Hsp27 and Hsp60 in human submandibular salivary gland: Quantitative patterns in healthy and cancerous tissues with potential implications for differential diagnosis and carcinogenesis.

Tumors of the submandibular salivary gland (SMG) are uncommon but sufficiently frequent for the physician to consider them in routine examinations and for the pathologist to be prepared to differentiate them from other tissue abnormalities. However, scarcity of specimens makes training difficult, a situation compounded by the lack of accepted universal diagnostic guidelines. Furthermore, there is little information on the chaperone system (CS) of the gland, despite the increasing evidence of its participation in carcinogenesis as a biomarker for diagnosis and patient follow up, and in the mechanisms by which the tumor cells thrive. We are investigating this aspect of various tumors, and here we describe standardized methods for assessing the tissue levels of two chaperones, Hsp27 and Hsp60, in normal SMG and its tumors. We present illustrative results obtained with immunohistochemistry (IHC) and immunofluorescence-confocal microscopy (IF-CM), which we propose as a platform onto which a data base could be built by adding new information and which would provide material for developing guidelines for tumor identification and monitoring. The initial findings are encouraging in as much as the tumors surveyed showed quantitative patterns of Hsp27 and Hsp60 that distinguished tumoral from normal tissue and certain tumors from the others, and the results from IHC were confirmed by IF-CM.

The quantitative analysis of the human papillomavirus DNA load in submandibular gland lesions with droplet digital polymerase chain reaction.

BACKGROUND: The relationship between infection with human papillomavirus (HPV) and tumorigenesis of salivary gland remains controversial. OBJECTIVES: This study explored the relationship between HPV and salivary gland lesions as well as that of the HPV infection status and p16INK4A immunoreactivity. The HPV DNA loads were also quantitatively evaluated. MATERIALS AND METHODS: Tissue samples from 31 submandibular gland lesions were evaluated. p16INK4A immunohistochemical (IHC) staining, nested polymerase chain reaction (PCR), DNA sequencing, and droplet digital PCR (ddPCR) were performed. RESULTS: Non-neoplastic lesion, benign tumors, and malignant tumors were noted in 9, 16, 6 cases, respectively. p16INK4A immunoreactivity was higher in malignant tumors than in benign tumors (50.0% vs. 6.3%). Single PCR with MY09/11 found that all samples were negative. Nevertheless, nested PCR revealed a high HPV-DNA positivity rate of 96.8%. No relationship between the HPV status and p16INK4A immunoreactivity was shown. HPV-18 was the only subtype identified in this study. ddPCR showed significantly lower HPV-18 DNA loads in submandibular gland lesions than in oropharyngeal cancers. CONCLUSIONS: HPV-DNA positivity and p16INK4A-immunoreactivity were not correlated in submandibular gland lesions. The loads of HPV DNA detected in this study were small. HPV positivity therefore may not be associated with tumorigenesis of the submandibular gland.

Prognostic factors and outcome analysis of salivary duct carcinoma.

OBJECTIVE: Salivary duct carcinoma (SDC) is highly aggressive, with high rates of recurrence and nodal and distant metastases. The aim of the current study was to evaluate the clinical implication of EGFR and HER2 expression for predicting prognosis and to identify the factors associated with outcome. METHODS: The medical records of 28 patients with SDC underwent surgery and adjuvant RT. Expression of c-erbB-2 and EGFR was determined immunohistochemically on the 25 SDC specimens. Disease-free survival (DFS), overall survival (OS) and distant metastasis-free survival (DMFS) were analyzed. RESULTS: Three-year DFS, OS and DMFS rates were 38.3%, 78.1% and 45.7%, respectively. Expression of c-erbB-2 and EGFR was seen in 64% and 40%. c-erbB-2 and EGFR expression did not correlate with recurrence or metastasis. Advanced N classification and perineural invasion (PNI) were significant predictors of DFS and DMFS. CONCLUSION: c-erbB-2 and EGFR expression did not correlate with recurrence or metastasis. Despite aggressive surgery and RT, approximately 50% of SDCs failed systemically. More effective therapy to inhibit distant metastases in patients with advanced N classification and PNI should be considered.

Primary Merkel Cell Carcinoma of the Submandibular Gland: When CK20 Status Complicates the Diagnosis.

Merkel cell carcinoma is a neuroendocrine tumor that occurs predominantly on the sun-exposed skin, with rare cases in the extracutaneous sites. It represents one of the extremely rare malignant neuroendocrine tumors of the salivary glands. We report a case of primary Merkel cell carcinoma of the right submandibular gland. The preoperative diagnosis was doubtful and the definitive histological diagnosis proved to be very difficult considering the extreme rarity of this tumor. The intraoperative evaluation of the macroscopic characteristics of the lesion led to an elective lymph node dissection. The extreme aggressiveness of the disease has resulted in the necessity of a new post-operative staging and in a multimodal treatment. This is the first primary submandibular gland Merkel cell carcinoma described in the literature. Differential diagnosis may be challenging and proper hematoxylin-eosin staining and immunohistochemical studies are mandatory.

