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Background: Canonical transient receptor potential channels play a crucial role in cancer cell proliferation. While TRPC6 subtype detection in submandibular glands and the relevance of some TRPC channels in this gland have been shown in animal models, its histological detection in human lacrimal and submandibular glands, as well as related tumors, lacks systematic study. Studying TRPC6 in humans could lead to new therapeutic options. This research aimed to immunohistochemically detect TRPC6 in human samples of physiological lacrimal and submandibular glands and of adenoid cystic carcinoma and mucoepidermoid carcinoma., Methods: Seven fixed body donors and samples of six cancer patients were examined. The ten tissue samples collected from the submandibular and lacrimal glands were then processed into histological slides and stained with hematoxylin-eosin. Tumor samples were provided as sections. TRPC6 presence was determined by immunohistochemistry, which was performed by indirect detection with a primary TRPC6 antibody, a secondary HRP-conjugated antibody and the chromogen diaminobenzidine., Results: Results confirm TRPC6 expression in all ten physiological gland samples: all samples showed a immunohistochemical signal with varying intensity. No significant gender-specific differences could be observed. TRPC6 was detected in four of six submandibular adenoid cystic carcinoma and the mucoepidermoid carcinoma samples, especially in tumor cells' cytoplasma and nuclei. Excretory ducts consistently showed TRPC6. Mucous tubules, their nuclei and the nuclei of adipocytes generally showed no signal while serous acini and their nuclei showed a weak TRPC6 signal., Conclusion: The discovery of TRPC6 in glandular tissue indicates a role in salivary gland function and calcium homeostasis is a basis for further research into its significance for tumor development in adenoid cystic carcinoma and mucoepidermoid carcinoma of salivary glands. TRPC6 could be used as a target for treatment of these tumors. However, the correlation between TRPC6 and submandibular and lacrimal gland diseases requires further exploration., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objective: While several studies reported epidermal growth factor receptor (EGFR) expression in salivary gland cancer (SGC), results varied due to a lack of unified definition of EGFR positivity. In this study, we assessed the EGFR expression level using both EGFR positive score and cumulative EGFR score in the patients with SGC., Methods: Between January 2010 and April 2021, 102 patients with SGC who underwent surgical resection were reviewed retrospectively by immunohistochemistry. The membrane staining intensity was scored as follows: no staining (0), weak staining (1+), intermediate staining (2+), and strong staining (3+). The cumulative EGFR score was determined on a continuous scale of 0-300 using the formula:1 × (1+: percentage of weakly stained cells) + 2 × (2+: percentage of moderately stained cells) + 3 × (3+: percentage of strongly stained cells)., Results: EGFR expression in SGC varied widely even among the same as well as different histopathological types. The average EGFR positive scores were 46.0 %, 55.7 %, 51.6 %, 1.0 %, 26.8 %, 50 %, and 76.8 % for mucoepidermoid carcinoma (MEC), salivary duct carcinoma (SDC), adenoid cystic carcinoma (AdCC), acinic cell carcinoma (AcCC), adenocarcinoma NOS (ACNOS), carcinoma ex pleomorphic adenoma (CAexPA), and squamous cell carcinoma (SqCC), respectively. The average cumulative EGFR scores were 82, 91, 80, 1, 52, 93, and 185 for MEC, SDC, AdCC, AcCC, ACNOS, CAexPA, and SqCC, respectively., Conclusions: EGFR positive scores and cumulative EGFR scores in SGCs varied among the various histological types, and even in the same histological type. These scores may predict the clinical outcome of SGC treated with EGFR-targeting therapies, such as head and neck photoimmunotherapy, and need to be evaluated in future studies., (Copyright © 2024. Published by Elsevier B.V.)

There is a limited amount of data on the role of programmed cell death ligand (PD-L) -1 and PD-L2 in salivary gland carcinomas. We aimed to evaluate the prognostic value of PD-L1 and PD-L2 expressions, which are closely related to immune mechanisms, with respect to salivary gland tumor types and stages. Data from patients with salivary gland masses surgically removed between 2006 and 2021, diagnosed with a malignant salivary gland neoplasm, were retrospectively analyzed. Immunoreactivity for PD-L1 and PD-L2 was performed on resection materials. The mean age of 90 patients was 52.1±18.8 and 46.7% were male. Overall, 55.6% of patients were diagnosed with adenoid cystic carcinoma (ACC), 23.3% with mucoepidermoid carcinoma (MEC), 16.7% with acinic cell carcinoma (AciCC), 3.3% with ductal carcinoma (DC), and 1 patient with pleomorphic adenoma ex carcinoma (PA-ex-CA). In all, 52% of ACC, 12% of AciCC, 24% of MEC, and 12% of DC cases were at stage IV. The tumor diameter, frequencies of lymphovascular invasion, metastasis, positive surgical margin, recurrence, and mortality rates of patients at stages III and IV were significantly larger than those at stages I and II ( P <0.05). The percentages of tumor cell score (TCS) and immune cell score (ICS) for PD-L1 were significantly higher among patients with MEC compared with those with other types of tumors ( P =0.0011). However, the percentages of combined score (CS) for PD-L1 and tumor cell score for PD-L2 were comparable among tumor types ( P >0.05). No significant difference was found in these scores for PD-L1 between tumor stages ( P >0.05), but for PD-L2, all patients at stage I had TCS <1% for PD-L2, while all patients at stages II and III, and 92% of patients at stage IV had TCS ≥1% ( P <0.0001). High expression of PD-L1 was mostly observed in MEC cases ( P =0.0016), while all patients with AciCC had a low PD-L1 expression level ( P =0.0206). The mean tumor diameter, rate of lymphovascular invasion, perineural invasion, metastasis, positive surgical margin, recurrence, type of treatment, mortality, and TILs ratio did not differ significantly according to PD-L1 expression level ( P >0.05). The percentage of tumor-infiltrating lymphocytes was comparable among negative and positive PD-L1 scores according to both 1% and 5% threshold values ( P >0.05). High PD-L1 expression is rare in AciCC, while PD-L1 expression is high in MEC. Our findings underline the importance of future screening for PD-L1 and PD-L2 before patients undergoing immunotherapies in all salivary gland tumors., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFβ signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFβ signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFβ cascade., Design: Thirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR., Results: Cases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFβ signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC., Conclusions: Our study demonstrated the differential expression of TGFβ cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background and Objectives : Mucin has been implicated via various mechanisms in the development and growth of tumour cells. However, mucin expression studies in salivary gland tumours are limited, especially with samples from minor salivary glands. This study aims to investigate and compare mucin expression in benign and malignant salivary gland tumours of minor and major salivary gland origins. Materials and Methods : Special stains were used to stain neutral mucin (Periodic acid Schiff), sialomucin (Alcian Blue) and sulfomucin (Aldehyde Fuschin) within tissues from six normal salivary glands and 73 salivary gland tumours including 31 pleomorphic adenomas, 27 mucoepidermoid carcinomas, and 15 adenoid cystic carcinomas. A semi-quantitative approach was used to evaluate mucin expression within ductal lumens. Sialomucin was the most expressed mucin in all salivary gland tumours, regardless of origin. Results : A significant difference was observed in the mucin expression between benign and malignant salivary gland tumours, as pleomorphic adenoma showed three times significantly higher expression of sialomucin compared to mucoepidermoid carcinoma and adenoid cystic carcinoma ( p = 0.028). Pleomorphic adenomas of major glands showed 42 times significantly higher expression of sialomucin compared to those of minor glands ( p = 0.000). Conclusions : Sialomucin content in pleomorphic adenomas of major glands was vastly increased compared to that in minor glands. Differential sialomucin expression in benign and malignant salivary gland tumours suggests a role in diagnosing of borderline salivary gland tumours., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper.

