Adenoid Cystic Carcinoma Tissues Research Articles

Anti-NOTCH1 therapy with OMP-52 M51 inhibits salivary adenoid cystic carcinoma by depressing epithelial-mesenchymal transition (EMT) process and inducing ferroptosis

Salivary adenoid cystic carcinoma (ACC) is a common type of salivary gland cancer, and the mechanisms underlying its progression still remain poorly understood without efficient therapies. NOTCH1, an evolutionally conserved cell-cell signaling pathway, is involved in the progression of ACC. In our study, we attempted to explore whether NOTCH1 suppression using the monoclonal anti-NOTCH1 antibody OMP-52 M51 could be of potential for ACC treatment. Here, we identified NOTCH1 elevation in human ACC tissues compared with the matched normal samples. Patients with metastasis expressed much higher NOTCH1. We then found that OMP-52 M51 markedly reduced the expression of NOTCH1 and its intracellular active form NICD1 (NOTCH1 intracellular domain). Importantly, OMP-52 M51 markedly reduced the proliferation, migration and invasion of ACC cells. RNA-Seq and in vitro studies further showed that OMP-52 M51 significantly induced ferroptosis in ACC cells, indicated by the increased cellular malondialdehyde (MDA), iron contents and lipid ROS production, and decreased glutathione (GSH) levels. Further, remarkable glutathione peroxidase 4 (GPX4) reduction was detected in ACC cells with OMP-52 M51 treatment. However, promoting NOTCH1 expression markedly abolished the function of OMP-52 M51 to induce ferroptosis. Intriguingly, low-dose OMP-52 M51 strongly facilitated the capacity of ferroptosis inducer erastin to trigger ferroptotic cell death, revealing that OMP-52 M51 could improve the sensitivity of ACC cells to ferroptosis. In vivo, OMP-52 M51 administration suppressed tumor growth and induced ferroptosis in the constructed ACC xenograft mouse model. Collectively, our findings demonstrated that NOTCH1 inhibition by OMP-52 M51 represses the proliferation and epithelial-mesenchymal transition (EMT) in ACCs, and promotes ferroptosis, revealing the potential therapeutical application of OMP-52 M51 in ACC.

Single-cell RNA sequencing reveals the heterogeneity and microenvironment in one adenoid cystic carcinoma sample

Adenoid cystic carcinoma (ACC) is one of the most common malignancy of the major salivary glands with a high recurrence rate and poor prognosis. Determining tumor heterogeneity and factors in the microenvironment may provide novel therapeutic targets for ACC. We performed single-cell RNA sequencing of one ACC sample and normal salivary gland tissues from a patient to analyze tumor heterogeneity, immunosuppressive landscape, and intercellular communication networks. The heterogeneity of epithelial cells in ACC tissues was significantly higher compared with that in normal tissues, whereas immune cells were almost absent. We found four malignant cell clusters in ACC and explored their characteristics and function. In tumor tissues, CD8 + cytotoxic T cells and CD4 + T helper cells were significantly decreased, whereas IgA + plasma cells were absent. There were two clusters of macrophages, one representing IL1B macrophages and the other consisted of a cluster of macrophages associated with the epithelial mesenchymal transition (EMT). Both were significantly different from the normal tissue composition. In addition, the communication between epithelial cells and other cells in the tumor tissue was enhanced. MIF-CD74 and APP-CD74 were significantly upregulated. We comprehensively described the heterogeneity of ACC and the tumor microenvironment (TME) from a single cell perspective including cell characteristics, immune cell infiltration, and cell communication. CLINICAL RELEVANCE: This study provided further insights into ACC and may lead to new treatment strategies.

Transcriptomic insights into adenoid cystic carcinoma via RNA sequencing.

Background: The aim of this study was to investigate the underlying mechanisms of adenoid cystic carcinoma (ACC) at the transcriptome level. Materials and methods: We obtained paired tumor and normal salivary gland tissues from 15 ACC patients, which were prepared for RNA sequencing. Results: Gene enrichment analysis revealed that the upregulated pathways were mainly involved in axonogenesis, and the downregulated pathways were mainly related to leukocyte migration, the adaptive immune response, lymphocyte-mediated immunity, and the humoral immune response. T-cells, B-cells and NK cells showed low infiltration in ACC tissues. In addition to the gene fusions MYB-NFIB and MYBL1-NFIB, a new gene fusion, TVP23C-CDRT4, was also detected in 3 ACC tissues. PRAME was significantly upregulated in ACC tissues, while antigen-presenting human leukocyte antigen (HLA) genes were downregulated. Conclusion: We found a new gene fusion, TVP23C-CDRT4, that was highly expressed in ACC. PRAME may be an attractive target for ACC immunotherapy.

