Normal Salivary Gland Tissue Research Articles

The prognostic impact of human leukocyte antigen (HLA) class I antigen abnormalities in salivary gland cancer. A clinicopathological study of 288 cases

To study abnormalities of proteins of the major histocompatibility complex class I in a series of 288 salivary gland carcinomas, and to correlate findings with patients' overall survival (OS). Protein expression of human leukocyte antigen (HLA)-A, heavy chain (HC)-10, β2 -microglobulin, low molecular weight polypeptides (LMP) 2 and 7, transporters associated with antigen processing (TAP) 1 and 2, calnexin, calreticulin, endoplasmic reticulum (ER) p57 and tapasin was evaluated by immunohistochemistry and semiquantitatively analyzed. As compared with normal salivary gland tissue, HLA-A, LMP7, TAP2 and HLA class I were significantly down-regulated in salivary gland carcinomas, whereas β2 -microglobulin, calnexin, LMP2, and TAP1 were upregulated. Expression of calreticulin, ERp57 and tapasin was unaltered. In univariate Kaplan-Meier analyses, low expression of LMP7 (P = 0.005) and high expression of β2 -microglobulin (P = 0.028), HLA-A (P < 0.001), TAP1 (P = 0.01), and tapasin (P < 0.001) were significantly associated with shorter OS. In multivariate analysis incorporating tumour stage, nodal/distant metastasis, and grade, HLA-A (P = 0.014), LMP7 (P = 0.033), and tapasin (P = 0.024), as well as distant metastasis (P = 0.012) and high tumour grade (P < 0.001), remained statistically significant. The prognostic influence of up-regulated HLA-A and tapasin and down-regulated LMP7 may provide a rationale for targeting these specific components of the antigen processing and presentation pathway in salivary gland carcinomas.

PP061: Genetic expressions of ER/PR and HER2/neu among salivary gland tumour patients at UCH Ibadan

Acinar and ductal structures are found in normal breast and salivary gland tissue, also similar histopathological findings were revealed by some neoplasm from these tissues therefore the role of ER/PR and HER2/neu as molecular targets in breast cancer were projected to salivary gland tumours (SGT’s). The purpose of this study was to determine the levels of expression of these genes which may be indicators for targeted therapy in SGT’s. The study utilised sections of hematoxylin-eosin (H&E) and IHC sections of antibody panels to estrogen (ERα), progesterone (PR) and Human Epithelial Receptor (HER2) as specified by the manufacturer (Dako Cytomation, USA). 40 salivary gland tumour specimen from 19 males (47.5%) and 21 females (52.5%) were utilised (age range was 6–75 years; mean 45.4 ± 16.6 years) there were 15 benign and 25 malignant cases. There was a definite increased positive expression of oestrogen receptor with increasing clinical stage ( x 2 = 5.6; p = 0.00) and higher histological grades irrespective of sex and histological type ( X 2 = 4.2; p = 0.04 Fisher’s exact) while the positive expression of PR was generally low irrespective of sex, clinical stage, histological type and grade of malignant SGT’s. There was lack of positive expression of HER2/neu among all benign SGT’s while only one high grade acinic cell carcinoma showed minimal positive expression among the malignant SGT’s. ERα receptor may be a suitable molecular target for therapy of high grade malignant SGT that show positive expression, the role of PR receptor targeted therapy is doubtful in malignant SGT’s. None of the SGT’s showed convincing positive HER2/neu expression.

Abstract 1952: Deregulated microRNA expression in the progression of salivary gland cancer.