Background: Mucoepidermoid carcinoma is a rare salivary gland malignant tumour. This study aimed to investigate inflammatory and immune signatures of mucoepidermoid carcinoma by identifying potential proteo-transcriptomic biomarkers towards the development of precision immuno-oncology treatment strategies., Methods: A total of 30 biopsies obtained from patients diagnosed with mucoepidermoid carcinoma between 2013 and 2022 were analysed after H&E staining for scoring of histological inflammatory stroma subtypes and inflammatory hotspots with QuPath. Multiplex immunofluorescence staining and NanoString nCounter PanCancer IO 360™ panel were used to assess stroma and tumour inflammation signatures in high grade mucoepidermoid carcinoma cases in the tumour microenvironment via proteomics and transcriptomics, respectively., Results: Inflammatory cells within the histological inflammatory stroma inflammatory (HIS-INF/hot) tumour neighbourhoods were greater compared to the histological inflammatory stroma-immune desert (HIS-ID/cold) (p = 0.001). A similar trend was observed between treatment non-responders and responders in stroma neighbourhoods (p = 0.0625) and in stroma-to-interface inflammatory hotspots (p = 0.0081), indicating an augmented inflammatory response in hot tumours and non-responders. Furthermore, there were striking differences in the expression of pan-immune leukocyte marker CD45 between responders and non responders particularly in the tumour neighbourhoods (p = 0.0341), but such were not robust for PD-1 and macrophage fractions. Additionally, transcriptomic analysis revealed key differences in leukocyte activation profiles between responders and non-responders., Conclusion: This preliminary report unveils the importance of assessing immune leukocyte cellular fractions and pathways for future prognostic biomarker discoveries in mucoepidermoid carcinoma as per the involvement of CD45-driven inflammatory and immune mediators in high grade mucoepidermoid carcinoma in non-responders to treatment. These findings will potentially contribute to the development of novel personalised immunotherapies., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Carcinomas of the salivary gland are known to be aggressive in nature, making them difficult to manage. The therapeutic options offered include excision of the gland (maxillectomy in cases of palatal tumours), with or without lymph node dissection, proceeded with radiotherapy. Chemotherapy has not produced promising outcomes and has a minimal impact as a therapeutic alternative. Targeted therapy against human epidermal growth factor receptor 2 (HER-2), which is a commonly used treatment modality for their mammary analogues, is not being offered to these patients since scant literature is available showing its usefulness and no promising evidence is present regarding their efficacy and efficiency in such cases. The study aimed to evaluate and quantify the immunohistochemical expression of HER-2 in cases of adenoid cystic carcinoma (AdCC), mucoepidermoid carcinoma (MEC) and salivary duct carcinoma (SDC), which are analogues of similar tumours arising in breast tissue., Methods: A retrospective, cross-sectional study was carried out in the department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi, duration of which was six months. A total of 45 cases (15 of each tumour) were taken, and sampled using non-probability convenience technique. The immunohistochemical marker, monoclonal HER-2 antibody (Leica microsystem Germany) was applied on appropriate blocks of all included cases. The staining pattern and intensity were recorded after visualizing the slides under a light microscope., Results: Seven cases of salivary duct carcinoma and a single case of mucoepidermoid carcinoma expressed positivity for HER-2, while no expression could be seen in the case of adenoid cystic carcinoma. A statistically significant difference was seen when HER-2 expression was compared among the aforementioned tumours., Conclusions: The use of targeted therapy against HER-2 is limited to patients of salivary duct carcinoma and a fraction of patients suffering from mucoepidermoid carcinoma.

Objective: This study aimed to compare the immunoexpression profile of tumor stem cell (TSC) biomarkers CD44, aldehyde dehydrogenase 1 (ALDH1), OCT4, and SOX2 in salivary gland tumors (SGTs)., Study Design: Sixty tissue specimens of SGTs, including 20 pleomorphic adenomas, 20 adenoid cystic carcinomas (ACCs), and 20 mucoepidermoid carcinomas, in addition to 4 samples of normal glandular tissue, were subjected to immunohistochemistry. The expression of the biomarkers in the parenchyma and stroma was evaluated. Data were analyzed statistically by nonparametric tests (P < .05)., Results: Higher parenchymal expression of ALDH1, OCT4, and SOX2 was observed in pleomorphic adenomas, ACCs, and mucoepidermoid carcinomas, respectively. Most ACCs did not express ALDH1. Higher immunoexpression of ALDH1 in major SGTs (P = .021) and of OCT4 in minor SGTs (P = .011) was found. Immunoexpression of SOX2 was related to lesions without myoepithelial differentiation (P < .001) and malignant behavior (P = .002). Furthermore, OCT4 was related to myoepithelial differentiation (P = .009). CD44 expression was related to a better prognosis. Stromal immunoexpressions of CD44, ALDH1, and OCT4 were higher in malignant SGTs., Conclusions: Our findings suggest the participation of TSCs in the pathogenesis of SGTs. We emphasize the need for further investigations into the presence and role of TSCs in the stroma of these lesions., Competing Interests: Conflict of Interest None., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: Mucoepidermoid carcinoma (MEC) is a poorly understood salivary gland malignancy with limited therapeutic options. Cancer stem cells (CSC) are considered drivers of cancer progression by mediating tumor recurrence and metastasis. We have shown that clinically relevant small molecule inhibitors of MDM2-p53 interaction activate p53 signaling and reduce the fraction of CSC in MEC. Here we examined the functional role of p53 in the plasticity and self-renewal of MEC CSC., Experimental Design: Using gene silencing and therapeutic activation of p53, we analyzed the cell-cycle profiles and apoptosis levels of CSCs in MEC cell lines (UM-HMC-1, -3A, -3B) via flow cytometry and looked at the effects on survival/self-renewal of the CSCs through sphere assays. We evaluated the effect of p53 on tumor development (N = 51) and disease recurrence (N = 17) using in vivo subcutaneous and orthotopic murine models of MEC. Recurrence was followed for 250 days after tumor resection., Results: Although p53 activation does not induce MEC CSC apoptosis, it reduces stemness properties such as self-renewal by regulating Bmi-1 expression and driving CSC towards differentiation. In contrast, downregulation of p53 causes expansion of the CSC population while promoting tumor growth. Remarkably, therapeutic activation of p53 prevented CSC-mediated tumor recurrence in preclinical trials., Conclusions: Collectively, these results demonstrate that p53 defines the stemness of MEC and suggest that therapeutic activation of p53 might have clinical utility in patients with salivary gland MEC., (©2022 American Association for Cancer Research.)

Mucoepidermoid Carcinomas (MEC) represent the most common malignancies of salivary glands. Approximately 50% of all MEC cases are known to harbor CRTC1/3-MAML2 gene fusions, but the additional molecular drivers remain largely uncharacterized. Here, we sought to resolve controversy around the role of human papillomavirus (HPV) as a potential driver of mucoepidermoid carcinoma. Bioinformatics analysis was performed on 48 MEC transcriptomes. Subsequent targeted capture DNA sequencing was used to annotate HPV content and integration status in the host genome. HPV of any type was only identified in 1/48 (2%) of the MEC transcriptomes analyzed. Importantly, the one HPV16+ tumor expressed high levels of p16, had high expression of HPV16 oncogenes E6 and E7, and displayed a complex integration pattern that included breakpoints into 13 host genes including PIK3AP1 , HIPI, &nbsp; OLFM4, SIRT1 , ARAP2 , TMEM161B-AS1, and EPS15L1 as well as 9 non-genic regions. In this cohort, HPV is a rare driver of MEC but may have a substantial etiologic role in cases that harbor the virus. Genetic mechanisms of host genome integration are similar to those observed in other head and neck cancers.

Advanced salivary gland carcinomas (SGC) often lack therapeutic options. Agents targeting CD138 have recently shown promising results in clinical trials for multiple myeloma and a preclinical trial for triple-negative breast cancer. Immunohistochemistry for CD138 was performed for all patients who had undergone primary surgery for SGC with curative intent. Findings were validated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging. Overall, 111 primary SGC and 13 lymph node metastases from salivary duct carcinomas (SaDu) were evaluated. CD138 expression was found in 60% of all SGC with differing expression across entities (p < 0.01). A mean of 25.2% of the tumor cells in mucoepidermoid carcinoma (MuEp) were positive, followed by epithelial-myoepithelial carcinoma (20.9%), acinic cell carcinoma (16.0%), and SaDu (15.2%). High-/intermediate-grade MuEp showed CD138 expression in a mean of 34.8% of tumor cells. For SaDu, lymph node metastases showed CD138 expression in a mean of 31.2% of tumor cells which correlated with CD138 expression in their primaries (p = 0.01; Spearman’s ρ = 0.71). MALDI-MS imaging confirmed the presence of the CD138 protein in SGC. No significant association was found between clinicopathological data, including progression-free survival (p = 0.50) and CD138 expression. CD138 is expressed in the cell membrane of different entities of SGC and SaDu lymph node metastases and therefore represents a potential target for CD138 targeting drugs.

