the spindle cells and occasional luminal cells. Epithelial-myoepithelial carcinoma and tubular adenoid cystic carcinoma (ACC) showed intense expression in all cells. Cribriform ACC evidenced only few positive cells of the luminal type, while solid subtype showed rare positive cells. Normal salivary gland tissue has shown low levels of maspin positivity. CONCLUSIONS: Maspin has small participation in normal salivary gland, is increased in PA, and decreases as the histological malignancy raises. Hence, in salivary gland, its expression is not exclusive of myoepithelial cells; thus, it should not be used as a marker for this cell. Nevertheless, we believe it is an important marker of biological behaviour in these tumours.

cGMP phosphodiesterase activity evaluation in human carcinoma of salivary glands.

The aim of this study was to evaluate differences of cGMP-PDE activity in salivary glands, between a control group and different benign tumour groups and, where present, with malign tumour groups. Endogen cGMP was evaluated too. The enzymatic reaction used the method of Spoto et al., with minor variations. The samples were organized in six groups: A (Adenolymphoma and Warthins tumour); B (Pleomorphic Adenoma); C (Basaloid Adenoma); D (Myoepitelioma). The control group was represented by healthy patients. In A and B groups, we have analyzed malign pathologies (Adenocarcinoma and Parotid Lymphoma) The benign tumours have more activity than controls, especially in Myoepitelioma (D) but with a decrement in the C group, which presents lower values of cGMP than the other three groups, where the concentration is similar. Between A and B groups, the activity values of malign tumours are similar, higher than controls and than the other benign pathologies, but not higher than in myoepitelioma. The cyclic concentration is similar for malign pathologies, with concentrations lower than controls, similar to Basaloid Adenoma (C).

Centrosomal abnormalities, multipolar mitoses, and chromosomal instability in head and neck tumours with dysfunctional telomeres.

Carcinomas of the head and neck typically exhibit complex chromosome aberrations but the underlying mutational mechanisms remain obscure. Evaluation of cell division dynamics in low-passage cell lines from three benign and five malignant head and neck tumours revealed a strong positive correlation between multipolarity of the mitotic spindle and the formation of bridges at anaphase in both benign and malignant tumours. Cells exhibiting a high rate of mitotic abnormalities also showed several chromosome termini lacking TTAGGG repeats and a high frequency of dicentric chromosomes. Multicolour karyotyping demonstrated a preferential involvement in structural rearrangements of chromosomes with deficient telomeres. The majority of malignant, mitotically unstable tumours expressed the reverse transcriptase subunit of telomerase. These data indicate that some of the genomic instability in head and neck tumours is initiated by telomere dysfunction, leading to the formation of dicentric chromosomes. These form chromosome bridges at mitosis that could prevent the normal anaphase-telophase transition. In turn, this may cause an accumulation of centrosomes and mitotic multipolarity. Telomerase expression does not confer total stability to the tumour genome but could be crucial for moderating the rate of chromosomal evolution.

[Detection of telomerase activity in salivary tumor and its significance].

OBJECTIVE: To investigate telomerase activity in human salivary cancer and corresponding adjacent tissues and to explore the possibility of telomerase as a tumor marker and its clinical significance. METHODS: Twenty-eight salivary cancers, 28 adjacent peritumoral tissues, 10 mixed tumors, 6 adenolymphomas, and 5 normal salivary tissues were examined for telomerase activity by the silver-staining TRAP assay based on PCR. RESULTS: Twenty-five of the 28 salivary cancers and 2 of the 28 adjacent peritumoral tissue specimens were positive for telomerase activity with a positive rate of 89.3% and 6.3%, respectively. Telomerase activity was negative in the 10 mixed tumors, 6 adenolymphomas, and 5 normal salivary tissues. There was no correlation between the clinical stage of salivary cancer and its expression of telomerase activity (P > 0.05). CONCLUSION: Positive telomerase activity occurs in the majority of salivary cancers examined. It can be used as a tumor marker in the diagnosis of salivary cancer. Detection of telomerase activity in the adjacent peritumoral tissues can be used as a monitoring marker after treatment.

Immunohistochemical study of the distribution of endogenous biotin and biotin-binding enzymes in ductal structures of salivary gland tumours.

To clarify the pathologic value of endogenous biotin in the salivary gland, we examined in a series of neoplasms of the salivary gland by immunohistochemical staining the distribution of endogenous biotin and of biotin-binding enzymes, namely, acetyl CoA carboxylase (AC), which is a cytosolic enzyme, and pyruvate carboxylase (PC), which is a mitochondrial enzyme. In pleomorphic adenoma, we found biotin and PC in ductal epithelial elements, while AC was found mainly in myoepithelial elements. Carcinoma ex pleomorphic adenoma, adenocarcinoma and mucoepidermoid carcinoma were frequently immunopositive for biotin, PC and AC, while adenoid cystic carcinoma was rarely immunopositive for biotin, PC or AC. These results indicate that endogenous biotin might be associated with the mitochondrial enzyme, which is present at high levels in ductal cells of the salivary gland. However, the neoplastic cells in adenoid cystic carcinoma seemed to have an unusual expression of biotin and related enzymes.

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.

Are myoepithelial cells responsible for the widespread expression of inducible nitric oxide synthase in pleomorphic adenoma? An immunohistochemical study.

Many of the actions of nitric oxide (NO) are still poorly understood. Recently, it has been shown that the inducible isoform of the enzyme nitric oxide synthase, iNOS, is expressed in both salivary ducts and pleomorphic adenoma. The current immunohistochemistry study determined whether or not this distribution correlated with smooth muscle actin (SMA) expression, thereby suggesting the expression by myoepithelial cells in both sites. Twenty cases of histologically confirmed pleomorphic adenoma, the sections of which contained adjacent normal salivary gland tissue, were stained for iNOS and smooth muscle actin (clone 1A4). The salivary ducts of all cases were stained intensely by both antibodies, with smooth muscle actin staining also being noted around acini in the normal gland parenchyma. Moderate or heavy staining for iNOS was found in all specimens of pleomorphic adenoma, with smooth muscle actin being distributed in a similar manner in 19 cases. Smooth muscle actin, but not iNOS, was also noted in blood vessels of both normal glands and tumours. The correlation between iNOS and SMA in pleomorphic adenoma was significant (P<0.001). The presence of iNOS in normal salivary ducts and pleomorphic adenoma is most likely due to expression by myoepithelial cells.

Immunohistochemical detection of DNA topoisomerase type II alpha and Ki-67 in adenoid cystic carcinoma and pleomorphic adenoma of the salivary gland.

Immunohistochemical detection of cell proliferation-associated antigens was investigated in 28 cases of adenoid cystic carcinoma (ACC) and 20 cases of pleomorphic adenoma (PA), using antibodies against DNA topoisomerase type II alpha (topo-II) (Ki-S1) and Ki-67 (MIB-1). The correlation of staining indices with clinicopathological data, histological features and prognosis was also studied. The topo-II value was significantly higher in ACC than in PA (P<0.0001), and highest in the solid growth pattern of ACC. In addition, significant relationships were found between topo-II values and clinical features such as local recurrence, surgical margins, and distant metastases. By log-rank test, the topo-II index was also correlated significantly with patient survival (P<0.01). The values of topo-II index paralleled those of Ki-67 index in ACC, and a correlation coefficient of 0.97 was obtained. Topo-II may be considered an additional marker for estimating the proliferating fraction of cells and for predicting the short-term prognosis for patients with salivary gland tumors.