Abstract Salivary gland cancer constitutes one of the five main subsets of cancer in the head and neck region. Nonresectable malignant tumors demonstrate a low 20-year survival rate due to radioresistance and a high propensity for metastasis. The molecular mechanisms underlying pathogenesis of salivary cancer remains poorly elucidated, and thus necessitates further investigation. MicroRNAs (miRNA) are small noncoding RNAs that are predicted to regulate up to 30% of protein-encoding genes. Signature miRNA expression profiles have been identified in various malignancies, implicating miRNAs in the progression of human cancer. In order to better understand the role of miRNAs in the progression of salivary cancer, we profiled the miRNA expression levels of normal salivary tissue, benign salivary tumor, and salivary cancer. A total of 17 formalin-fixed paraffin-embedded salivary tissues, including 4 normal, 4 pleomorphic adenoma (PA), 3 squamous cell carcinoma (SCC), 3 mucoepidermoid carcinoma (ME) and 3 adenocarcinoma (AC), were collected and the endogenous expression of 95 miRNAs was analyzed by microarray. A large number of miRNAs were observed to be aberrantly expressed and generally down-regulated in salivary cancer and salivary tumor tissues with respect to normal salivary gland tissues. An unsupervised clustering and a student t-test were performed with a threshold p-value less than 0.05, resulting in the identification of 22 miRNAs differentially expressed at a statistically significant level. Eight candidate miRNAs were selected and further validated by RT-qPCR. The results suggest three miRNAs in SCC (miR-200a, let-7-family, and miR-192) and three miRNAs in AC (miR-107, miR-15b and miR-200a) to be highly involved in the carcinogenetic process of salivary cancers. Furthermore, the up-regulated expression pattern of miR-203 is indicative of a tumorigenetic function in salivary tumors. Interestingly, miR-200b was found to be consistently and significantly down regulated (p=0.01, 19.03 fold decrease) in the three types of salivary cancer under investigation. Recent studies have implicated miR-200b in EMT and invasion in cancer cells by directly targeting E-cadherin transcriptional repressors. The miRNAs identified in this study may serve as potential biomarkers and targets for future miRNA based therapy. Citation Format: Hao Zheng, Alan Kiang, Selena Z. Kuo, Elham Rahimy, Jessica Wang-Rodriguez, Weg M. Ongkeko. Deregulated microRNA expression in the progression of salivary gland cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1952. doi:10.1158/1538-7445.AM2013-1952

LINE-1 and Alu hypomethylation in mucoepidermoid carcinoma

BackgroundMucoepidermoid carcinoma (MEC) can be classified into low-, intermediate-, and high-grade tumors based on its histological features. MEC is mainly composed of three cell types (squamous or epidermoid, mucous and intermediate cells), which correlates with the histological grade and reflects its clinical behavior. Most cancers exhibit reduced methylation of repetitive sequences such as Long INterspersed Element-1 (LINE-1) and Alu elements. However, to date very little information is available on the LINE-1 and Alu methylation status in MEC. The aim of this study was to investigate LINE-1 and Alu element methylation in MEC and compare if key differences in the methylation status exist between the three different cell types, and adjacent normal salivary gland cells, to see if this may reflect the histological grade.MethodsLINE-1 and Alu element methylation of 24 MEC, and 14 normal salivary gland tissues were compared using Combine Bisulfite Restriction Analysis (COBRA). Furthermore, the three different cell types from MEC samples were isolated for enrichment by laser capture microdissection (LCM), essentially to see if COBRA was likely to increase the predictive value of LINE-1 and Alu element methylation.ResultsLINE-1 and Alu element methylation levels were significantly different (p<0.001) between the cell types, and showed a stepwise decrease from the adjacent normal salivary gland to the intermediate, mucous and squamous cells. The reduced methylation levels of LINE-1 were correlated with a poorer histological grade. In addition, MEC tissue showed a significantly lower level of LINE-1 and Alu element methylation overall compared to normal salivary gland tissue (p<0.001).ConclusionsOur findings suggest that LINE-1 methylation differed among histological grade mucoepidermoid carcinoma. Hence, this epigenetic event may hold value for MEC diagnosis and prognostic prediction.