Aim: To evaluate the potential use of Cephaeline as a therapeutic strategy to manage mucoepidermoid carcinomas (MEC) of the salivary glands., Material and Methods: UM-HMC-1, UM-HMC-2, and UM-HMC-3A MEC cell lines were used to establish the effects of Cephaeline over tumor viability determined by MTT assay. In vitro wound healing scratch assays were performed to address cellular migration while immunofluorescence staining for histone H3 lysine 9 (H3k9ac) was used to identify the acetylation status of tumor cells upon Cephaeline administration. The presence of cancer stem cells was evaluated by the identification of ALDH enzymatic activity by flow cytometry and through functional assays using in vitro tumorsphere formation., Results: A single administration of Cephaeline resulted in reduced viability of MEC cells along with the halt on tumor growth and cellular migration potential. Administration of Cephaeline resulted in chromatin histone acetylation as judged by the increased levels of H3K9ac and disruption of tumorspheres formation. Interestingly, ALDH levels were increased in UM-HMC-1 and UM-HMC-3A cell lines, while UM-HMC-2 showed a reduced enzymatic activity., Conclusion: Cephaeline has shown anti-cancer properties in all MEC cell lines tested by regulating tumor cells' viability, migration, proliferation, and disrupting the ability of cancer cells to generate tumorspheres., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This study is a systematic review and meta-analysis to assess the overexpression rate of HER2 in patients with salivary gland tumors. We included peer-reviewed publications from 1995 to 2020, indexed in medical databases, using search terms such as "human epidermal growth factor receptor 2 (HER2)" and "salivary gland tumors", and extracted relevant data. The extracted data were analyzed with RevMan 5.3 software. Intra-and intergroup post hoc analyses of outcome variables were performed using t-tests, and the rates of HER2 positivity among studies were evaluated. 80 studies were included in the analysis. The positive rates of HER2 ranged from 3.3% to 84.0% and 1% to 9% in malignant and benign subtypes, respectively. The highest HER2 overexpression rate among malignant tumors was in salivary ductal carcinomas (SDC), with a 45% positive rate (CI 95%: 21.9-70.3%). Mucoepidermoid carcinoma (MEC) had the highest positive rate of 84% (CI 95%: 74.1-90.0%). Among benign salivary gland tumors, the highest rate was found in myoepithelioma, with a positive rate of 9% (CI 95%: 1.7-33.6%). The highest rate of HER2 overexpression is present in malignant subtypes of salivary gland tumors, more specifically in salivary ductal carcinoma, mucoepidermoid carcinomas, salivary duct carcinoma in situ, and carcinoma ex pleomorphic adenoma., (© 2022 JOURNAL of MEDICINE and LIFE.)

Background: Mucoepidermoid carcinoma and adenoid cystic carcinoma are the two most common malignancies of salivary gland. Our study aims to explore the role of human endogenous Retrovirus-H long terminal repeat-associating protein 2, transmembrane and immunoglobulin domain-containing 2, and glucocorticoid-induced tumor necrosis factor receptor in adenoid cystic carcinoma and mucoepidermoid carcinoma, and the relationship between human endogenous Retrovirus-H long terminal repeat-associating protein 2, transmembrane and immunoglobulin domain-containing 2, glucocorticoid-induced TNF receptor, oncogenic signaling molecules, and cluster of differentiation 8., Methods: Custom-made human salivary gland tissue microarrays included 81 Adenoid cystic carcinoma, 52 mucoepidermoid carcinoma, 76 normal salivary gland, and 14 pleomorphic adenoma samples. Immunohistochemical analysis of human endogenous Retrovirus-H long terminal repeat-associating protein 2, transmembrane and immunoglobulin domain-containing 2, and glucocorticoid-induced TNF receptor, oncogenic phosphorylated Erk 1/2 , the epithelial-mesenchymal transition (EMT) molecule transforming growth factor β1, and cluster of differentiation 8 was performed with salivary gland tissue microarray of human samples., Results: According to a digital pathological system, we analyzed the correlation of immunostaining. The expression levels of human endogenous Retrovirus-H long terminal repeat-associating protein 2, transmembrane and immunoglobulin domain-containing 2, and glucocorticoid-induced TNF receptor were significantly enhanced in the adenoid cystic carcinoma and mucoepidermoid carcinoma, compared with those of pleomorphic adenoma and NSG samples. However, the expression levels of human endogenous Retrovirus-H long terminal repeat-associating protein 2, transmembrane and immunoglobulin domain-containing 2, and glucocorticoid-induced TNF receptor were independent of the pathological grade of malignancy of mucoepidermoid carcinoma and histological pattern of adenoid cystic carcinoma. They were closely related to phosphorylated Erk1/2 and transforming growth factor β1, but negligibly related to cluster of differentiation 8., Conclusions: These results described that certain immune checkpoint molecules, namely, human endogenous Retrovirus-H long terminal repeat-associating protein 2, transmembrane and immunoglobulin domain-containing 2, and glucocorticoid-induced TNF receptor were overexpressed in Adenoid cystic carcinoma and mucoepidermoid carcinoma, but were independent of pathological grade, and may relate to transforming growth factor β1, phosphorylated Erk1/2, and cluster of differentiation 8., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: Cancer stem cells (CSCs) are a small group of cells resistant to therapy and play a major role in tumor progression, recurrence, and poor clinical outcomes of patients. This study aimed to evaluate the association of CSC markers with clinicopathologic features and survival in patients with salivary gland carcinomas (SGCs)., Materials and Methods: The medical records of 48 patients affected by mucoepidermoid carcinoma (MEC) and 47 patients with adenoid cystic carcinoma (AdCC) were reviewed retrospectively. SOX2, CD133, and CD44 expression was appraised by immunohistochemistry and statistically analyzed to weigh the correlation between these markers and patients' clinicopathologic features and tumor outcomes., Results: In AdCC patients showing poor outcomes, a trend toward a high expression of CD133 and CD44 and low expression of SOX2 was observed, while in MEC patients experiencing the same outcomes, there was a trend toward a high expression of CD44 and low expression of CD133 and SOX2. Only the increase of MEC histopathologic grade was statistically significant with decreased SOX2 expression. Distant metastasis in AdCC patients, tumor grade, lymph node involvement, and local recurrence in MEC patients had significant correlations with patients' survival., Conclusion: Besides the significant association between low SOX2 expression and higher grades of MEC, we found no statistically significant correlation between the studied CSC markers and patients' survival or clinicopathologic features. Therefore, a larger sample size with long-term follow-up is beneficial for thorough investigations toward the main role of CSCs in patients with SGCs., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Parotid cancer is relatively rare, and malignancy varies; therefore, novel markers are needed to predict prognosis. Recent advances in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS), useful for visualization of lipid molecules, have revealed the relationship between cancer and lipid metabolism, indicating the potential of lipids as biomarkers. However, the distribution and importance of phospholipids in parotid cancer remain unclear., Objective: This study aimed to use MALDI-IMS to comprehensively investigate the spatial distribution of phospholipids characteristically expressed in human parotid cancer tissues., Methods: Tissue samples were surgically collected from two patients with parotid cancer (acinic cell carcinoma and mucoepidermoid carcinoma). Frozen sections of the samples were assessed using MALDI-IMS in both positive and negative ion modes, with an m/z range of 600-1000. The mass spectra obtained in the tumor and non-tumor regions were compared and analyzed. Ion images corresponding to the peak characteristics of the tumor regions were visualized., Results: Several candidate phospholipids with significantly different expression levels were detected between the tumor and non-tumor regions. The number of unique lipid peaks with significantly different intensities between the tumor and non-tumor regions was 95 and 85 for Cases 1 and 2, respectively, in positive ion mode, and 99 and 97 for Cases 1 and 2, respectively, in negative ion mode. Imaging differentiated the characteristics that phospholipids were heterogeneously distributed in the tumor regions., Conclusion: Phospholipid candidates that are characteristically expressed in human parotid cancer tissues were found, demonstrating the localization of their expression. These findings are notable for further investigation of alterations in lipid metabolism of parotid cancer and may have potential for the development of phospholipids as biomarkers., Competing Interests: The authors have declared that no competing interests exist.