Expression of Lymphoma-2 Gene and NF-κB in Human Adenoid Cystic Carcinoma Tissue and Its Relation with Disease Prognosis

Background: Adenoid cystic carcinoma (ACC) is a common tumor of the head and neck. It is characterized by slow growth, strong invasiveness, easy recurrence and distant organ metastasis. Bcl-2 and NF-κB are indicated to relate to tumor growth, proliferation and apoptosis. This study aims to analyze the relationship between Bcl-2, NF-κB and ACC. Methods: With ELISA method to detect Bcl-2 and NF-κB expression in the blood of patients with ACC, patients with benign adenoma and healthy controls, and the expression of Bcl-2 and NF-κB in tissues was detected by immunohistochemistry, and their relationship with ACC was analyzed. relevance. Relationships between clinicopathological features. Results: The levels of Bcl-2 (4.16±1.34 ng/ml) and NF-κB (4.47 ± 1.37 ng/ml) as well as positive rates of Bcl-2 and NF-κB (87.5% and 85%) were significantly elevated in the ACC group (P <0.05). Bcl-2 was positively correlated with NF-κB (P <0.05). Of note, Bcl-2 and NF-κB expressions were correlated with the incidence frequency, histological type, clinical stage, lymph node metastasis and distant metastasis of ACC patients (P < 0.05) as the distant metastasis is an independent risk factor. Conclusion: In summary, Bcl-2 and NF-κB expression in ACC patients is associated with the clinicopathological features of ACC.

Functional profiles of curatively treated adenoid cystic carcinoma unveil prognostic features and potentially targetable pathways

Adenoid cystic carcinoma (ACC) of salivary gland is a slowly growing tumor showing a propensity for delayed recurrence, with decreased survival rates. The identification of poor prognosis patients may help in defining molecular-based targeted strategies in this rare disease orphan of new treatments. Through a gene expression microarray-based approach followed by GSE functional analysis the expression profile of 46 primary untreated ACC samples and of ACC (h-TERT) tumor cells was analyzed. Patients who experienced early relapse showed enrichment in proliferation-related gene sets, including the G2-M checkpoint, E2F and myc targets, and in gene sets related to IFN signaling and aberrant proteostasis (FDR < 0.1), indicating increased mitotic and transcriptional activity in aggressive ACC. Similar functions were enriched in ACC samples classified by immunohistochemical staining as p63-negative, which exhibited increased protein burden and activation of pro-survival stress response pathways compared to p63-positive tumors. Compared to ACC tissues, ACC (h-TERT) cells share transcriptional features of aggressive p63-negative tumors. These data suggest association of specific pathway alterations with histopathological features of ACC, as recapitulated by p63 testing in patient prognostic stratification, anticipating new avenues for therapeutic intervention.

The expression and significance of vascular endothelial growth factor A in adenoid cystic carcinoma of palatal salivary gland.

To explore the VEGF-A expression in salivary gland adenoid cystic carcinoma tissues and detect the relationship between the mechanism of occurrence, development and metastasis of jaws with salivary gland adenoid cystic carcinoma and VEGF-A expression. Paraffin samples from 58 cases of SACC of the palate and ten cases of normal salivary gland tissues were collected. The expression levels of VEGF-A protein were detected using the immunohistochemistry EnVision system. Among the 58 cases, there were 20 cases of the cribriform type, 17 cases of the tubular type, and 21 cases of the solid type. There were 9 cases with lymph node metastasis and 21 cases without lymph node metastasis. And there were 8 cases of T1, 15 cases of T2, and 7 cases of T3/T4. The positive expression rate of VEGF-A in SACC of the palate was 74.1%, which was higher than that found in normal salivary gland tissues (10%). The VEGF-A was localized in the cytoplasm/cell membrane. VEGF-A is highly expressed in SACC of the palate. The level of expression is closely related to the pathological grade, lymph node metastasis, and clinical stage of the tumor, and it can thus be used as an important indicator for judging the biological behavior of SACC of the palate.