Primary lymphoepithelioma-like carcinoma of salivary glands: a clinicopathological study of 21 cases.

Lymphoepithelioma-like carcinoma (LELC) of salivary glands is a rare kind tumor. In this study, the authors evaluated 21 patients with LELC of salivary glands who had long-term follow-up. Clinical characteristics, Epstein-Barr virus (EBV) infection, immunohistochemical features, oncoprotein expression, treatments, and outcomes were analyzed. All patients were Chinese. Their ages ranged from 20 to 73 years. All tumors showed the typical syncytial growth pattern of undifferentiated epithelial cells with a significant lymphocyte reaction. All of patients were found by in situ hybridization to have the EBV genome. All tumors showed positive immunostaining of AE1/AE3, CK5/6 and p63. Nearly all cases had bcl-2 oncoprotein expression, but the detection rate of p53, and c-erb B-2 expression was extremely low. LELC of salivary glands is a distinct entity of salivary cancer. LELC of salivary glands can receive multimodality treatment and has a better prognosis similar to that of nasopharyngeal carcinoma.

Salivary duct carcinoma with rhabdoid features: report of 2 cases with immunohistochemical and ultrastructural analyses.

BACKGROUND: Salivary duct carcinoma with rhabdoid features is extremely rare. METHODS: We report 2 cases of salivary duct carcinoma with rhabdoid features treated at our institution. RESULTS: Case 1 was a 44-year-old Japanese man who had swelling in the left parotid region. This tumor consisted of residual pleomorphic adenoma and widely invasive carcinoma, which showed a diffuse growth pattern by atypical rhabdoid cells. Case 2 was a 66-year-old Japanese man who had swelling of the right cervical region. This submandibular tumor was also composed of both residual pleomorphic adenoma region and invasive adenocarcinoma components, whereas some metastatic lesions were purely composed of rhabdoid cells. Such cells were strongly and diffusely positive for cytokeratins (CKs), gross cystic disease fluid protein-15 (GCDFP), and androgen receptor (AR). Case 1 was also positive for Her-2 and p53. CONCLUSION: Both patients were diagnosed with carcinoma ex pleomorphic adenoma and their carcinomatous components were composed of salivary duct carcinoma with rhabdoid features, which is a highly aggressive tumor, similar to salivary duct carcinoma.

Most nonparotid "acinic cell carcinomas" represent mammary analog secretory carcinomas.

Acinic cell carcinoma (ACC) is a low-grade salivary gland malignancy characterized by serous acinar differentiation. Most ACCs arise in the parotid gland, but ACCs have been reported to originate in nonparotid salivary glands where serous acini are less abundant. Given the recent discovery of mammary analog secretory carcinoma (MASC)-a salivary malignancy that histologically mimics ACC-a retrospective reevaluation of nonparotid ACCs is warranted. The surgical pathology archives of The Johns Hopkins Hospital were searched for all ACCs arising outside of the parotid gland. For each case, the histologic slides were reviewed; immunohistochemical analysis (mammaglobin, S100 protein) was performed; and confirmatory ETV6 breakapart fluorescence in situ hybridization assay was completed. Demographic and clinical data were obtained from the medical records. Fourteen extraparotid tumors diagnosed as ACC were identified. Eleven of 14 (79%) tumors harbored the ETV6 translocation (oral cavity=9 of 11; submandibular gland=2 of 2). The translocation-positive tumors occurred in 7 women and 4 men ranging in age from 20 to 86 years (mean, 56 y) and usually presented as painless masses. Immunohistochemistry for mammaglobin and S100 was positive in all 11 translocation-positive tumors but negative in the 3 translocation-negative tumors. Histologically, the translocation-positive tumors exhibited uniform cells with vacuolated cytoplasm, microcystic/cystic and papillary architecture, and intraluminal secretions; however, the presence of basophilic cytoplasmic granules was conspicuously absent. Basophilic cytoplasmic granules, indicative of true serous acinar differentiation, were present in the 3 translocation-negative tumors. Of the translocation-positive tumors, only 1 locally recurred, and none metastasized. Most alleged ACCs of nonparotid origin actually represent misclassified MASCs. The impact of diagnostic error is mitigated by the low-grade nature of MASC that, like ACCs, do not appear to be clinically aggressive.

Sclerosing mucoepidermoid carcinoma with eosinophilia of the salivary gland: case report and review of the literature.