Mucoepidermoid carcinoma (MEC) is generally characterized by an admixture of mucous, epidermoid and intermediate type cells. Numerous variants morphologies are described and defined by stromal and/or cytoplasmic tinctorial characteristics. We now report 11 cases of MEC with serous acinar differentiation, reflecting a distal intercalated duct/acinar phenotype, which we designate as mucoacinar carcinomas. Seven patients were female while 4 were male with a mean age of 55 years (range: 21 to 72 y). Ten cases were from the parotid while 1 was from the submandibular gland. Mean size of the tumors was 1.8 cm (range: 0.7 to 4.5 cm). Three cases were low grade, 7 were intermediate grade, and 1 was high grade. Low to intermediate grade cases demonstrated prominent clear to vacuolated cells with focal serous acinar differentiation. The high-grade case showed a distinctive scattering of acinar cells interspersed between epidermoid cells. Periodic acid Schiff after diastase (9/9), SOX-10 (9/9), and DOG-1 (9/10) highlighted the acinar component. Six of 7 cases showed a focal acinar predominant NR4A3 expression. MAML2 fluorescence in situ hybridization was positive in all cases, in both acinar and mucoepidermoid components. Two cases tested by next generation sequencing showed standard CRTC1-MAML2 fusions. MSANTD3 and NR4A3 fluorescence in situ hybridization on the other hand were negative. Evidence thus suggests that mucoacinar carcinoma represents an acinar variant morphology in MEC, rather than a true MEC-acinic cell carcinoma hybrid, or collision tumor. The acinar differentiation, SOX-10, DOG-1, and even focal NR4A3 reactivity may thus be diagnostic pitfalls., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article, (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Cytosolic 5'-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.

Mucoepidermoid carcinoma (MEC) is the most common malignant salivary gland tumour in both adults and children. Histological grading of MEC is subjective, but plays an important role in predicting patient prognosis. Epithelial mucin (MUC) status may aid in establishing a more accurate grade. This study aimed to investigate the expression of various mucins (MUC1, MUC2, MUC4 and MUC5AC) in MECs to determine a possible correlation with tumour grade. Fifteen cases of each tumour grade (low-, intermediate-, and high-grade) were retrieved from the pathology archives of the Department of Oral Pathology and Oral Biology at the University of Pretoria. The patients included 23 men and 22 women, and ranged from 13 to 85 years (mean 49.8 years). Sections from formalin-fixed paraffin-embedded (FFPE) tissue were used for fluorescence in situ hybridization (FISH) for MAML2 rearrangements and MUC immunohistochemical analysis. The percentage immunohistochemical expression of the neoplastic mucous cells was evaluated first, followed by the overall percentage expression of all tumour cells. The results indicated that MUC1 overexpression may be a reliable marker of high-grade MECs, whereas MUC4 overexpression may be more indicative of low-grade tumours. MUC5AC expression was considered an unreliable marker in determining grade. MUC2 was only expressed in a single case of MEC and may be considered a useful marker to exclude MEC as a diagnostic possibility. This study demonstrates that MECs show an altered MUC expression pattern that can be used for diagnostic purposes and to aid in establishing a more accurate tumour grade.

Context.—: Both salivary lymphadenomas (LADs) and Warthin-like mucoepidermoid carcinoma (MEC) contain components of epithelium and lymphoid stroma and their differential diagnosis can be difficult on the basis of morphology alone., Objective.—: To clarify whether Warthin-like MEC was diagnosed as a LAD, and to compare their clinicopathologic features., Design.—: A total of 16 LAD cases were analyzed for MAML2 rearrangement by using fluorescence in situ hybridization, and the clinical, histologic, immunohistochemical, and prognostic features were compared between MAML2 rearrangement-positive and MAML2 rearrangement-negative groups., Results.—: Among the 16 cases investigated, 9 harbored a MAML2 rearrangement and were reclassified as Warthin-like MEC. The remaining 7 cases were classified as LADs with 1 nonsebaceous and 6 sebaceous cases. The patients with Warthin-like MEC had a wider age range (10-75 years) than the patients with LADs (36-68 years). Histologically, 2 of the 9 Warthin-like MECs (22.2%) showed focal invasion, whereas all the LADs had complete capsules. Warthin-like MECs exhibited a diverse epithelial cell morphology, including basaloid, glandular, cuboidal, epidermoid, with mucinous cells, although these cytologic features were seen only focally in some cases. Nonsebaceous LAD was composed of basaloid and glandular epithelial cells predominantly. In sebaceous LAD, the epithelial cells were composed of basaloid and large foamy sebaceous cells. In all cases, the stroma was composed mainly of lymphocytes accompanied by lymphoid follicles, although plasma cell infiltration could be much heavier in Warthin-like MEC. All the patients had a good outcome after a longer follow-up (3-166 months)., Conclusions.—: Warthin-like MEC can be misdiagnosed as a LAD owing to overlap in clinicopathologic features of the 2 entities. Careful histologic evaluation and detection of MAML2 rearrangement can facilitate their differential diagnosis.

Background: Treatment regimens for patients with metastatic or recurrent post-radiation, locoregional, unresectable salivary cancer are limited. An inverse correlation between somatostatin receptor 2 (SSTR2) and the proliferating marker Ki-67 in neuroendocrine tumors has enabled a treatment plan for metastatic disease, utilizing peptide receptor radionuclide therapy. Interestingly, healthy salivary glands express high levels of SSTR2. In this study, the presence of SSTR2, its correlation with Ki-67 in glandular salivary carcinomas and the clinical applicability thereof was determined., Methods: In the retrospective part of this study, 76 adequate tumor tissue specimens obtained from patients diagnosed with primary or metastatic salivary carcinomas between 1988 and 2016, were collected for tissue array and histologically classified. Immunohistochemistry was performed to determine the presence, relative expression and potential correlation of SSTR2 and Ki-67. The clinical significance of SSTR2 expression was determined by prospectively assessing 68 Ga-DOTATATE uptake using PET-CT imaging, in patients diagnosed with metastatic salivary gland malignant tumors between 2015 and 2016., Results: Sixty-three primary cancer tumors and 14 metastatic tumors were tested. All tumor subtypes were found to express SSTR2 to some extent. The highest expression was seen in Mucoepidermoid carcinoma (MEC) tissues where the majority of specimens (86.4%) expressed SSTR2. A relatively strong immunohistochemical staining score for SSTR2 was observed in MEC, adenoid cystic carcinoma and polymorphous adenocarcinoma. Interestingly, an inverse correlation between SSTR2 and Ki-67 expressions was observed (44%) in MEC tissue. Uptake of 68 Ga-DOTATATE was visualized using PET-CT imaging in 40% of patients, across metastatic MEC and ACC. All observations were found to be statistically significant., Conclusion: This study confirms the expression of SSTR2 in glandular salivary carcinomas and an inverse correlation in expression levels between SSTR2 and Ki-67. This lays a foundation for novel treatment options in salivary metastatic cancers where SSTR2 may be a potential novel therapeutic target.

