Nerve Growth Factor and Vascular Endothelial Growth Factor: Retrospective Analysis of 63 Patients with Salivary Adenoid Cystic Carcinoma

Aim To detect the expression of nerve growth factor （NGF） and vascular endothelial growth factor （VEGF） in salivary adenoid cystic carcinoma （SACC） tissues, as well as to determine the correlation between growth factor expression and prognosis in SACC. Methodology Medical records of 63 patients surgically treated for SACC between January 1988 and October 2005 were reviewed. Immunohistochemistry was performed to examine the expression of NGF and VEGF in tumor tissues. Kaplan-Meier analysis and Cox＇s proportional hazard regression model were applied to assess predictors of survival. Results NGF and VEGF were overexpressed in SACC tissues, compared with those in normal salivary tissues （P〈0.05）, and the staining intensity of these two factors was stronger in groups of solid subtype, advanced TNM stage, perineural invasion and recurrence. Patients with high- expression of NGF and VEGF, solid subtype, advanced stage, perineural invasion, recurrence and extended resection alone had worse survival rates （P〈0.05）. Conclusion NGF and VEGF are expressed increasingly in the tissues of SACC cases with invasion and metastasis. NGF expression and VEGF expression are independent prognosis factors for survival.

ANTIMETASTATIC EFFECT OF INTEGRIN IIb/IIIa INHIBITORSON SALIVARY ADENOID CYSTIC CARCINOMA

Objectives: To investigate the relation between metastatic potential of salivary adenoid cystic carcinoma (SACC) and tumor cell-platelet adhesion, and the antimetastatic effect of integrin IIb/IIIa inhibitor on SACC. Methods: Tumor cell-platelet adhesion of highly metastatic SACC-LM, non-highly metastatic SACC-83 and effect of aspirin, arginine-aspartate (RD), magnesium acetylsalicylate on adhesion were studiedin vitro. Antimetastafic effect of aspirin, RD, magnesium acetysalicylate on experimental metastasis of SACC was observedin vivo. Results: The tumor cell-platelet adhesion was stronger in SACC-LM than in SACC-83. Aspirin, RD and magnesium acetylsalicylate could inhibit the adhesion of SACC-LM at the concentration of 1, 5 and 25 μg/ml. RD can inhibit experimental metastasis of SACC. Conclusion: Metastasis of SACC is related to platelet-tumor cell adhesion, RD could inhibit metastasis of SACC.

Expression of cyclin-dependent kinase inhibitor 2A 16,tumour protein 53 and epidermal growth factor receptor in salivary gland carcinomas is not associated with oncogenic virus infection

It is known that human papillomavirus （HPV） infection can cause squamous cell neoplasms at several sites, such as cervix uteri carcinoma and oral squamous carcinoma. There is little information on the expression of HPV and its predictive markers in tumours of the major and minor salivary glands of the head and neck. We therefore assessed oral salivary gland neoplasms to identify associations between HPV and infection-related epidermal growth factor receptor （EGFR）, cyclin-dependent kinase inhibitor 2A （CDKN2A/p16） and tumour protein p53 （TP53）. Formalin-fixed, paraffin-embedded tissue samples from oral salivary gland carcinomas （n=51） and benign tumours （n=26） were analysed by polymerase chain reaction （PCR） analysis for several HPV species, including high-risk types 16 and 18. Evaluation of EGFR, CDKN2A, TP53 and cytomegalovirus （CMV） was performed by immunohistochemistry. Epstein-Barr virus （EBV） was evaluated by EBV-encoded RNA in situ hybridisation. We demonstrated that salivary gland tumours are not associated with HPV infection. The expression of EGFR, CDKN2A and TP53 may be associated with tumour pathology but is not induced by HPV. CMV and EBV were not detectable. In contrast to oral squamous cell carcinomas, HPV, CMV and EBV infections are not associated with malignant or benign neoplastic lesions of the salivary glands.