Expression and clinical significance of FAK, ILK, and PTEN in salivary adenoid cystic carcinoma

Conclusion: Focal adhesion kinase (FAK) and integrin-linked kinase (ILK) are highly expressed in salivary adenoid cystic carcinoma (SACC), especially metastatic SACC, suggesting their potential role as prognostic markers for SACC. Objectives: This study aimed to investigate the expression of FAK, ILK, and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in SACC tissues and the clinical significance. Methods: PAK and ILK expression in samples from 50 cases of SACC and 12 subjects with normal salivary glands was detected by immunohistochemical analysis. PAK and ILK expression in SACC cell lines was detected by RT-PCR and Western blot analysis. Results: The positive rate of PAK and ILK staining was 94% (47/50) and 48% (24/50) in SACC, respectively, significantly higher than in normal salivary gland tissues (p < 0.05). However, the positive rate of PTEN staining was 30% (15/50), significantly lower than in normal salivary gland tissues (p < 0.05). Pearson analysis showed that FAK expression was positively correlated with ILK expression but negatively correlated with PTEN expression in SACC tissues. FAK and ILK expression was positively associated with advanced stage, solid histological subtype, perineural invasion, and distant metastasis of SACC (p < 0.05). In addition, FAK and ILK expression at both mRNA and protein levels was significantly higher in highly metastatic SACC-LM cells than low-metastatic SACC-83 cells.

Suprabasin Is Hypomethylated and Associated with Metastasis in Salivary Adenoid Cystic Carcinoma

BackgroundSalivary gland adenoid cystic carcinoma (ACC) is a rare cancer, accounting for only 1% of all head and neck malignancies. ACC is well known for perineural invasion and distant metastasis, but its underlying molecular mechanisms of carcinogenesis are still unclear.Principal FindingsHere, we show that a novel oncogenic candidate, suprabasin (SBSN), plays important roles in maintaining the anchorage-independent and anchorage-dependent cell proliferation in ACC by using SBSN shRNA stably transfected ACC cell line clones. SBSN is also important in maintaining the invasive/metastatic capability in ACC by Matrigel invasion assay. More interestingly, SBSN transcription is significantly upregulated by DNA demethylation induced by 5-aza-2′-deoxycytidine plus trichostatin A treatment and the DNA methylation levels of the SBSN CpG island located in the second intron were validated to be significantly hypomethylated in primary ACC samples versus normal salivary gland tissues.Conclusions/SignificanceTaken together, these results support SBSN as novel oncogene candidate in ACC, and the methylation changes could be a promising biomarker for ACC.

Expression of Ki67 and CD105 as Proliferation and Angiogenesis Markers in Salivary Gland Tumors

To investigate the association between CD105 and tumor cell proliferation in salivary gland tumors. In this study, 59 samples of salivary tumors from Khalili Hospital archive, including 20 cases of pleomorphic adenoma (PA), 20 cases of mucoepidermoid carcinoma (MEC) and 19 cases of adenoid cystic carcinoma, as well as 10 cases of normal salivary gland tissue, were reviewed by immunohistochemistry (IHC) for CD105 and Ki67 staining. CD105 positive vessels were absent in normal salivary gland tissue in the vicinity of tumors (51.6% of all tumors were positive). There was a statistically significant difference in frequency of CD105 staining between PA and malignant tumors and between four groups of different lesions (p<0.000) being highest in MEC. Intratumoral microvessel density was also elevated in malignant neoplasms (2.61 ± 3.1) as compared to PA (0.46 ± 0.6). Normal salivary glands did not express Ki67. There was a statistically significant difference in frequency and percentage of Ki67 immunoreactivity in malignant neoplasms (86.5% and 10.7 ± 10.8 respectively) compared to PA (50% and 0.78 ± 0.2) and among the four groups values were highest in MEC (p<0.000). n this study, it was observed a higher rate of angiogenesis and cellular proliferation was noted in malignant tumors compared to benign tumors, but no correlation was observed between these two markers.