Proteomic analysis reveals the heterogeneity of metabolic reprogramming in lacrimal gland tumors

Lacrimal gland adenoid cystic carcinoma (ACC) is associated with high recurrence and mortality rates. Many recent studies have focused on the clinical features of the disease, and a better understanding of its underlying molecular mechanisms may help guide future treatment strategies. For proteomics quantitation, we analyzed normal tissues, benign tumor tissues and ACC tissues by LC-MS/MS with Tandem mass tags (TMTs) labeling. Bioinformatics analysis of the KEGG pathway found that, compared with normal tissues, the expression levels of major proteins related to cell metabolism were lower in benign tumors and cancer tissues of the lacrimal gland. In addition, we also performed IHC staining to verify the expression of representative proteins in tissue samples. All of these results indicated that compared with normal tissues, lacrimal gland tumors had unique metabolic reprogramming characteristics. Further Short Time-series Expression Miner (STEM) analysis revealed that glycine, serine and threonine metabolism in ACC tissues was significantly enhanced compared with that in normal tissues and benign tumor tissues. This finding suggested that glycine, serine and threonine metabolism might be the key to the malignant transformation of ACC; thus, assessing the metabolism in these tissues could be an effective approach enabling the early diagnosis of ACC, and the proteins involved in these metabolic pathways could represent therapeutic targets.

Antizyme inhibitor 2 (AZIN2) associates with better prognosis of head and neck minor salivary gland adenoid cystic carcinoma.

The key regulator of the polyamine biosynthetic pathway is ornithine decarboxylase (ODC). ODC is activated by antizyme inhibitor 1 (AZIN1) and 2 (AZIN2). AZIN1 and recently AZIN2 have been related to cancer; however, their functions in adenoid cystic carcinoma (ACC) have not been studied. We performed immunohistochemical study on minor salivary and mucous gland ACC tissue samples of patients treated at the Helsinki University Hospital (Helsinki, Finland) during 1974-2012. We scored AZIN1 and 2 immunoexpression in 42 and 45 tumor tissue samples, respectively, and correlated them with clinicopathological factors and survival. Enhanced AZIN2 expression was associated with better survival. In addition, both AZINs were seen more commonly in cribriform and tubular than in solid growth patterns. AZIN1 expression did not correlate with the studied clinicopathological factors. It seems that AZIN2 expression is higher in cancer tissue with secretory functions. In ACC tissue, high AZIN2 expression could be related to well-differentiated histological type which still has a functioning vesicle transportation system. Thus, AZIN2 could be a prognostic factor for better survival of ACC patients.

The NOTCH1-HEY1 pathway regulates self-renewal and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells.

Our previous study demonstrated a close relationship between the NOTCH signaling pathway and salivary adenoid cystic carcinoma (SACC). Its receptor gene, NOTCH1, and its downstream gene, HES1, contribute to the proliferation, invasion and metastasis of SACC. Accumulating evidence supports HEY1 as another effector of the signaling pathway. The purpose of this study was to explore the effects of the NOTCH1-HEY1 pathway on the proliferation, invasion and metastasis of SACC cells. Our results verified that HEY1 is a specific molecular target of the NOTCH signaling pathway in SACC cells and that its expression in carcinoma is much higher than that in paracarcinoma tissues. The expression of NOTCH1 and HEY1 are positively correlated in the salivary adenoid cystic carcinoma tissues. NOTCH1 is significantly related to the activation of HEY1 in SACC, and that HEY1 reciprocally regulates NOTCH1 expression in SACC. HEY1 promotes cell proliferation and spheroid formation and inhibits cell apoptosis in vitro. In addition, HEY1 enhances the tumorigenicity of SACC in vivo. Furthermore, HEY1 increases cell invasion and metastasis by driving the expression of epithelial-mesenchymal transition (EMT)-related genes and MMPs. The results of this study indicate that the NOTCH1-HEY1 pathway is specifically upregulated in SACC and promotes cell proliferation, self-renewal, invasion, metastasis and the expression of EMT-related genes and MMPs. Our findings suggest that a NOTCH1-HEY1 pathway inhibitor might therefore have potential therapeutic applications in treating SACC patients by inhibiting cancer cell growth and metastasis.