RNAi knockdown of C-erbB2 expression inhibits salivary gland adenoid cystic carcinoma SACC-83 cell growthin vitro

Objective：To knockdown the C-erbB2 gene in salivary gland adenoid cystic carcinoma SACC-83 cells using RNA interference,and determine the effect of silencing C-erbB2 on cell proliferation.Methods：C-erbB2-siRNA was transfected into SACC-83 cells.RT-PCR and immunohistochemistry were used to detect C-erbB2 expression in SACC-83 cells.Cell proliferation was measured by the MTT assay and gene knockdown was achieved by RNA interference.Apoptosis was analyzed by flow cytometry.Results：Compared with the control,C-erbB2 mRNA expression was decreased in the C-erbB2-siRNA transfection group,and immunohistochemical analysis indicated that C-erbB2 protein expression was decreased.After C-erbB2-siRNA was transfected for 48 h,absorbance at 570 nm （MTT）was 0.185±0.021 compared with 0.354±0.034,0.299±0.053,and 0.314±0.049 in the blank control,liposome control and negative control siRNA groups,respectively.The differences were statistically significant （P〈0.05）between the C-erbB2-siRNA group and the control groups.Following the C-erbB2 knockdown,the percentage of apoptotic cells was 5.63%compared with 2.04%,2.85%,and 2.98%in the three control groups,respectively.Proliferation of SACC-83 cells was inhibited,and early apoptotic cells were increased.Conclusion： RNA interference can effectively silence C-erbB2 gene expression and inhibit growth of SACC-83 cells,which indicates the potential of targeting this gene as a novel gene therapy approach for the treatment of salivary gland adenoid cystic carcinoma.

Postoperative radiotherapy for thymus salivary gland carcinoma:A case report

BACKGROUND Salivary gland cancer is a rare disease in which cancer cells form in the tissues of the salivary glands.It mostly occurs in the glands that have secretion functions,such as the parotid gland,sublingual gland and submandibular gland.This is very rare when it occurs in other nonsecreting glands.Here,we report one case of salivary gland carcinoma occurring in the thymus and discuss related diagnoses and treatment progress.CASE SUMMARY One 33-year-old middle-aged man presented with a thymus mass without any clinical symptoms when he underwent regular physical examination.Later,the patient was admitted to the hospital for further examination.Computed tomography(CT)showed that there was a mass of 3 cm×2.8 cm×1.5 cm in the thymus area.The patient had no symptom of discomfort or tumor-related medical history before.After completing the preoperative examinations,it was confirmed that the patient had indications for surgery.The surgeon performed a transthoracoscope"thymectomy+pleural mucostomy"for him.During the operation,the tumor tissue was quickly frozen,and the symptomatic section showed a malignant tumor.The final pathological result suggested thymus salivary gland carcinoma-mucoepidermoid carcinoma(MEC).In the second month after surgery,we performed local area radiotherapy for the patient,with a total radiation dose of 50.4 Gy/28Fx.After 12 mo of surgery,the patient underwent positron emission tomography-CT examination,which indicated that there was no sign of tumor recurrence or metastasis.After 16 mo of operation,CT scan re-examination showed that there was no sign of tumor recurrence or metastasis.As of the time of publication,the patient was followed up for one and a half years.He had no sign of tumor recurrence and continued to survive.CONCLUSION The incidence of MEC in the thymus is low,and its diagnosis needs to be combined with clinical features and imaging methods.Histopathological analysis plays a key role in the diagnosis of the disease.Patients with early-stage disease have a good prognosis and long survival period.In contrast,patients with advanced-stage disease have a poor prognosis and short survival period.Combining radiotherapy and chemotherapy in inoperable patients may prolong survival.

Salivary duct carcinoma of the submandibular gland presenting a diagnostic challenge: A case report

BACKGROUND Salivary duct carcinoma(SDC)is a rare,extremely aggressive malignancy that arises in the submandibular gland.It can metastasize locally early and therefore is an important differential diagnosis of metastatic disease in cervical lymph nodes or specific lymphadenitis such as tuberculous cervical lymphadenitis.CASE SUMMARY We report a case of SDC in the submandibular gland that presented diagnostic difficulty.The lesion was coincidentally discovered through examination of the radiolucent area of the maxilla.Imaging failed to confirm the possibility of specific inflammation,leading us to execute an open biopsy to verify the diagnosis.The surgical specimen showed that the submandibular gland was primarily replaced with a calcified body.Following histological analysis and confirmation,we performed surgical resection,radiotherapy,and various chemotherapies.CONCLUSION Radiographic imaging characteristics of lymph node metastases of salivary gland cancer,especially of SDC,may resemble other cervical lymphadenitis;calcification at the submandibular gland is the landmark of SDC occurring at the submandibular gland.