P63 Expression Can Be Used in Differential Diagnosis of Salivary Gland Acinic Cell and Mucoepidermoid Carcinomas

Differentiation of salivary gland acinic cell carcinoma from mucoepidermoid carcinoma can be diagnostically challenging as both may have prominent mucin production. P63 is a p53 homologue required for limb and epidermal morphogenesis. Itis expressed in basal and myoepithelial cells of normal salivary gland tissues. In this immunohistochemical study, we examined the expression of p63 in salivary gland acinic cell and mucoepidermoid carcinomas (MEC) and its use in differentiating these two entities. A search was performed and appropriate cases were selected from Lifespan Hospital System archives as well as the consult archives of one author (DRG). 31 salivary gland acinic cell carcinomas (ACC) and 24 MEC were examined for p63 expression by immunohistochemistry. The nuclear immunoreactivity was examined by both authors and was graded semi-quantitatively with negative being less than 10% of cells staining. Positive staining was graded as follows: 10-25% of tumor cells staining was weakly positive, 26-75% of tumor cells staining was moderately positive, and 76-100% of tumor cells staining was strongly positive. Negative nuclear staining of the tumor cells was seen in 30/31 (96%) of salivary gland ACC while 1/31 (3%) showed diffuse nuclear staining of the tumor cells. This latter case was later reclassified as mammary analogue secretory carcinoma following confirmatory molecular testing for the ETV6-NTRK3 fusion gene. Strong positive nuclear staining of the tumor cells was seen in 24 (100%) of salivary gland MEC cases. P63 is an immunohistochemical stain that can potentially aid in differentiating unusual ACC with prominent mucin production from MEC of the salivary gland. According to this study, acinic cell carcinoma is always negative for p63 immunoreactivity while mucoepidermoid carcinoma is always positive.

TrkC signaling is activated in adenoid cystic carcinoma and requires NT-3 to stimulate invasive behavior

Treatment options for adenoid cystic carcinoma (ACC) of the salivary gland, a slowly growing tumor with propensity for neuroinvasion and late recurrence, are limited to surgery and radiotherapy. Based on expression analysis performed on clinical specimens of salivary cancers, we identified in ACC expression of the neurotrophin-3 receptor TrkC/NTRK3, neural crest marker SOX10, and other neurologic genes. Here, we characterize TrkC as a novel ACC marker, which was highly expressed in 17 out of 18 ACC primary-tumor specimens, but not in mucoepidermoid salivary carcinomas or head and neck squamous cell carcinoma. Expression of the TrkC ligand NT-3 and Tyr-phosphorylation of TrkC detected in our study suggested the existence of an autocrine signaling loop in ACC with potential therapeutic significance. NT-3 stimulation of U2OS cells with ectopic TrkC expression triggered TrkC phosphorylation and resulted in Ras, Erk 1/2 and Akt activation, as well as VEGFR1 phosphorylation. Without NT-3, TrkC remained unphosphorylated, stimulated accumulation of phospho-p53 and had opposite effects on p-Akt and p-Erk 1/2. NT-3 promoted motility, migration, invasion, soft-agar colony growth and cytoskeleton restructuring in TrkC-expressing U2OS cells. Immunohistochemical analysis demonstrated that TrkC-positive ACC specimens also show high expression of Bcl2, a Trk target regulated via Erk 1/2, in agreement with activation of the TrkC pathway in real tumors. In normal salivary gland tissue, both TrkC and Bcl2 were expressed in myoepithelial cells, suggesting a principal role for this cell lineage in the ACC origin and progression. Sub-micromolar concentrations of a novel potent Trk inhibitor AZD7451 completely blocked TrkC activation and associated tumorigenic behaviors. Pre-clinical studies on ACC tumors engrafted in mice showed efficacy and low toxicity of AZD7451, validating our in vitro data and stimulating more research into its clinical application. In summary, we describe in ACC a previously unrecognized pro-survival neurotrophin signaling pathway and link it with cancer progression.