Salivary gland neoplasms are an uncommon and widely heterogeneous group of tumors. In recent years, there has been considerable progress in efforts to reveal the molecular landscape of these tumors, although it is still limited and appears to be only the tip of the iceberg. Genomic aberrations, especially specific chromosomal rearrangements including CRTC1-MAML2 and CRTC3-MAML2 in mucoepidermoid carcinoma, MYB-NFIB and MYBL1-NFIB fusions in adenoid cystic carcinoma, PLAG1 and HMGA2 alterations in pleomorphic adenoma and carcinoma ex pleomorphic adenoma, ETV6-NTRK3 and ETV6-RET in secretory carcinoma, EWSR1-ATF1 and EWSR1-CREM in clear cell carcinoma, provide new insights into the molecular pathogenesis of various salivary gland neoplasms and help to better classify them. These genetic aberrations primarily serve as diagnostic tools in salivary gland tumor diagnosis; however, some also have promise as prognostic or predictive biomarkers. This review summarizes the latest developments in molecular pathology of salivary gland tumors with a focus on distinctive molecular characteristics., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Mucoepidermoid carcinoma (MEC) is a life-threatening salivary gland cancer that is driven primarily by a transcriptional coactivator fusion composed of cyclic AMP-regulated transcriptional coactivator 1 (CRTC1) and mastermind-like 2 (MAML2). The mechanisms by which the chimeric CRTC1/MAML2 (C1/M2) oncoprotein rewires gene expression programs that promote tumorigenesis remain poorly understood. Here, we show that C1/M2 induces transcriptional activation of the non-canonical peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) splice variant PGC-1α4, which regulates peroxisome proliferator-activated receptor gamma (PPARγ)-mediated insulin-like growth factor 1 (IGF-1) expression. This mitogenic transcriptional circuitry is consistent across cell lines and primary tumors. C1/M2-positive tumors exhibit IGF-1 pathway activation, and small-molecule drug screens reveal that tumor cells harboring the fusion gene are selectively sensitive to IGF-1 receptor (IGF-1R) inhibition. Furthermore, this dependence on autocrine regulation of IGF-1 transcription renders MEC cells susceptible to PPARγ inhibition with inverse agonists. These results yield insights into the aberrant coregulatory functions of C1/M2 and identify a specific vulnerability that can be exploited for precision therapy., Competing Interests: Declaration of interests A.L.A. is a GAB member and paid consultant for LG Chem Life Sciences Innovation Center., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: The aim of the present study was to investigate the expression of claudin-1, -3, -4, -5 and -7 proteins in mucoepidermoid carcinoma of oral cavity and analyze whether EGF may interfere in the expression of the genes that encode claudins using in vitro models., Material and Methods: Using immunohistochemistry, the expression of claudins was searched in 36 histologically graded cases of mucoepidermoid carcinoma. The association of expression of claudins with clinical-pathological parameters was evaluated. An in vitro step investigated the influence of EGF on gene expression of claudins by real time RT-PCR technique., Results: Claudin-1, -3, -4, -5, and -7 were highly expressed in most mucoepidermoid carcinomas. These expressions were compared with clinicopathological parameters. High expression of claudin-1 was associated with patients over 40 years-old (p = 0.05) and Caucasians (p = 0.024). In vitro experiments demonstrated a tendency for Claudin gene expression increase after EGF stimulus., Conclusions: The expression of claudins is maintained in mucoepidermoid carcinoma cells and EGF could be related with this expression. Our results point out to a fundamental biological importance to CLDNs in normal and neoplastic tissue. The expression patterns of CLDNs does not yet allow a clinical application, but the biological knowledge will ground evidence to new studies towards possible target-therapies., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Nitidine chloride (NC) was recently reported to exhibit a wide range of pharmacological properties for several diseases, including cancer. Here we report for the first time that NC is a potential therapeutic agent for mucoepidermoid carcinoma (MEC) occurring in the head and neck because it suppresses X chromosome-linked inhibitor of apoptosis protein (XIAP) in human MEC in vitro and in vivo. The antitumor effects of NC were evaluated by trypan blue exclusion assay, western blotting, live/dead assay, 4',6-diamidino-2-phenylindole (DAPI) staining, human apoptosis antibody array, immunofluorescence staining, immunohistochemistry, small interfering RNA assay, transient transfection of XIAP overexpression vector, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and histopathological examination of organs. NC inhibited cell viability and induced caspase-dependent apoptosis in vitro. A human apoptosis antibody array assay showed that XIAP is suppressed by NC treatment. XIAP was overexpressed in oral squamous cell carcinoma (OSCC) tissues that arose from the head and neck, and high XIAP expression was correlated with poor prognosis in OSCC patients. XIAP depletion significantly increased apoptosis, and ectopic XIAP overexpression attenuated the apoptosis induced by NC treatment. NC suppressed tumor growth in vivo at a dosage of 5 mg/kg/day. The number of TUNEL-positive cells increased and the protein expression of XIAP was consistently downregulated in NC-treated tumor tissues. In addition, NC caused no histopathological changes in the liver or kidney. These findings provide new insights into the mechanism of action underlying the anticancer effects of NC and demonstrate that NC is a promising therapeutic agent for the treatment of human MEC of the head and neck. KEY MESSAGES: • Nitidine chloride induces caspase-dependent apoptosis in MEC of the head and neck. • High XIAP expression correlates with poor prognosis of OSCC patients. • Nitidine chloride suppresses tumor growth in vivo without any systemic toxicities. • Targeting XIAP is a novel chemotherapeutic strategy for MEC of the head and neck.

Diagnosis of salivary gland tumors on small biopsy can be difficult because of overlapping morphology, limited tissue availability, and technical artifact. Although a specific diagnosis is not feasible in all cases, a cautious and thoughtful approach to the differential diagnosis and a keen awareness of clinical consequences can facilitate the most complete and useful classification possible. In this review, we present a general strategy for the evaluation of small salivary biopsies, including consideration of clinical and radiographic information, systematic assessment of histologic patterns, and judicious use of immunohistochemistry and molecular studies. We then focus on the distinctive differential diagnoses raised by 6 specific histologic patterns: tubular and cribriform architecture, squamous differentiation, mucin and other secretions, high-grade cytology, epithelial and lymphoid elements, and oncocytic features. Throughout this systematic and pattern-based approach, we focus on practical and cost-effective strategies to overcome the most common diagnostic challenges in limited material., (Copyright © 2020 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Oridonin, an active diterpenoid isolated from Rabdosia rubescens, has been reported to exhibit anticancer activities in several tumors. The aim of the present study was to investigate the anticancer effects and molecular mechanisms of oridonin in mucoepidermoid carcinoma (MEC). Treatment with oridonin induced the apoptosis of MC‑3 and YD‑15 cell and inhibited the expression of myeloid cell leukemia‑1 (MCL‑1) through the regulation of the protein level through post‑translational regulation in these cell lines. Oridonin significantly increased the expression level of truncated Bid (t‑Bid) as a downstream target of MCL‑1 and subsequently decreased the mitochondrial membrane potential. The ectopic expression of MCL‑1 protein was sufficient to reverse the induction of apoptosis and the increased t‑Bid expression induced by oridonin in both cell lines. Taken together, these results suggest that oridonin exerts an apoptotic effect through the modulation of MCL‑1 and t‑Bid in human MEC cell lines and may thus be a potential anticancer drug candidate for the treatment of human MEC.

Salivary gland aquaporins (AQPs) are essential for the control of saliva production and maintenance of glandular structure. However, little is known of their role in salivary gland neoplasia. Salivary gland tumors comprise a heterogeneous group of lesions, featuring variable histological characteristics and diverse clinical behaviors. Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. The aim of this study was to evaluate the expression of AQP1, AQP3, and AQP5 in 24 MEC samples by immunohistochemistry. AQP1 expression was observed in vascular endothelium throughout the tumor stroma. AQP3 was expressed in epidermoid and mucosal cells and AQP5 was expressed in mucosal cells of MEC. These proteins were expressed in the human MEC cell line UH-HMC-3A. Cellular ultrastructural aspects were analyzed by electron microscopy to certificate the tumor cell phenotype. In summary, our results show that, despite the fact that these molecules are important for salivary gland physiology, they may not play a distinct role in tumorigenesis in MEC. Additionally, the in vitro model may offer new possibilities to further investigate mechanisms of these molecules in tumor biology and their real significance in prognosis and possible target therapies.

Mucoepidermoid carcinoma (MEC) is the most common tumor in the salivary glands, often presenting with recurrence and metastasis due to its high invasive capacity. Metallothionein (MT), a zinc storage protein that supplies this element for protease activity, is probably related to mucoepidermoid carcinoma behavior. This prompted us to characterize a cell line derived from mucoepidermoid carcinoma and to correlate metallothionein expression with transforming growth factor-α (TGF-α), tumor necrosis factor-α (TNF-α) and matrix metalloproteinases (MMPs). Transcriptomic analysis and cytogenetic assays were performed to detect the expression of genes of interest and cellular chromosomal alterations, respectively. MEC cells with a depleted metallothionein 2A ( MT2A ) gene were subjected to Western blot to correlate metallothionein expression with growth factors and MMPs. Additionally, cells with depleted MT were subjected to migration and invasion assays. The transcriptomic study revealed reads mapped to cytokeratins 19 and AE1/AE3, α-smooth muscle actin, vimentin, and fibronectin. Cytogenetic evaluation demonstrated structural and numerical alterations, including the translocation t(11;19)(q21;p13), characteristic of MEC. Metallothionein depletion was correlated with the decreased expression of TGF-α and MMP-9, while TNF-α protein levels were augmented. Migration and invasion activity were diminished after metallothionein silencing. Our findings suggest an important role of MT in MEC invasion, through the regulation of proteins involved in this process., Competing Interests: The authors declare no conflict of interest.