Accumulated cytotoxicity of CDK inhibitor dinaciclib with first-line chemotherapy drugs in salivary adenoid cystic carcinoma cells.

Adenoid cystic carcinoma (ACC) is one of the most common salivary gland malignant tumors. Its treatment failure is partly due to the limitations of chemotherapeutic agents and their adverse effects. The objective of this study was to determine the potential additive anti-cancer effect of a novel CDK inhibitor dinaciclib with first-line chemotherapy drugs in ACC. Protein expression of phosphorylated CDK2 (p-CDK2) in paraffin-embedded tissue specimens of ACC from 17 patients was investigated by immunohistochemistry (IHC). Cell Counting Kit (CCK-8), clone formation assay, and flow cytometry were used to test the proliferation and apoptosis of ACC-2 cells treated with dinaciclib with or without other first-line chemotherapy drugs. Protein expression was also determined by Western blot. Interestingly, we discovered that p-CDK2 protein was expressed in both cytoplasmic and nucleus in salivary ACC tissues, which was higher than that in normal salivary tissues, indicating that agents targeting CDK2 may be potential therapeutic strategies against this type of tumor. As expected, CDK inhibitor dinaciclib significantly induced ACC-2 cells apoptosis. Moreover, it sensitized cells to the chemotherapeutic agents such as cisplatin, pemetrexed, and etoposide (VP-16), and this effect by dinaciclib may induce cell cycle arrest via abrogating CDK2 activity. Therefore, combinational therapy of CDK inhibitor dinaciclib with first-line chemotherapy drugs may be a promising strategy in the treatment of salivary ACC.

MYB-NFIB gene fusions identified in archival adenoid cystic carcinoma tissue employing NanoString analysis: an exploratory study

BackgroundAdenoid cystic carcinoma (ACC) is a slow growing salivary gland malignancy that is molecularly characterized by t(6:9)(q22–23;p23–24) translocations which predominantly result in MYB-NFIB gene fusions in nearly half of tumours. Detection of MYB-NFIB transcripts is typically performed with fresh ACC tissue using conventional RT-PCR fragment analysis or FISH techniques, which are prone to failure when only archival formalin fixed paraffin embedded (FFPE) tissue is available. The purpose of this pilot study was to evaluate the utility of NanoString probe technology for the detection of MYB-NFIB transcripts in archival ACC tissue.MethodsA NanoString probeset panel was designed targeting the junctions of three currently annotated MYB-NFIB fusion genes as well as 5′/3′ MYB probesets designed to detect MYB gene expression imbalance. RNA isolated from twenty-five archival ACC specimens was profiled and analyzed. RT-qPCR and sequencing were performed to confirm NanoString results. MYB protein expression was analyzed by immunohistochemistry.ResultsOf the 25 samples analyzed, 11/25 (44%) expressed a high degree of MYB 5′/3′ imbalance and five of these samples were positive for at least one specific MYB-NFIB variant in our panel. MYB-NFIB variant detection on NanoString analysis was confirmed by direct cDNA sequencing. No clinical correlations were found to be associated with MYB fusion status.ConclusionWe conclude that the application of NanoString digital probe counting technology is well suited for the detection and quantification of MYB-NFIB fusion transcripts in archival ACC specimens.