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

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

High expression of the autophagy gene Beclin‐1 is associated with favorable prognosis for salivary gland adenoid cystic carcinoma

Although autophagy is universally involved in tumorigenesis and tumor progression, the roles of autophagy and autophagy-regulating genes in salivary gland adenoid cystic carcinoma (ACC) remain unknown. In this study, we investigated the expression of the autophagy-regulating genes Beclin-1, death-associated protein kinase-1, ultraviolet radiation resistance-associated gene, and phosphatase and tensin homolog in salivary gland ACC samples. Immunohistochemistry and real-time polymerase chain reaction were used to analyze the expression of these genes in 89 ACC samples and normal salivary gland tissue samples. The relationship of their expression with clinicopathological features was analyzed. The data showed significantly lower expression of these genes in the tumor samples than in normal salivary gland tissue samples. Furthermore, Beclin-1 expression was significantly correlated with histological pattern of ACC (P<0.05), and high expression of ultraviolet radiation resistance-associated gene was associated with distant metastasis (P<0.05). Most importantly, univariate and multivariate survival analyses suggested that Beclin-1 protein and mRNA expression in cancer cells were independent prognostic indicators for ACC. Our results suggest that autophagy-regulating genes may participate in the pathogenesis of salivary gland ACC. Further research will be required to gain a better understanding of autophagy in ACC.

Abstract 5017: DNA methylation array screening in salivary gland adenoid cystic carcinoma

Abstract Objective: DNA methylation alterations, with either gain or loss of CpG methylation in the promoter region, can alter the expression of tumor suppressor genes or oncogenes. This contributes to the tumorigenesis of human cancers, including salivary adenoid cystic carcinoma (ACC). In this study, we used a DNA methylation array (Illumina HumanMethylation27 BeadChip, San Diego, CA) to screen for novel oncogene and tumor suppressor gene (TSG) candidates under the control of promoter methylation in ACC. Methods: The Illumina HumanMethylation27 BeadChip Array was used to analyze the DNA methylation patterns of ∼14.000 genes at 27,578 CpG sites in 21 ACC and 13 normal salivary gland tissues. With our basic screening algorithm, we narrowed the list down to ∼100 tumor suppressor gene candidates (hypermethylated in tumor) and oncogene candidates (hypomethylated in tumor). Next, the differential methylation levels in the promoter region of candidates were validated by bisulfite genomic sequencing in four primary ACC and four normal salivary gland tissues. Candidate genes that showed differential methylation were then confirmed by analyzing gene re-expression in seven cell lines treated with 5-Aza deoxycytidine, a global demethylating agent. Furthermore, the expression levels of the candidate genes were examined by quantitative reverse transcription PCR (qRT-PCR) in a separate ACC cohort. Lastly, the DNA methylation levels of promising candidate genes were validated by quantitative methylation specific PCR (qMSP) in a larger ACC cohort. Results: With the methylation array, we initially identified 28 oncogene candidates which displayed relative hypomethylation and 64 TSG candidates that showed relative hypermethylation in ACC compared to normal tissues. After the above mentioned additional validation steps were performed, we settled on two promising candidates, PDE9A and TP53TG3, that were significantly hypomethylated and overexpressed in primary ACC tumors compared to normal salivary gland tissue. Furthermore, the expression of PDE9A and TP53TG3 demonstrated consistent upregulation after 5-aza-cytidine treatment of cancer cell lines, suggesting that promoter methylation does play in a role in controlling gene expression. Conclusions: The Illumina HumanMethylation27 BeadChip array is a reliable and efficient tool for analyzing global gene methylation. It is useful in screening for novel oncogene and TSG candidates controlled by promoter methylation in salivary gland ACC. PDE9A and TP53TG3 were identified as promising novel oncogene candidates in salivary gland ACC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5017. doi:1538-7445.AM2012-5017

Abstract 5352: Investigation of stem-cell markers in pleomorphic adenoma: Immunohistochemical and molecular study