Epithelial membrane antigen (EMA) and DOG1 are used as marker of epithelial cells, particularly the luminal cells, of salivary gland tumours. The aim of this study was to compare the EMA and DOG1 expression in tumours of minor salivary glands. Cases of pleomorphic adenoma (PA), basal cell adenoma (BCA), canalicular adenoma (CA), adenoid cystic carcinoma (ACC), polymorphous adenocarcinoma (PAC), mucoepidermoid carcinoma (MEC) and epithelial-myoepithelial carcinoma (EMC) were submitted to immunohistochemistry for EMA and DOG1. In PA and BCA, EMA and DOG1 were observed in luminal cells, while in CA the tumour cells were negative for both proteins. The EMA and DOG1 pattern expression detected in EMC was similar to that one observed in benign tumours. In ACC, both myoepithelial e epithelial expressed EMA and DOG-1. PAC tumour cells were only positive for DOG1, whereas MEC were only positive for EMA. In conclusion, EMA and DOG1 expression in benign salivary gland tumours was similar to normal salivary gland tissue and can be used as good marker of tumoral cells derived from intercalated ducts or its progenitor cells, while in malignant salivary gland tumours EMA expression is, however, better used as an indicator of aggressive behavior than a marker of luminal cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Malignant salivary gland tumors (MSGTs) present different phenotypic characteristics and various clinical outcomes, which proved to be a diagnostic challenge. Considering the heterogeneity of MSGT, this study aims to identify molecule related to the nature of MSGT., Methods: For screening, proteomic analysis comparing MSGT with pleomorphic adenoma (PA) and salivary gland was performed. The MSGT-associated protein which presented in the higher number in the Gene Expression Omnibus (GEO) database was selected. To validate the data, immunohistochemistry (IHC) was performed in 14 patients with PA, 22 patients with MSGT, and 14 controls., Results: 16 proteins were associated with MSGT. ANXA2 was the primary protein, according to GEO database analyses. ANXA2 was most expressed in the cell membrane. However, some ANXA2 staining was also observed in the cytoplasm and nucleus. ANXA2 was highly expressed in MSGT in comparison with control. Also, ANXA2 has a higher expression in adenocarcinoma not otherwise specified (ANOS) and myoepithelial carcinoma (MC) in comparison with PA., Conclusion: In conclusion, this study demonstrated that MSGT presented higher levels of ANXA2 in comparison with normal salivary glands. Also, ANXA2 might be interesting as a molecular marker of ANOS and MS., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Mucoepidermoid carcinoma is the most common malignant tumor of salivary glands. Apoptosis plays an important role in organogenesis of glandular structures, and aberrations of apoptotic mechanisms is associated with a wide array of pathologic conditions., Methods: The immunoexpression of proteins associated with apoptosis and proliferation was evaluated in 40 mucoepidermoid carcinoma cases., Results: Par-4, Survivin, MUC1, PHLDA1, Fas, and Ki-67 were predominantly expressed in mucoepidermoid carcinoma. FasL was rarely expressed, and Caspase-3 expression was observed in almost 50% of the cases. SPARC expression was associated with low-grade tumors, and Ki-67 expression was associated with lymph node metastasis. Expression of Fas and decreased expression of Ki-67 and Caspase-3 were associated with better overall cancer-specific survival rates., Conclusions: The association of SPARC and Ki-67 expression with pathological features and the association of Fas, Caspase-3, and Ki-67 with survival probabilities suggest that these proteins may be useful prognostic markers for mucoepidermoid carcinoma., (© 2019 Wiley Periodicals, Inc.)

Background: CRTC1-MAML2 fusion is often detected in low- or intermediate-grade salivary mucoepidermoid carcinoma (MEC), and it is associated with a favorable clinical course. Primary MEC of the liver is an extremely rare, aggressive tumor, and no study has investigated CRTC1-MAML2 fusion., Case Presentation: A 79-year-old Japanese female presented with an approx. 5-cm hepatic mass lesion. We surgically resected the lesion under the clinical diagnosis of intrahepatic cholangiocarcinoma. The histological and immunohistochemical findings were consistent with high-grade MEC, consisting of squamoid, mucin-producing, and intermediate tumor cells. Our RT-PCR analysis revealed the presence of CRTC1-MAML2 fusion. This fusion gene was further confirmed by direct sequencing. The patient is still alive almost 10 years after the surgery., Conclusion: This is the first case report of primary MEC of the liver with CRTC1-MAML2 fusion, with long survival. The present case has significant implications for the entity of primary MEC of the liver which should be distinguished from adenosquamous carcinoma.

TW-37 is a small-molecule inhibitor of Bcl-2 family proteins, which can induce anti-cancer activities in various types of cancer. In the current study, we investigated the potential molecular mechanism underlying the differential response to TW-37-induced apoptosis in two human mucoepidermoid carcinoma (MEC) cell lines. The differential response and underlying molecular mechanism of human MEC cells to TW-37 was evaluated by trypan blue exclusion assay, western blotting, 4', 6-diamidino-2-phenylindole staining, annexin V/propidium iodide double staining, analysis of the sub-G1 population, human apoptosis array, and measurements of intracellular reactive oxygen species (ROS). TW-37 decreased cell viability and induced apoptosis in YD-15 cells, but not in MC3 cells. Proteome profiling using a human apoptosis array revealed four candidate proteins and of these, heme oxygenase-1 (HO-1) was mainly related to the differential response to TW-37 of YD-15 and MC3 cells. TW-37 also led to a significant increase in intracellular levels of ROS in YD-15 cells, which is associated with apoptosis induction. The ectopic expression of HO-1 recovered YD-15 cells from TW-37-induced apoptosis by reducing intracellular levels of ROS. The expression of HO-1 was reduced through both transcriptional and post-translational modification during TW-37-mediated apoptosis. We conclude that HO-1 is a potential indicator to estimate response to TW37-induced apoptosis in human MEC.

Purpose: Unique cells characterized by multipotency, self-renewal, and high tumorigenic potential have been recently discovered in mucoepidermoid carcinomas. These cells are defined by high aldehyde dehydrogenase activity and high CD44 expression (ALDH high CD44 high ) and function as cancer stem cells (CSC). It has been recently shown that p53 regulates cell differentiation, suggesting that induction of p53 by therapeutic blockade of the MDM2-p53 interaction may constitute a novel strategy to ablate CSCs. Here, we evaluated the effect of a small-molecule inhibitor of MDM2-p53 interaction (MI-773) on the fraction of CSCs in mucoepidermoid carcinoma., Experimental Design: Human mucoepidermoid carcinoma cells (UM-HMC-1,-3A,-3B) were used to assess the effect of MI-773 on cell survival, cell cycle, fraction of CSCs, and expression of p53, p21, MDM2, and Bmi-1 (key regulator of self-renewal). Mice bearing xenograft tumors generated with these mucoepidermoid carcinoma cells were treated with MI-773 to determine the effect of MDM2-p53 inhibition on CSCs in vivo ., Results: MDM2 is highly expressed in human mucoepidermoid carcinoma tissues. MI-773 induced expression of p53 and its downstream targets p21 and MDM2, caused G 1 cell-cycle arrest, and induced mucoepidermoid carcinoma tumor cell apoptosis in vitro . Importantly, a marked decrease in expression of Bmi-1 and in the fraction of ALDH high CD44 high (CSCs) was caused by MI-773 in vitro and in mice harboring mucoepidermoid carcinoma xenografts., Conclusions: Collectively, these data demonstrate that MI-773 reduces the fraction of CSCs, suggesting that patients with mucoepidermoid carcinoma might benefit from therapeutic inhibition of the MDM2-p53 interaction., (©2018 American Association for Cancer Research.)

Salivary gland tumors (SGTs) are rare tumors of the head and neck with different clinical behavior. Preoperative diagnosis, based on instrumental and cytologic examinations, is crucial for their correct management. The identification of molecular markers might improve the accuracy of pre-surgical diagnosis helping to plan the proper treatment especially when a definitive diagnosis based only on cytomorphology cannot be achieved. miRNAs appear to be new promising biomarkers in the diagnosis and prognosis of cancer. Studies concerning the useful of miRNA expression in clinical decision-making regarding SGTs remain limited and controversial.The expression of a panel of 798 miRNAs was investigated using Nanostring technology in 14 patients with malignant SGTs (6 mucoepidermoid carcinomas, 4 adenoid cystic carcinomas, 1 acinic cell carcinoma, 1 ductal carcinoma, 1 cystadenocarcinoma and 1 adenocarcinoma) and in 10 patients with benign SGTs (pleomorphic adenomas). The DNA Intelligent Analysis (DIANA)-miRPath v3.0 software was used to determinate the miRNA regulatory roles and to identify the controlled significant Kyoto Encyclopedia of Genes and Genomes (KEGG) molecular pathways. Forty six miRNAs were differentially expressed (False Discovery Rate-FDR<0.05) between malignant and benign SGTs. DIANA miRPath software revealed enriched pathways involved in cancer processes as well as tumorigenesis, cell proliferation, cell growth and survival, tumor suppressor expression, angiogenesis and tumor progression. Interestingly, clustering analysis showed that this signature of 46 miRNAs is able to differentiate the two analyzed groups. We found a correlation between histological diagnosis (benign or malignant) and miRNA expression profile.The molecular signature identified in this study might become an important preoperative diagnostic tool., Competing Interests: The authors have declared that no competing interests exist.