EXPRESSION OF EPIDERMAL GROWTH FACTOR RECEPTOR IN DIFFERENT SALIVARY ADENOID CYSTIC CARCINOMA CELL LINES

Objective： To investigate the expression of epidermal growth factor receptor, a receptor tyrosine protein kinase, in the subcellular fractions of human salivary adenoid cystic carcinoma cell lines SACC-83 and SACC-LM. Methods： Low metastatic and high metastatic cells of the adenoid cystic carcinoma, SACC-83 and SACC-LM, were cultured. Their subcellular fractions were extracted. The expression of epidermal growth factor receptor was detected with Western blot method, and the results of protein expression were quantitatively analyzed by FluorChem V2.0 software. Results： The results of Western blot analysis indicated that, EGFR expression on the membrane of SACC-83 cells was significantly higher than that of SACC-LM cells, but its expression in cytoplasm was significantly less in the former than the later （P〈0.01）. In SACC-83 cell line, EGFR was over-expressed in membrane （P〈0.01）, but in SACC-LM cell line, EGFR was over-expressed in cytoplasm （P〈0.01）. Conclusion： The results suggest that the obtaining of metastasis ability is related to the high expression of EGFR protein in cytoplasm, so the molecular targeting therapy to EGFR may be an ideal treatment for the invasion and metastasis of salivary adenoid cystic carcinoma.

Inhibition of Salidroside on Salivary Gland Adenoid Cystic Carcinoma

To evaluate the role of salidroside on proliferation,apoptosis and invasiveness of salivary gland adenoid cystic carcinoma cells（SACC）,immunocytochemical staining was employed to detect proliferating cell nuclear antigen（PCNA）,caspase 3 and caspase 8 expression in SACC-2 cells.Modified Boyden chamber assay combined with laser confocal microscopy（LSCM） was used to evaluate the invasion and migration abilities of SACC-2 cells at different time point.Immunohistochemistry staining revealed that the expression of PCNA was significantly decreased（P0.01） after salidroside treatment.In contrast,salidroside treatment led to increased caspase 3 and caspase 8 in SACC-2 cells.Cell migration depth and number of cells that penetrated Boyden chamber were also decreased by salidroside.Salidroside potently inhibits the proliferation and simultaneously induces the apoptosis of SACC-2 cells.Migration and invasion of SACC-2 cells are also inhibited.Our data throw light on potential clinical application of salidroside to the patients with SACC.

CT and MRI Imaging Characteristics of Salivary Ductal Carcinoma

Objective:To investigate the CT and MRI features of salivary ductal carcinoma(SDC).Method:The imaging,clinical and pathological data of 32 patients with SDC confirmed by histomathology and operation were retrospectively analyzed.The location,size,shape,boundary,relationship with surrounding tissues,density,signal,enhancement mode,calcification,cystic degeneration and metastasis were observed.Result:Of the 32 patients with SDC,31 cases were isolated,17 were located in the parotid gland,8 in the submandibular gland,2 in the sinuses,2 in the orbit,1 in the part of the eye,and 1 in the sublingual gland.One case had multiple lesions located in the parotid gland.The maximum diameter of tumor was 1.5-7.2cm,and the median diameter was 3.0cm.The tumor showed diffuse growth in 11 cases and focal growth in 21 cases.The boundary was clear in 24 cases and unclear in 8 cases.The lesion may invade parapharyngeal space,soft palate,facial nerve,auditory nerve,skin,surrounding muscle and bone;There were 15 cases(47%)with lymph node metastasis and 1 case with lung metastasis.MRI showed that the solid part of the tumor was dominated by isointensity and low intensity on T1 WI,mixed high intensity on T2 WI,low intensity on T1 WI and high intensity on T2 WI.CT showed uneven tumor density,with equal or low density in 15 cases,high density in 4 cases,and calcification in 7 cases.Contrast-enhanced scan showed moderate to significant enhancement of the solid part.Conclusion:SDC is mostly single,prone to cystic necrosis and calcification,with strong aggressiveness and frequent lymph node metastasis.Understanding the imaging findings of SDC is helpful to improve the accuracy of preoperative diagnosis.