Abstract Background: Salivary gland neoplasms are originated from the various salivary gland compartments and are histologically related to these structures. Several protein and molecular markers are employed in research and diagnosis in order to classify and understand histogenesis and other aspects of salivary gland neoplastic lesions. Pleomorphic adenoma is a benign salivary gland neoplasm composed by epithelial and myoepithelial cells and a complex stroma. Its varied structural and architectural aspects suggest the participation of stem cells in its composition. Additionally, pleomorphic adenoma originates from intercalated duct of salivary glands, a region reputed to host the regenerative/ stem-cell compartment of these glands. The present work investigated stem-cell markers (CD24, CD44) in pleomorphic adenoma and in specimens of developing human salivary glands using immunohistochemistry and real-time RT-PCR. Material and Methods: 101 cases of pleomorphic adenomas were used - 55 cases were FFPE and 60 specimens were fresh frozen tissue. From the total, 14 were pared (FFPE and frozen tissue). Salivary gland specimens dissected from 20 human foetuses were also included. Results: All cases of pleomorphic adenomas were positive for the stem-cell markers studied. Neoplastic luminal structures were positive for CD24. Modified myoepithelial cells were positive for CD44. In foetal salivary glands, these markers were restricted to the intercalated duct region. Increased expression of CD44 stem-cell marker was observed in pleomorphic adenoma specimens using real-time RT-PCR technique when compared to normal salivary gland controls. An specific expression pattern was not observed for CD24 when compared to the normal salivary gland tissue - in some cases of pleomorphic adenoma there was an increased expression of the protein, whilst in other cases the expression was decreased or normal-like. Conclusion: Pleomorphic adenoma cells share similar markers with stem-cells; it is not possible to confirm that neoplastic cells bear the same characteristics of multipotency. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5352. doi:1538-7445.AM2012-5352

Expression and significance of E-cadherin in adenoid cystic carcinoma of salivary glands

To examine the expression of E-cadherin and the methylation status of CDH1 and explore their clinical significance in salivary adenoid cystic carcinoma (SACC). The expression of E-cadherin was detected by the immunohistochemical method. And the methylation of CDH1 gene promoter 5'-CpG island was analyzed by real-time methylation-specific polymerase chain reaction (real-time MSP) in salivary adenoid cystic carcinoma and normal salivary gland tissue respectively. The expression rate of E-cadherin was lower in SACC than that in normal salivary gland tissue (100.0% vs 55.6%, P < 0.05). And the expression of E-cadherin was associated with different histopathological types, T-stage, nerve invasion, lymphatic and distant metastasis (P < 0.05). However, there was no correlation between the expression of E-cadherin and gender, age and tumor location. Partial methylation of CDH1 was detected in 3 of 30 cases with a positive expression of E-cadherin and full methylation of CDH1 in 23 of 24 cases with a negative expression of E-cadherin. There was a negative correlation between the expression of E-cadherin and the methylation of CDH1 in salivary adenoid cystic carcinoma (r = -0.483, P < 0.001). The down-regulation of E-cadherin, as modulated by the methylation of CDH1, may contribute to nerve invasion, lymphatic and distant metastasis in SACC. Thus it may be used as a biological indicator of malignancy and prognosis.

Immunohistochemical correlation of epidermal growth factor receptor and c-erbB-2 with histopathologic grading of mucoepidermoid carcinoma