Objective: Sodium butyrate (NaBu) is a histone deacetylase inhibitor that possesses an apoptotic ability. However, the molecular mechanism by which NaBu induces apoptosis in human oral mucoepidermoid carcinoma (MEC), a type of salivary gland tumor, remains unclear., Materials and Methods: The anticancer effects of NaBu and its related molecular mechanisms were determined by trypan blue exclusion assay, 4'-6-diamidino-2-phenylindole staining, live/dead assay, human apoptosis array, RT-PCR, western blotting, immunocytochemistry, preparation of nuclear fractions, and nude mice tumor xenograft., Results: In this study, we found that NaBu inhibited growth and induced apoptosis in the human oral MEC cell lines MC3 and YD15 with acetylation of histone proteins H2A and H3. NaBu apparently down-regulated survivin protein, as evidenced by the results of the human apoptosis antibody array, and modulated it at the post-translational process. Interestingly, NaBu caused nuclear translocation of survivin protein in both cell lines. NaBu also resulted in decreased expression levels of Bcl-xL mRNA and protein, leading to induction of caspase-dependent apoptosis in human oral MEC cell lines. In addition, NaBu administration inhibited tumor growth in vivo at a dosage of 500 mg/kg/day, but it did not cause any hepatic or renal toxicity., Conclusion: This study provides new insights into the molecular mechanism of apoptotic actions by NaBu in human oral MEC and the basis of its clinical application for the treatment of human oral MEC., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Gefitinib is well-known as a tyrosine kinase inhibitor targeting non-smalllung- cancer (NSCLC) containing EGFR mutations. However, its effectiveness in treating mucoepidermoid carcinoma (MEC) without such EGFR mutations suggests additional targets., Objective: The CRTC1-MAML2 (C1-M2) fusion typical for MEC has been proposed to be a gefitinib target., Methods: To test this hypothesis, we developed a set of siRNAs to down-regulate C1-M2 expression. RNA-seq and Western blot techniques were applied to analyze the effects of gefitinib and siC1-M2 on the transcriptome of and the phosphorylation of tyrosine kinases in a MEC cell line H292., Results: Deep-sequencing transcriptome analysis revealed that gefitinib extensively inhibited transcription of genes in JAK-STAT and MAPK/ERK pathways. Both siC1-M2 and gefitinib inhibited the phosphorylation of multiple signaling kinases in these signaling pathways, indicating that gefitinib inhibited JAK-STAT and MAPK/ERK pathways activated by C1-M2 fusion. Moreover, gefitinib inhibition of EGFR and MAPK/ERK was more effective than that of AKT, JAK2 and STATs, and their dependence on C1-M2 could be uncoupled. Taken together, our results suggest that gefitinib simultaneously represses phosphorylation of multiple key signaling proteins which are activated in MEC, in part by C1-M2 fusion. Gefitinib-repressed kinase phosphorylation explains the transcriptional repression of genes in JAK-STAT and MAPK/ERK pathways., Conclusion: These findings provide new insights into the efficacy of gefitinib in treating mucoepidermoid carcinoma, and suggest that a combination of gefitinib and other inhibitors specifically against C1-M2 fusion could be more effective., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Salivary gland cancers represent a rare group of tumors composed by over 20 histological subtypes. Initially treated as one single disease, its diagnosis, prognosis, and treatment are currently being stratified based on morphology. More recently, insight has been provided on the molecular characterization of each subtype, further improving diagnostic accuracy and paving the way for personalized therapy. In this article, we provide a comprehensive review of recent breakthroughs, preliminary results of novel therapy, and future directions on the treatment of these complex malignancies., (© 2018 Wiley Periodicals, Inc.)

Objective: Intratumoral hypoxia (IH) occurs during cellular proliferation of malignant tumors. This phenomenon is characterized by a decrease in oxygen levels in the neoplastic microenvironment. Throughout this condition, the proteins HIF-1α, NOTCH1, ADAM12, and HB-EGF can be activated, triggering signaling pathways associated with tumor invasiveness through invadopodia formation. This study aimed to evaluate the immunostaining of HIF-1α, NOTCH1, ADAM12, and HBEGF in 19 cases of mucoepidermoid carcinoma (MEC) and 10 samples of salivary glands (control group)., Study Design: The immunoperoxidase technique was employed to detect the proteins of interest. The Student t test was used to compare immunoexpression between MEC samples and the control group., Results: Protein immunostaining was statistically significantly higher in MEC samples than in the control group (P < .01), and the proteins were especially overexpressed in epidermoid cells of MEC., Conclusions: We suggest that there is an association between the NOTCH1 signaling pathway activated by IH and the biologic behavior of MEC., (Copyright © 2019. Published by Elsevier Inc.)

Mucoepidermoid carcinoma (MEC) of salivary gland is a disease characterized by high rate of diatant metastasis, and associated with poor outcomes. However, the molecular mechanisms underlying the MEC remain poorly understand. Here, we simultaneously detected, for the first time, the expression profiles of mRNAs, lncRNAs, and circRNAs in four pairs of MEC and matched non-carcinoma tissues by microarrays. A total of 3612 mRNA, 3091 lncRNAs, and 284 circRNAs were altered during the pathogenesis of MEC. The functions of these differentially expressed RNAs were predicted by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Co-expression networks of lncRNA-mRNA and circRNA-miRNA were conducted to uncovered the hidden ceRNA mechanisms. Moreover, NONHSAT154433.1 that associated with ADAM12 and hsa&#95;circ&#95;0012342 were further screened and confirmed using qRT-PCR analysis. In conclusion, this study provides a systematic perspective on the potential function of non-coding RNAs (ncRNAs) in the molecular mechanisms of MEC. Among these, NONHSAT154433.1 and hsa&#95;circ&#95;0012342 might be served as potential prognostic biomarkers and therapeutic target of MEC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: Salivary gland tumors are complex and have a great histomorphological diversity; more than 30 histological subtypes are currently described and the study of proteins that help understand and differentiate these tumors is essential. We aimed to analyze the immunoexpression of cyclooxygenase-2 (COX-2) and cyclin D1 proteins in pleomorphic adenomas (PA), mucoepidermoid carcinomas (MEC) and adenoid cystic carcinomas (AdCC) of salivary glands., Methods: A total of 38 PA, 12 AdCC and 12 MEC underwent immunohistochemical study by the polymeric biotin-free technique. Immunopositive cells were analyzed semi-quantitatively. For statistical analysis, a significance level was set at p ≤ 0.05., Results: Overall, these tumors were more prevalent in women (n = 37). The mean age of these patients was 58-year-old and the parotid gland was the most affected anatomic site (n = 33). All cases of AdCC and MEC showed immunopositivity to cyclin D1; however, 39.5% of the PAs were negative (p < 0.001). Regarding COX-2 immunoexpression, we observed that all cases of CME were positive, whereas 60.5% of the PA and 75% of the CAC analyzed were completely negative (p = 0.042)., Conclusions: The overexpression of COX-2, observed only in MEC, emphasizes that salivary gland tumors have different profiles. Cyclin D1 is more immunoexpressed in malignant tumors. Together, these immunohistochemical findings may be useful in differentiating the studied tumors.

Recently, it has been reported that deregulation of the receptor activator of NFkB ligand (RANKL)/RANK signaling axis results in salivary gland tumor development in a mouse transgenic model. The aim of this study was to ascertain RANKL and RANK protein expression in a series of primary parotid gland carcinomas and to correlate it with clinicopathologic parameters. Formalin-fixed paraffin-embedded tumor samples from 46 consecutive cases of parotid gland carcinoma were selected for this study. For comparison, we examined a group of 40 randomly chosen parotid gland adenomas, including 20 pleomorphic adenomas, 10 myoepitheliomas, and 10 Warthin tumors. Immunohistochemical analysis for RANK and RANKL was conducted on tissue microarrays. Overall, 33 carcinomas (71.7%) were scored as positive for RANK and 25 (54.3%) for RANKL. The expression of both RANK and RANKL was significantly higher in carcinomas than in adenomas as only 6 (15%) adenomas were positive for RANK, and RANKL was negative in all benign tumors (P<0.001 for both, Fisher exact test). Some histologic types, including salivary duct carcinoma, mucoepidermoid carcinoma, and carcinoma ex-pleomorphic adenoma presented a high frequency of RANK and RANKL expression. No significant correlation was observed between RANK/RANKL expression and clinical parameters. Our study indicates that the expression of RANK and RANKL in parotid gland neoplasms is associated with the acquisition of a malignant phenotype and this pathway may represent an attractive therapeutic target in patients with parotid gland carcinomas.

