Immunohistochemical expression of the cation channel TRPC6 in the submandibular and lacrimal gland and in salivary gland tumors.

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.

Differential expression of epidermal growth factor receptor in various pathological types of salivary gland cancers.

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.

PD-L1 and PD-L2 Expression in Different Tumor Stages and Types of Malignant Salivary Gland Neoplasms: A Single-center Experience.

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.

Differential Expression of Mucin in Salivary Gland Tumours.

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.

TGFß signaling pathway in salivary gland tumors.

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 TGFß signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFß signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFß 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 TGFß 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 TGFß 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.

Identifying potential immuno-oncology targets in salivary gland mucoepidermoid carcinoma based on inflammatory status and treatment response.

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.

Immunohistochemical Expression Of Human Epidermal Growth Factor Receptor-2 (Her-2) In Common Salivary Gland Carcinomas.

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.

Immunohistochemical comparative analysis of tumor stem cell biomarkers in pleomorphic adenoma, adenoid cystic carcinoma and mucoepidermoid carcinoma of salivary glands.

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.

Analysis of Human Papilloma Virus Content and Integration in Mucoepidermoid Carcinoma.

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, 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.

p53 Inhibits Bmi-1-driven Self-Renewal and Defines Salivary Gland Cancer Stemness.

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.

CD138 Is Expressed in Different Entities of Salivary Gland Cancer and Their Lymph Node Metastases and Therefore Represents a Potential Therapeutic Target.

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.

Evaluation of HER2/neu expression in different types of salivary gland tumors: a systematic review and meta-analysis.

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.

Expression of HHLA2, TMIGD2, and GITR in salivary gland adenoid cystic carcinoma and mucoepidermoid carcinoma.

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 Erk1/2 , the epithelial-mesenchymal transition (EMT) molecule transforming growth factor ß1, 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 ß1, 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 ß1, phosphorylated Erk1/2, and cluster of differentiation 8.

Prognostic Significance of Cancer Stem Cell Markers in Patients With Salivary Gland Carcinomas.

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.

Cephaeline is an inductor of histone H3 acetylation and inhibitor of mucoepidermoid carcinoma cancer stem cells.

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.

Short communication: Distribution of phospholipids in parotid cancer by matrix-assisted laser desorption/ionization imaging mass spectrometry.

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.

Cytosolic 5'-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway.

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.

Mucoacinar Carcinoma: A Rare Variant of Mucoepidermoid Carcinoma.

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.

CRTC1/MAML2 directs a PGC-1α-IGF-1 circuit that confers vulnerability to PPARγ inhibition.

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.

The potential of somatostatin receptor 2 as a novel therapeutic target in salivary gland malignant tumors.

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 68Ga-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 68Ga-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.