Mucoepidermoid carcinoma is the most common salivary gland malignancy with highly variable biologic potential that correlates with the histopathologic grade of the tumor. Therefore, identification of the histopathologic grade of the mucoepidermoid carcinoma is very important in the treatment and determination of the final prognosis. The present study was performed to survey immunohistochemically Epidermal Growth Factor ReceptorEGFR and c-erbB-2 expression in different grades of mucoepidermoid carcinoma. This retrospective study included 46 formalin-fixed, paraffin-embedded blocks of mucoepidermoid carcinoma. Based on histopathologic parameters, samples were classified into three grades. Then new sections were made and stained by immunohistochemistry (IHC) method for EGFR and c-erbB-2. Finally, EGFR and c-erbB-2 expression and their correlation with histopathologic grading were statistically analyzed by ANOVA. Nineteen samples of normal salivary gland tissue were also chosen as control group. The means of EGFR and c-erbB-2 were 71%, 71%, respectively. Statistically significant correlation was found between EGFR expression and histopathologic grading of mucoepidermoid carcinoma of salivary glands (P < 0.001). There was no statistically significant correlation between histopathologic grading of salivary gland mucoepidermoid carcinoma and c-erbB-2 expression (P = 0.60). There is a parallelism between an increase in EGFR expression and increase in the histopathologic grading of salivary gland mucoepidermoid carcinoma. Therefore, the biologic behavior of salivary gland mucoepidermoid carcinoma can be determined by EGFR expression and it is a useful technique for determination of tumor grades and probably their prognosis.

Astrocyte elevated gene-1 (AEG-1) is a marker for aggressive salivary gland carcinoma

BackgroundAstrocyte elevated gene-1 (AEG-1) is associated with tumorigenesis and progression in diverse human cancers. The present study was aimed to investigate the clinical and prognostic significance of AEG-1 in salivary gland carcinomas (SGC).MethodsReal-time PCR and western blot analyses were employed to examine AEG-1 expression in two normal salivary gland tissues, eight SGC tissues of various clinical stages, and five pairs of primary SGC and adjacent salivary gland tissues from the same patient. Immunohistochemistry (IHC) was performed to examine AEG-1 protein expression in paraffin-embedded tissues from 141 SGC patients. Statistical analyses was applies to evaluate the diagnostic value and associations of AEG-1 expression with clinical parameters.ResultsAEG-1 expression was evidently up-regulated in SGC tissues compared with that in the normal salivary gland tissues and in matched adjacent salivary gland tissues. AEG-1 protein level was positively correlated with clinical stage (P < 0.001), T classification (P = 0.008), N classification (P = 0.008) and M classifications (P = 0.006). Patients with higher AEG-1 expression had shorter overall survival time, whereas those with lower tumor AEG-1 expression had longer survival time.ConclusionsOur results suggest that AEG-1 expression is associated with SGC progression and may represent a novel and valuable predictor for prognostic evaluation of SGC patients.

Decrease in thyroid adenoma associated (THADA) expression is a marker of dedifferentiation of thyroid tissue

BackgroundThyroid adenoma associated (THADA) has been identified as the target gene affected by chromosome 2p21 translocations in thyroid adenomas, but the role of THADA in the thyroid is still elusive. The aim of this study was to quantify THADA gene expression in normal tissues and in thyroid hyper- and neoplasias, using real-time PCR.MethodsFor the analysis THADA and 18S rRNA gene expression assays were performed on 34 normal tissue samples, including thyroid, salivary gland, heart, endometrium, myometrium, lung, blood, and adipose tissue as well as on 85 thyroid hyper- and neoplasias, including three adenomas with a 2p21 translocation. In addition, NIS (sodium-iodide symporter) gene expression was measured on 34 of the pathological thyroid samples.ResultsResults illustrated that THADA expression in normal thyroid tissue was significantly higher (p < 0.0001, exact Wilcoxon test) than in the other tissues. Significant differences were also found between non-malignant pathological thyroid samples (goiters and adenomas) and malignant tumors (p < 0.001, Wilcoxon test, t approximation), anaplastic carcinomas (ATCs) and all other samples and also between ATCs and all other malignant tumors (p < 0.05, Wilcoxon test, t approximation). Furthermore, in thyroid tumors THADA mRNA expression was found to be inversely correlated with HMGA2 mRNA. HMGA2 expression was recently identified as a marker revealing malignant transformation of thyroid follicular tumors. A correlation between THADA and NIS has also been found in thyroid normal tissue and malignant tumors.ConclusionsThe results suggest THADA being a marker of dedifferentiation of thyroid tissue.