PFKFB3促进腺样囊性癌ACC-2细胞增殖、迁移、侵袭和转移

Objective To evaluate the expression of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) in adenoid cystic carcinoma and the effects of PFKFB3 on the proliferation, migration, invasion and tumorigenesis in vivo of adenoid cystic carcinoma cells. Methods Immunohistochemistry and Western blotting were applied to detect the expression of PFKFB3 in 29 cases of adenoid cystic carcinoma tissues and 10 cases of para-carcinoma normal salivary gland tissues. PFKFB3 in adenoid cystic carcinoma ACC-2 cells was silenced by adenovirus vector. The effects of PFKFB3 silence on cell proliferation, migration, invasion and tumorigenesis in vivo and distant metastasis of adenoid cystic carcinoma cells were evaluated by cell proliferation assay, wound healing assay, Transwell assay, xenografted mice model and lung metastasis mice model. Results The results of immunohistochemical staining showed that the positive expression rate of PFKFB3 in adenoid cystic carcinoma tissues was 93.1% (27/29), and the positive expression rate of PFKFB3 in normal salivary gland tissues was 20.0% (2/10), with a significant difference (χ2=20.84, P<0.001). The results of methyl thiazolyl tetrazolium (MTT) assay showed that, compared with ACC-2 group, the proliferation activity of cancer cells with silence of PFKFB3 (PFKFB3--ACC-2) was significantly suppressed for 72 h (1.8±0.2 vs. 4.7±0.8, t=3.582, P=0.001). The results of wound healing assay showed that after scraping cells away for 24 h, the number of cells in the scratch area was 99.8±13.2 in the PFKFB3--ACC-2 group, which was significantly less than that in the ACC-2 group (263.0±97.4, t=2.868, P=0.029). Transwell results indicated that the number of cells passing through matrigel was 17.6±2.1 in the PFKFB3--ACC-2 group, which was less than that in the ACC-2 group (28.6±3.8, t=4.452, P=0.004). The tumor volume in the PFKFB3--ACC-2 [(623.5±134.1) mm3] was smaller than that in the ACC-2 group [(1 621.0±278.1) mm3,t=4.213, P=0.001]. More pulmonary metastases were found in the ACC-2 group than PFKFB3--ACC-2 group (18.1±3.2 vs. 4.1±2.2, t=6.322, P=0.001). Conclusion The expression of PFKFB3 is higher in adenoid cystic carcinoma tissue than normal salivary gland tissue, and the highly expressed PFKFB3 plays a driving role in the proliferation, migration, invasion and metastasis in adenoid cystic carcinoma. Key words: Carcinoma, adenoid cystic; Glycolysis; PFKFB3; Tumor development

Abstract 778: Potential drug targets for adenoid cystic carcinoma elucidated by proteogenomic analysis

Abstract Background: Adenoid cystic carcinoma (ACC) is a rare cancer of secretory glands accounting for 10% of salivary gland malignancies and 1% of head and neck cancers. About 1,200 new cases are diagnosed annually in the United States. ACC is typically chemoresistant and clinical trials of multiple targeted agents found few responders. Studies of ACC oncogenesis have described frequent fusion of MYB-NFIB genes and other infrequent genomic mutations. ACC has not been proteomically characterized. We hypothesized that proteogenomic analysis of ACC tumor tissues would identify clinically relevant molecular differences between ACC and other tumor types. The objective of this study was to identify altered disease pathways and potential drug targets in ACC. Methods: Clinical tumor samples of ACC and squamous cell carcinoma (SCC) of the head and neck were analyzed using the GPS Cancer diagnostic test, which includes whole genome and transcriptome sequencing, and proteomic expression analysis using mass spectrometry. To quantify proteins, tumor areas of formalin-fixed paraffin-embedded tissue sections were marked by a pathologist, microdissected and solubilized. The resulting lysate was analyzed with mass spectrometry to quantitate 30 clinically relevant proteins. Samples were further analyzed with a global proteomics platform intended to discover targetable protein biomarkers. Proteins that were overexpressed or underexpressed (defined as a 1.5-fold difference between ACC and SCC) were subjected to pathway analysis to identify perturbed pathways and potential drug targets. Finally, the mRNA expression in ACC tumors was compared with RNA-seq data from various solid tumor types using the k-nearest neighbors algorithm. Results: In 14 tumor samples, unsupervised hierarchical clustering analysis of 4,002 proteins revealed a clear separation between ACC (n=8) and SCC (n=6) tissues. Four of 8 ACC samples harbored an MYB-NFIB fusion and single samples had fusions of MYBL-NFIB or AHI1-NFIB. Mutational burden in ACC and SCC samples was 1.35 and 3.53 mutations per megabase, respectively. Pathway analysis found enrichment of ACC genes in essential cell cycle processes. CDK6 protein expression was 4-fold higher in ACC samples than SCC samples by mass spectrometry (p=0.0036) and 3-fold higher at the mRNA level. Expression of p16 protein was 3-fold lower in ACC than in SCC (p=0.0289) and corresponding p16 mRNA levels were 17-fold lower in ACC than SCC. All ACC samples harbored intact retinoblastoma (RB1) gene. Expression of mRNA in 15 ACC samples was compared with that of 25 different tumor types from TCGA and the authors' clinical laboratory; breast cancer was found to be the nearest neighbor to ACC. Conclusions: Proteogenomic analysis revealed CDK6 overexpression in ACC. The combination of CDK6 overexpression, p16 underexpression and RB1 proficiency suggests that ACC tumors may respond to treatment with CDK6 inhibitors. Citation Format: Sheeno P. Thyparambil, Yeoun Jin Kim, Andrew G. Chambers, Dongyao Yan, Shankar Sellappan, Andrew J. Sedgewick, Yulia Newton, J Zachary Sanborn, Charles J. Vaske, Stephen C. Benz, Fabiola Cecchi, Hyunseok Kang, Todd A. Hembrough. Potential drug targets for adenoid cystic carcinoma elucidated by proteogenomic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 778.