Sclerosing mucoepidermoid carcinoma with eosinophilia (SMCE) of the salivary gland is a rare variant of mucoepidermoid carcinoma. We report a case of SMCE in the right submandibular gland of a 79-year-old man. Fine needle aspiration cytology revealed cohesive clusters of atypical squamous epithelial cells admixed with cells containing intracytoplasmic mucin and eosinophils. Histologically, the tumor was composed of epithelial nests with keratinizing cells occasionally present at the center, as well as peripherally located atypical basaloid cells, and some mucin-containing cells embedded in a fibrosclerotic stroma, which were accompanied by a prominent lymphoplasmacytic and eosinophilic infiltrate. Inflammatory infiltrate and stromal fibrosclerosis also were seen in the non-neoplastic salivary gland tissue adjacent to the tumor. Immunohistochemically, many plasma cells were IgG4-positive. The postoperative serum IgG4 level was elevated. Our reverse transcription-polymerase chain reaction using formalin-fixed, paraffin-embedded tumor tissue failed to detect any fusion-gene transcripts which are specifically identified in ordinary mucoepidermoid carcinoma. The findings of the present case suggest that this rare type of salivary gland carcinoma may be associated with a chronic inflammatory condition such as IgG4-related sclerosing disease. Only 23 cases of sclerosing mucoepidermoid carcinoma with or without eosinophilic infiltratie have been reported to date in such an anatomical location.

A mucin-rich variant of salivary duct carcinoma with a prominent mucinous component, a tumor that mimics mucinous adenocarcinoma.

The mucin-rich variant of salivary duct carcinoma (mSDC) is a rare type of salivary duct carcinoma. mSDC usually has both conventional SDC and mucinous adenocarcinoma-like areas. This article describes a first case of mSDC in which 95% of the tumor consisted of a mucinous area without no solid conventional SDC, so that the tumor mimicked mucinous adenocarcinoma. A 55-year-old man was evaluated for a 14 mm mass in the left submandibular gland. The tumor showed that floating tumor nests in a prominent mucinous lake. Some floating tumor nests had focal cribriform pattern with comedo necrosis, and all tumor cells had immunoreactivity for androgen receptor, gross cystic disease fluid protein 15, and Her-2/neu. A diagnosis of mSDC was rendered. mSDC with prominent mucinous component sometimes resembles mucinous adenocarcinoma. Identifying specific histological and immunohistochemical features of floating tumor nests in the mucinous area are important for the diagnosis.

Cytomegalovirus-induced salivary gland pathology: AREG, FGF8, TNF-α, and IL-6 signal dysregulation and neoplasia.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor originating in major and minor salivary glands (SGs). Although the precise multifactorial etiology of human SG-MEC is largely unknown, we have recently shown that cytomegalovirus (CMV) is an important component of MEC tumorigenesis. Despite the well-documented overexpression of the EGFR → ERK signaling pathway in SG-MEC, there has been limited to no clinical success with inhibition of this pathway. Using our previously characterized mouse model of CMV-induced SG dysplasia/neoplasia, we report that inhibitors of the EGFR → ERK pathway do not ameliorate or rescue well-established pathology, either singly or in combination, but they do inhibit the evolution of progressive pathogenesis ("disease tolerance") in the face of mounting CMV burden. Failure to rescue SG pathology, suggested a possible increase in the ligand levels of alternative pathways that share cell proliferation and survival effectors (e.g. ERK and PI3K). Here we present evidence of a highly significant upregulation of ligands for the EGFR, FGFR, IL-6R, and TNFR signaling pathways, all of which converge upon the Raf/MEK/ERK amplifier module. This explains our finding that even in the presence of the highest nontoxic dose of an ERK phosphorylation inhibitor, pERK is undiminished. Given the considerable pathway crosstalk, a deep understanding of subversion and dysregulation of the SG interactome by CMV is a priori quite daunting. Circumventing this dilemma, we present evidence that concurrent inhibition of ERK phosphorylation (U0126) and CMV replication (acyclovir) obviates progressive pathogenesis and results in complete SG rescue (tumor regression). These findings provide a mechanistic foundation for potential clinical trials that utilize similar concurrent treatment with extant FDA-approved drugs.

Signet-ring cell change in adenoid cystic carcinoma: a clinicopathological and immunohistochemical study of four cases.

AIMS: Signet-ring cell (SRC) change has not been reported in adenoid cystic carcinoma (ACC). The aim of this study was to describe the clinicopathological and immunohistochemical findings in four cases of ACC with SRCs (ACC-SRC), in which the relative proportion of the SRC component ranged from 25% to 50%. METHODS AND RESULTS: The median age was 58 years (range: 48-81 years), and all patients were women. The involved sites were sinonasal, lip, and submandibular. Two patients developed lung metastasis, and one died of disease 63 months after tumour resection. Neither mucinous nor lipid substances were detected in the SRCs. These showed positive staining for AE1/AE3, cytokeratin 14, and epithelial membrane antigen, which highlighted the intracytoplasmic vacuole borders. The SRC nests were surrounded by myoepithelial cells positive for α-smooth muscle actin and p63. The SRCs showed similar p53 positivity but lower Ki67 and mitotic indices than the conventional component. SRCs were c-Myb-negative. Ultrastructural examination revealed that the intracytoplasmic vacuoles were lumina lined by microvilli. CONCLUSIONS: ACC-SRC is a non-mucin-producing and non-lipid-producing phenomenon, possibly related to disturbed differentiation of ductal/luminal cells. This cellular modification in ACC apparently does not change the biological behaviour of the tumour, but it may cause significant diagnostic problems, particularly in incisional biopsies.