P135. Identification of candidate gene of pleomorphic adenoma in salivary gland by suppression subtractive hybridization

Introduction: Pleomorphic adenoma is the most common benign salivary gland neoplasm with a variety of histologic appearance. Due to its diversity, the precise pre-operative diagnosis through fine needle aspiration cytology is difficult. The aim of this study is to identify the differentially expressed genes in pleomorphic adenoma to contribute to the precise diagnosis and to discover the mechanism of tumorigenesis. Methods: To screen the differential gene expressed in pleomorphic adenoma, we performed suppressive subtractive hybridization (SSH) on pleomorphic adenoma cells and the corresponding normal salivary gland cells. Results: Four samples were subjected to DNA sequencing analysis. The human microfibril-associated protein 4 (MFAP-4), an extracellular matrix associated protein, was found to differentially expressed in the tumors compared with those of the normal tissues. The expression profiles of MFAP-4 were further confirmed in pleomorphic adenoma and the corresponding normal salivary gland tissues by quantitative real-time RT-PCR in 10 patients. Discussion: This study suggested that MFAP-4 is a potential biomarker of pleomorphic adenoma, and MFAP-4 is related to tumorigenesis of pleomorphic adenoma.

Association of clinicopathologic parameters with the expression of inducible nitric oxide synthase and vascular endothelial growth factor in mucoepidermoid carcinoma

The roles that inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) play in tumorigenesis have been given special attention. In many tumors, their expression is upregulated. In addition, iNOS can stimulate the expression of VEGF. This study was carried out to investigate the expression of iNOS and VEGF as well as their relationship with angiogenesis and the clinicopathological characteristics of mucoepidermoid carcinoma (MEC). The expression of iNOS and VEGF was detected by Streptavidin-peroxidase immunohistochemistry, and microvessel density (MVD) was determined by anti-CD34 antibody staining in 70 MEC cases and 40 normal salivary gland tissues (NSG). Follow-up was performed on the 70 patients with MEC. Non-parametric tests were performed for the comparison of iNOS and VEGF expression. The positive expression rates of iNOS and VEGF were successively enhanced in NSG, well-differentiated and poorly differentiated MEC (P < 0.05). MVD counts were positively correlated with the expression levels of iNOS and VEGF in MEC (P < 0.05). The expression of iNOS was positively correlated with the expression of VEGF (P < 0.05). iNOS and VEGF expression were significantly associated with tumor differentiation, size metastasis, and relapse (P < 0.05) but were not correlated lymph node metastasis and metastasis. Inducible nitric oxide synthase can stimulate the expression of VEGF, and their expression status may help assess tumor malignancy and patient prognosis.

Expression and significance of Cyr61 in distant metastasis cells of human primary salivary adenoid cystic carcinoma

The objective of this study was to examine the expression of cysteine-rich protein 61 (Cyr61) in the distant metastatic tumor cells of human primary salivary adenoid cystic carcinoma (SACC) and its relationship with tumor angiogensis and metastasis. The experimental group comprised 35 paraffin-embedded tumor specimens of distant metastasis from primary SACC, with their corresponding primary tumor tissues and matched normal salivary gland tissues used as the control groups. Immunohistochemical staining was used to detect the expression of Cyr61 and vascular endothelial growth factor in the experimental and control groups. Vascular endothelial cells were highlighted by the anti-CD34 antibody, and the Weidner method was used to quantify microvessel density (MVD). Cyr61 was overexpressed in distant metastatic tumor cells of primary SACC. Positive expression of Cyr61 and vascular endothelial growth factor (VEGF) progressively increased in normal salivary gland tissues, primary tumor tissues, and tumor tissues of distant metastasis (P < .05). Compared with primary tumor tissues, Cyr61 expression and VEGF expression showed significant increase in tumor tissues of distant metastasis (P < .05). Cyr61 expression significantly correlated with VEGF expression and MVD (P < .05). Cyr61 appeared to have a significant association with tumor angiogenesis and metastasis in SACC and may be an important target in tumor antiangiogenesis therapy.