The impact of interval between surgery and postoperative radiotherapy in major salivary gland carcinoma

Background:Radiotherapy following primary operation is strongly recommended for salivary gland carcinomas(SGCs)with adverse features.The interval between surgery and the initiation of radiotherapy(SRT)varied and a prolonged SRT may cause failure of cancer treatment.However,the association of SRT with survival is unclear in major SGCs.Methods:This retrospective study included a total of 346 patients who underwent radiotherapy after the pri-mary operation from Fudan University Shanghai Cancer Center from 2005 to 2020.The best cutoffvalue of the SRT was determined by the maximum log-rank statistic method.The primary endpoint of the study was over-all survival(OS).Correlations between variables and OS were conducted by the univariable analysis using the Log-rank method,and a multivariate Cox proportional hazards regression was performed to identify the indepen-dent prognostic factors associated with OS.The estimated survival rates were captured using the Kaplan-Meier method.Results:With a median follow-up time of 70.31 months,the estimated 5-year OS,LRFS,and DMFS were 83.3%,80.1%,and 75.9%,respectively.The cutoffvalue for SRT was 8.5 weeks,while age,T stage,N stage,perineural invasion(PNI),pathological aggression,chemotherapy,and SRT were associated with OS in the univariable analysis.The Cox regression analysis demonstrated that older age(P<0.001),T3-4 tumors(P=0.007),positive N stage(P<0.001),pathological aggression(P=0.014),and longer SRT(P=0.009)were independent prognostic factors for major SGCs.Using the stratification model,we observed that delay in the SRT was associated with worse OS(P=0.006)in the high-risk group,whereas no significant difference was observed in the low-risk subgroup(P=0.61).Conclusions:The delay in the initiation of postoperative radiotherapy may be a prognostic factor for patients with major SGCs.It was suggested that radiotherapy should be delivered within 8.5 weeks following the operation,especially for patients with≥2 risk factors,including older age,high pathological aggression,T3-4 tumors,and positive N stage.

Secretory carcinoma——impact of translocation and gene fusions on salivary gland tumor

Secretory carcinoma（SC）, previously described as mammary analogue secretory carcinoma（MASC）, is a recently described salivary gland tumor which morphologically resembles mammary secretory carcinoma. The first description of SC/MASC, reported by Skálová et al. in 2010, was as a rare salivary carcinoma imitating secretory carcinoma of the breast. SC/MASC is a unique salivary gland tumor with morphological overlap with acinic cell carcinoma（Aci CC）, mucoepidermoid carcinoma（MEC）, and adenocarcinoma not otherwise specified（ADCNOS）. SC/MASC shares similar clinicopathological features with Aci CC. As a critical difference between SC/MASC and Aci CC, SC/MASC characteristically has the chromosomal translocation t（12;15）（p13;q25） which leads to a fusion gene between the ETV6 gene on chromosome 12 and the NTRK3 gene on chromosome 15. This genetic background is an important differential diagnostic finding for excluding other salivary gland tumors and may be a critical factor determining the prognosis for patients with SC/MASC. Research in recent years has provided a large body of new data on SC/MASC and suggests the possibility that the ETV6-NTRK3 translocation could be a therapeutic target. Here, we review the morphological and clinicopathological features of SC/MASC and discuss new directions for therapy.

A20 inhibits human salivary adenoid cystic carcinoma cells invasion via blocking nuclear factor-kB activation

Background A20, also known as tumor necrosis factor α induced protein 3 （TNFaip3）, is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B （NF-κB） activity and prevents tumor necrosis factor （TNF）-mediated programmed cell death. NF-κB is a transcription factor that regulates expression of genes involved in cell proliferation, cell survival and anti-apoptosis. Several studies have implicated that the NF-κB signal pathway is associated with angiogenesis and clinico-pathological process of adenoid cystic carcinoma （ACC） of the salivary glands.Methods The ability of overexpression of A2.0 to influence the biological behavior and invasion of ACC cells was examined. The cells were stably transfected with full-length A20 cDNA. Stable gene transfer was verified by realtime-polymerase chain reaction （PCR） and Western blot analysis. The change of cell biological behavior was examined by methyl thiazolyl tetrazolium （MTT） and NF-κB luciferase reporter assay and the invasion of the cells was examined by a Matrigel invasion chamber.Results pEGPFN3-A2.0 gene was stably transferred into ACC-2 cells and overexpressed. When cells were treated with TNFα, the NF-κB activity of ACC-2-A20 cells could be down-regulated about 46.32% in contrast to ACC-2-GFP cells （P〈0.05）. A20 potently inhibited growth of A20 transfectant ACC-2-A20 compared with control vector transfected groups and the ACC-2 empty control group （P〈0.05）. The ACC-2-A20 cells showed significantly reduced ability to invade through Matdgei-coated filters compared to ACC-2-GFP and ACC-2 cells. The inhibition rate was up to 71.05% （P〈0.05）. Conclusions A2.0 gene transfer is associated with decreased tumor invasion, in part via the down-regulation of NF-κB expression, providing evidence for a potential application of A20 in designing a treatment modality for salivary gland cancers such as ACC.