Neuroendocrine tumors of the submandibular gland: literature review and report of a case.

Primary salivary neuroendocrine neoplasms are extremely rare, with only 1 case of carcinoid tumor and 7 cases of primary large-cell neuroendocrine carcinomas having been reported. Salivary small-cell neuroendocrine carcinomas are more frequent, accounting for less than 2% of all salivary malignancies. In addition to a literature review on the subject, the authors describe a rare case of malignant intermediate-grade neuroendocrine tumor of the submandibular gland.

Pleomorphic adenoma of the salivary glands with intravascular tumor deposits: a diagnostic pitfall.

The diagnosis of pleomorphic adenoma (PA) of salivary glands is usually straightforward posing few diagnostic problems for the general surgical histopathologist. The purpose of our investigation was to present a series of 22 cases of PA of major salivary glands, each of which contained small foci of tumor within vascular spaces. This feature has previously been described very rarely in PA and may represent a significant diagnostic pitfall. The patients included 12 women and 10 men, ranging in age at diagnosis from 17 to 82 years. Histopathologically, all 22 tumors displayed the features of PA with mixed epithelial and myoepithelial growth patterns and chondromyxoid areas. None of these neoplasms showed any cytologic evidence of malignancy. In all cases, there were multiple dilated thin-walled and/or muscular thick-walled blood vessels containing small intraluminal collections of neoplastic cells with or without myxoid stromal components. The intravascular tumor cells expressed cytokeratins, and in some cases they were also immunoreactive for S-100 protein, GFAP, D2-40, and p63 protein. The intravascular location of the neoplastic cells was confirmed by CD31, CD34, and factor VIII-related antigen immunostains. Reaction for D2-40 was negative in the endothelium of the involved vessel in all cases, confirming that they were vascular rather than lymphatic channels. Seven patients (36%) underwent fine-needle aspiration biopsy 25 days to several years before excision of the tumor. Follow-up of the patients in our series revealed no cases of recurrence or metastasis (range, 6 mo to 9.5 y; mean 3.8 y; median 3.5 y). The biological significance of intravascular tumor in PA is not clear, but there is growing evidence that it is an innocuous phenomenon that might be related to artifactual spillage caused by tumor injury presumably by either fine-needle aspiration or intraoperative trauma.

Muscarinic regulation of SCA-9 cell proliferation via nitric oxide synthases, arginases and cyclooxygenases. Role of the nuclear translocation factor-κB.

The submandibular gland-derived tumor cell line SCA-9 is considered a useful tool to study the signaling pathways involved in proliferation, and their regulation, triggered by different stimuli. It is proposed that the non neuronal cholinergic system: acethylcholine, the enzymes that synthesize and degrade it, and the nicotinic and muscarinic receptors, play a key role in tumorigenesis. Here, we investigate the role of muscarinic receptors in SCA-9 cell proliferation, and the modulation of cholinergic signaling pathways exerted by the nuclear transcription factor κB (NF-κB). The activation of cholinergic receptors by carbachol (10⁻9M) increased cell proliferation (P<0.001). This was prevented by preincubating cells with the muscarinic antagonist atropine but not by mecamylamine, a nicotinic receptor blocker. Phospholipase C (PLC)/nitric oxide synthase (NOS)/arginase pathway is involved in this effect, since carbachol stimulated nitric oxide production, increased NOS2 and NOS3 expressions, urea production, and arginase II expression (P<0.001). Also, phospholipase A2 (PLA2)/cyclooxygenase (COX) pathway is up-regulated in carbachol-induced SCA-9 cell proliferation, because prostaglandin E2 liberation (P<0.001) is increased and COX-1 expression is turned up (P<0.001). Interactions between PLC/NOS/arginases and PLA2/COX pathways via its metabolites were detected. SCA-9 cells exhibit a constitutive activation of NF-κB, which regulates carbachol-induced NOS2 and 3, arginase II and COX-1 expressions. In addition, protein kinase C is involved in the up-regulation of NOS2 and arginase II enzymes induced by carbachol via NF-κB. In conclusion, the activation of cholinergic receptors in SCA-9 tumor cells promotes proliferation via muscarinic effector enzymes, and reveals the participation of NF-κB at this step of tumorigenesis.