Expression of PIM-1 in salivary gland adenoid cystic carcinoma: Association with tumor progression and patients' prognosis.

Pim-1 proto-oncogene, serine/threonine kinase (PIM-1) phosphorylates a series of substrates to exert its oncogenic function in numerous malignancies. The present study investigated the clinical significance of the PIM-1 protein, apoptosis status and apoptosis-associated proteins, including forkhead box O3a (FOXO3a), B cell lymphoma-2 (BCL-2) and BCL-2-associted agonist of cell death (BAD), were investigated in salivary gland adenoid cystic carcinoma (ACC) tissues. PIM-1 expression levels in 4 pairs of ACC tissues and corresponding normal salivary gland tissues were determined by western blot analysis. PIM-1, FOXO3a, BAD and BCL-2 expression levels in 60 ACC tissues were evaluated by immunohistochemistry (IHC). A terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay was performed to detect the apoptosis status of ACC tissues. PIM-1 was revealed to be highly expressed in ACC tissues compared with adjacent normal tissues. IHC staining results demonstrated high expression ratios of PIM-1, FOXO3a, BCL-2 and BAD [33.33% (20/60), 51.67% (31/60), 51.67% (31/60) and 55% (33/60)], respectively, and significant correlations between the expression of PIM-1 and FOXO3a and BCL-2 (P<0.05). Apoptotic rates were significantly associated with PIM-1, FOXO3a, BCL-2 and BAD expression levels (P<0.05). PIM-1 expression levels were significantly associated with tumor size, lymph node involvement, nerve invasion, distant metastasis and weakly associated with tumor node metastasis stage. Kaplan-Meier survival curves revealed that PIM-1 expression level was significantly associated with disease-free survival of patients with ACC (P=0.009). Cox regression multivariate analysis results revealed that histotype, distant metastasis and apoptotic rate were independent prognosis factors for ACC. Assessment of PIM-1 may be useful in investigating the malignant behaviors of ACC and predicting the outcome of patients with ACC.

MYB fusions and CD markers as tools for authentication and purification of cancer stem cells from salivary adenoid cystic carcinoma

Cancer stem cells (CSC) are considered the major cause of aggressive tumor behavior, recurrence, metastases, and resistance to radiation, making them an attractive therapeutic target. However, isolation of CSC from tumor tissue and their characterization are challenging due to uncertainty about their molecular markers and conditions for their propagation. Adenoid cystic carcinoma (ACC), which arises predominantly in the salivary glands, is a slow-growing but relentless tumor that frequently invades nerves and metastasizes. New effective treatment approaches for ACC have not emerged over the last 40years. Previously, based on a highly conserved SOX10 gene signature that we identified in the majority of ACC tumors, we suggested the existence in ACC of SOX10+ cells with neural stem properties and corroborated this hypothesis via isolation from ACC tissue a novel population of CSC, termed ACC-CSC. These cells activated NOTCH1 signaling and co-expressed SOX10 and other ACC-intrinsic neural crest stem cell markers with CD133, a CSC cell surface marker, suggesting that ACC is driven by a previously uncharacterized population of SOX10+/CD133+ cells with neural stem cell properties. Here, we authenticated ACC identity of our primary cultures by demonstrating that most of them harbor MYB-NFIB fusions, which are found in 86% of ACC. We demonstrated using CyTOF, a novel mass cytometry technology, that these cells express high β-catenin and STAT3 levels and are marked by CD24 and CD44. Finally, to streamline development of ACC cell lines, we developed RT-PCR tests for distinguishing mouse and human cells and used immunomagnetic cell sorting to eliminate mouse cells from long-term cell cultures. Overall, this study describes a new population of CSC that activates signaling pathways associated with poor prognosis, validates their ACC identity, and optimizes approaches that can be used for purification of ACC-CSC and generation of cell lines.