Purpose: MDM2 inhibitors are promising anticancer agents that induce cell cycle arrest and tumor cells death via p53 reactivation. We examined the influence of Mycoplasma hyorhinis infection on sensitivity of human lung carcinoma cells NCI-H292 to MDM2 inhibitor Nutlin-3. In order to unveil possible mechanisms underlying the revealed effect, we investigated gene expression changes and signal transduction networks activated in NCI-H292 cells in response to mycoplasma infection., Methods: Sensitivity of NCI-Н292 cells to Nutlin-3 was estimated by resazurin-based cell viability assay. Genome-wide transcriptional profiles of NCI-H292 and NCI-Н292 Myc.h cell lines were determined using Illumina Human HT-12 v3 Expression BeadChip. Search for key transcription factors and key node molecules was performed using the geneXplain platform. Ability for anchorage-independent growth was tested by soft agar colony formation assay., Results: NCI-Н292 Myc.h cells were shown to be 1.5- and 5.2-fold more resistant to killing by Nutlin-3 at concentrations of 15 and 30 µM than uninfected NCI-Н292 cells (P < 0.05 and P < 0.001, respectively). Transcriptome analysis revealed differential expression of multiple genes involved in cancer progression and metastasis as well as epithelial-mesenchymal transition (EMT). Moreover, we have shown experimentally that NCI-Н292 Myc.h cells were more capable of growing and dividing without binding to a substrate. The most likely mechanism explaining the observed changes was found to be TLR4- and IL-1b-mediated activation of NF-κB pathway., Conclusions: Our results provide evidence that mycoplasma infection is an important factor modulating the effect of MDM2 inhibitors on cancer cells and is able to induce EMT-related changes.

Cationic protein is a cytotoxic protein secreted by eosinophils and takes part in the damage of airway epithelium in asthma. Poly-L-arginine (PLA), a synthetic cationic protein, is widely used to mimic the biological function of the natural cationic protein in vitro. Previous studies demonstrated the damage of the airway epithelial cells by cationic protein, but the molecular mechanism is unclear. The purpose of this study aimed at exploring whether PLA could induce apoptosis of human airway epithelial cells (NCI-H292) and the underlying mechanism. Methods . The morphology of apoptotic cells was observed by transmission electron microscopy. The rate of apoptosis was analyzed by flow cytometry (FCM). The expressions of the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Bcl-2/Bax, and cleaved caspase-3 were assessed by western blot. Results . PLA can induce apoptosis in NCI-H292 cells in a concentration-dependent manner. Moreover, the phosphorylation of the ERK1/2 and the unbalance of Bcl2/Bax, as well as the activation of caspase-3, were involved in the PLA-induced apoptosis. Conclusions . PLA can induce the apoptosis in NCI-H292 cells, and this process at least involved the ERK1/2 and mitochondrial pathway. The results could have some indications in revealing the apoptotic damage of the airway epithelial cells. Besides, inhibition of cationic protein-induced apoptotic death in airway epithelial cells could be considered as a potential target of anti-injury or antiremodeling in asthmatics.

Hyalinizing clear cell carcinoma of the bronchial glands is a very rare tumor. Since only five reports describing six tumors have been published to date, only a little is known about specific histologic findings and clinical features. Because of its rarity, hyalinizing clear cell carcinoma has not been described in the latest WHO classification of pulmonary tumors yet. Here we present three cases of bronchial hyalinizing clear cell carcinomas, confirmed by both fluorescence in situ hybridization (FISH) and RT-PCR, focusing on histologic and immunohistochemical characteristics in a comparison with three cases of salivary gland origin. In addition, we compared immunohistochemical features with bronchial mucoepidermoid carcinoma, a lesion that needs to be taken into account in differential diagnosis of hyalinizing clear cell carcinoma. All our bronchial hyalinizing clear cell carcinoma cases were surgically resected. Histologically, tumor cells showed clear to eosinophilic cytoplasm with hyalinizing stroma in various proportions, resembling those of salivary gland origin. Immunohistochemically, tumor cells were positive for CK7, CK5/6, p40, p63, and ATF1, while they were negative for TTF1, Napsin A, HMB45, and SOX10. The CK5/6 staining pattern varied in mucoepidermoid carcinomas, while that of hyalinizing clear cell carcinoma was uniformly positive. FISH revealed EWSR1-ATF1 fusion, and RT-PCR with sequencing confirmed specificity of the chimeric gene for hyalinizing clear cell carcinoma. Clinically, bronchial hyalinizing clear cell carcinoma was characterized by occurrence in the fourth to sixth decades, no link with smoking history, and a predilection for the right lung, in line with previous reports. In summary, our study confirmed that the bronchial hyalinizing clear cell carcinoma is a histologically and genetically identical tumor to that of salivary gland origin, and that gene rearrangement analysis can play a critical role in distinction from mucoepidermoid carcinoma.

Purpose: To investigate the effects of DNA methyltransferase in mucoepidermoid carcinoma of human salivary glands tissues., Methods: Forty-three samples from mucoepidermoid carcinoma of salivary glands and 17 normal salivary gland tissues were collected from January 2010 to September 2013. Immunohistochemistry and Western blot were used to detect the expression of Dnmt1 and Dnmt3b in normal tissues and specimen of mucoepidermoid carcinoma of salivary glands. The data were analysed with SPSS 22.0 software package., Results: The positive expression rate of Dnmt1 in mucoepidermoid carcinoma tissue was 37.21%, and that in normal salivary gland tissues was 17.65%, there was no significant difference between them; the positive expression rate of Dnmt3b in mucoepidermoid carcinoma tissue was 83.72%, which was significantly higher than that in normal salivary gland tissue (11.76%, P<0.01). However, there was no significant correlation between high expression of Dnmt1 and Dnmt3b and clinicopathological parameters., Conclusions: Dnmt3b may play a role in the tumorigenesis of mucoepidermoid carcinoma in salivary glands.

Background: Cancer stem cells (CSCs) may participate in angiogenesis by lining the wall of tumor vessels., Aim: The current study aimed to present the role of vimentin and CD44 in inducing vasculogenic mimicry (VM) and epithelial-mesenchymal transition (EMT) in different grades of mucoepidermoid carcinoma (MEC)., Materials and Methods: A total of 63 MEC samples were collected from the archive of Department of Pathology of Taleghani Educational Hospital, Tehran, Iran. Vimentin and CD44/periodic acid-Schiff double staining was performed., Statistical Analysis: Chi-square test was used to examine the differences with categorical variables. Significance level was set at 0.05. Pearson's correlation coefficient was used to assess the colocalization of the markers., Results: There were statistically significant differences between tumor grade and the expression levels of vimentin and CD44 (P = 0.000)., Conclusion: Our results may disclose a definite relationship between microvessl density (MVD), VM, EMT, and CSCs in MEC samples. Thus, it is reasonable to suggest that CSCs are related to angiogenesis and VM., Competing Interests: There are no conflicts of interest

Potentilla discolor has been used in traditional Chinese medicine for the treatment of hyperglycemia. However, the potential role of Potentilla discolor against cancer and its mode of action remain to be fully elucidated. The present study explored the apoptotic effect of methanol extract of Potentilla discolor (MEPD) in human mucoepidermoid carcinoma (MEC) cell lines of salivary glands. MEPD markedly suppressed the growth and induced apoptotic cell death in MC3 and YD15 cells. MEPD treatment significantly upregulated the expression of PUMA and reduced STAT3 phosphorylation. Overexpression of STAT3 partially recovered the growth of MEC cells inhibited by MEPD. In addition, dephosphorylation of STAT3 by cryptotanshinone (a potent STAT3 inhibitor) was sufficient to inhibit the growth of MEC cells and induce apoptosis via affecting PUMA protein. These results suggest that MEPD has a potential anticancer property via the STAT3/PUMA signaling axis in human MEC cells of salivary gland.