Histologic and immunohistochemical identification of cribriform adenocarcinoma

To assess whether tumors originally classified as adenocarcinoma not otherwise specified (Adca-NOS), adenoid cystic carcinoma (ACC), and polymorphous low-grade adenocarcinoma (PLGA) could be reclassified as cribriform adenocarcinoma of the tongue and minor salivary gland (CATMSG). Tumors diagnosed between 1992 and 2014 at Oral Pathology Laboratory, Inc. (New York Presbyterian Hospital) were selected. Each tumor was reviewed by 3 oral pathologists to confirm the diagnosis of CATMSG. After review, 11 of 70 Adca-NOS, 5 of 38 ACCs, and 5 of 23 PLGAs met the histologic criteria for CATMSG. One case diagnosed as CATMSG in 2014 was used as a control, and the following stains were completed: epithelial membrane antigen, Hector battifora mesothelial-1, p16, and CAM 5.2. Eleven Adca-NOS, 2 ACCs (tissue was available for only 2 of the 5 adenoid cystic carcinoma cases), and 5 PLGAs underwent immunohistochemical (IHC) staining. Positive HMBE-1, p16, and CAM 5.2 staining along with negative staining for epithelial membrane antigen were considered supportive of a diagnosis of CATMSG. Based on histologic features and IHC results, we were able to reclassify 10 tumors (8 Adca-NOS and 2 PLGA) as CATMSG. CATMSG should be distinguished from other salivary gland tumors with similar histologic features. The diagnosis of CATMSG can be made on hematoxylin and eosin staining alone, but IHC analysis can be useful for confirmation.

Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening.

Salivary gland adenoid cystic carcinoma (ACC) is a rare head and neck malignancy without molecular biomarkers that can be used to predict the chemotherapeutic response or prognosis of ACC. The regulation of gene expression of oncogenes and tumor suppressor genes (TSGs) through DNA promoter methylation may play a role in the carcinogenesis of ACC. To identify differentially methylated genes in ACC, a global demethylating agent, 5-aza-2′-deoxycytidine (5-AZA) was utilized to unmask putative TSG silencing in ACC xenograft models in mice. Fresh xenografts were passaged, implanted in triplicate in mice that were treated with 5-AZA daily for 28 days. These xenografts were then evaluated for genome-wide DNA methylation patterns using the Illumina Infinium HumanMethylation27 BeadChip array. Validation of the 32 candidate genes was performed by bisulfite sequencing (BS-seq) in a separate cohort of 6 ACC primary tumors and 6 normal control salivary gland tissues. Hypermethylation was identified in the HCN2 gene promoter in all 6 control tissues, but hypomethylation was found in all 6 ACC tumor tissues. Quantitative validation of HCN2 promoter methylation level in the region detected by BS-seq was performed in a larger cohort of primary tumors (n=32) confirming significant HCN2 hypomethylation in ACCs compared with normal samples (n=10; P=0.04). HCN2 immunohistochemical staining was performed on an ACC tissue microarray. HCN2 staining intensity and H-score, but not percentage of the positively stained cells, were significantly stronger in normal tissues than those of ACC tissues. With our novel screening and sequencing methods, we identified several gene candidates that were methylated. The most significant of these genes, HCN2, was actually hypomethylated in tumors. However, promoter methylation status does not appear to be a major determinant of HCN2 expression in normal and ACC tissues. HCN2 hypomethylation is a biomarker of ACC and may play an important role in the carcinogenesis of ACC.

Bmi-1 expression predicts prognosis in salivary adenoid cystic carcinoma and correlates with epithelial-mesenchymal transition-related factors.

Salivary adenoid cystic carcinoma (AdCC) is known for its high propensity to invade and metastasize. Bmi-1 acts as an oncogene by controlling cell cycle and self-renewal of adult stem cells, and its overexpression correlates with metastasis and poor prognosis in several cancers. Epithelial-mesenchymal transition (EMT) plays a central role in cancer metastasis. A key step in EMT is the down-regulation of E-cadherin that can be repressed by the transcriptional factors, such as Snail and Slug. In the present study, we investigated Bmi-1, Snail, Slug, and E-cadherin expression by immunohistochemistry in 102 patients with AdCC and analyzed statistically whether their expression correlated with clinicopathologic factors and prognosis. Reverse transcription-polymerase chain reaction was also performed in 22 tumor tissues and the adjacent noncancerous tissues to confirm Bmi-1 status in AdCCs. Our data demonstrated significant associations between the tumor metastasis and the expression of Bmi-1, Snail, Slug, and E-cadherin. Furthermore, a high level of Bmi-1 was not only correlated with the overexpression of Snail and Slug but also indicated an unfavorable metastasis-free survival and served as a high-risk marker for AdCC. In addition, Bmi-1 messenger RNA level was found much higher in AdCC tissues than in the adjacent noncancerous salivary gland tissues. Our results suggest that Bmi-1 may play a crucial role in AdCC progression by interaction with EMT-related markers and predict poor survival.

Expression of BNIP3 and its correlations to hypoxia-induced autophagy and clinicopathological features in salivary adenoid cystic carcinoma.

The expression of Bcl-2/adenovirus E1B 19 kDa-interacting protein3 (BNIP3) has been explored in many human malignancies, but not in adenoid cystic carcinoma (ACC). This study investigated the clinical significance of expression of BNIP3 in ACC tissues and cells and elucidated its correlations to hypoxia-induced autophagy. Immunohistochemical and immunofluorescence staining were used to explore BNIP3, HIF-1α and LC3 expression. BNIP3 was positively expressed in 41 cases (63.1%), and was significantly correlated with histological grade (P= 0.001). HIF-1α was positively expressed in 52 cases (80.0%) and was significantly correlated with TNM stage (P= 0.023) and histological grade (P= 0.024). LC3 was positively expressed in 37 cases (56.9%) and was significantly correlated with TNM stage (P= 0.019). The expression of BNIP3 was correlated with HIF-1α expression (P= 0.011). The overall survival in the negative BNIP3 expression group tended to be better than in the positive BNIP3 expression (P= 0.011). In vitro experiment, BNIP3 immunofluorescence staining was detected in cells treated with CoCl2 (for hypoxic condition). The data indicated that BNIP3 plays a vital role in the tumorigenesis of adenoid cystic carcinoma and could be a new target for gene therapy of adenoid cystic carcinoma.

Role of hypoxia-inducible factor-1α and CD146 in epidermal growth factor receptor-mediated angiogenesis in salivary gland adenoid cystic carcinoma.

Adenoid cystic carcinoma (AdCC) of the salivary gland in the head and neck is characterized by indolent yet persistent growth, multiple local recurrences and early hematogenous metastasis. Considering the possible association between the epidermal growth factor receptor (EGFR) signaling pathway and angiogenesis in various types of cancer and the overexpression of EGFR in AdCC, it is reasonable to examine the correlation between angiogenesis and the EGFR signaling pathway in this carcinoma. In the present study, the expression of EGFR, CD31, CD146 and hypoxia-inducible factor-1α (HIF-1α) were evaluated by immunohistochemical staining with tissue microarray containing normal salivary gland (NSG), pleomorphic adenoma (PMA) and AdCC tissues. Pearson's correlation coefficient was conducted to demonstrate the correlation between EGFR, CD31, CD146 and HIF-1α. To determine their similarity and intimacy, hierarchical analysis was performed with Cluster 3.0 and then visualized using TreeView software. Immunohistochemical results of tissue microarrays were quantified, revealing that the expression of EGFR, CD146 and HIF-1α increased in AdCC compared with in PMA and NSG tissues. The association between the expression of EGFR and CD31 was significant and positive. The expression of CD146 and HIF-1α was positively correlated with EGFR and CD31, respectively. These findings suggest that the EGFR signaling pathway has a vital role in AdCC progression and may be associated with HIF-1α-mediated angiogenesis. These results may enhance our understanding of the mechanism underlying AdCC progression and provide potential clinical therapeutic strategies based on the inhibition of EGFR.