Human papillomavirus and salivary gland neoplasia: a p16INK4 immunohistochemical and in situ hybridisation study.

OBJECTIVE: This study aimed to evaluate the association between human papillomavirus infection and salivary gland tumours in a Scottish cohort. METHODS: Specimens from a range of salivary gland tumours operated on between 1997 and 2012 were studied. A tissue microarray constructed from tissue blocks was subjected to p16INK4 (cyclin-dependent kinase inhibitor 2A) immunohistochemistry and in situ hybridisation using probes specific for human papillomavirus, including types 16 and 18. RESULTS: A total of 61 tumours (benign and malignant) were deemed suitable for the study. p16INK4 staining yielded three (4.9 per cent) positive samples: one small cell carcinoma, one squamous cell carcinoma and one poorly differentiated carcinoma. Human papillomavirus in situ hybridisation demonstrated a positive signal in the latter sample only (1.6 per cent). CONCLUSION: This study demonstrated a very low human papillomavirus detection rate in salivary gland tumours. It can therefore be concluded that human papillomavirus infection is unlikely to play a role in salivary gland neoplasia. Rare human papillomavirus positive cases should be carefully evaluated to exclude the possibility of a metastatic lesion.

CMV-induced pathology: pathway and gene-gene interaction analysis.

Mucoepidermoid carcinoma (MEC) is the most prevalent malignant tumor in major and minor salivary glands (SGs). We have recently identified human cytomegalovirus (hCMV) as a principle component in the multifactorial causation of SG-MEC. This finding is corroborated by the ability of the purified mouse CMV (mCMV) to induce malignant transformation of SG cells in a three-dimensional in vitro mouse model, using a similar oncogenic signaling pathway. Our prior studies indicate that the core tumor microenvironment (TME) is a key regulator of pathologic progression, particularly the cancer-associated fibroblast (CAF) component. Studies of early CAFs immunodetect aberrant expression of ECM components, as well as multiple growth factors, cytokines and transcription factors. Here we present the mechanistic insight derived from a mathematical structure ("wiring diagram") used to model complex relationships between a highly relevant (p=9.43×10(-12)) global "cancer network" of 32 genes and their known links. Detailed characterization of the functional architecture of the examined "cancer network" exposes the critical crosstalk and compensatory pathways that limit the efficacy of targeted anti-kinase therapies.

Mucoepidermoid carcinoma does not harbor transcriptionally active high risk human papillomavirus even in the absence of the MAML2 translocation.

High risk human papillomavirus (HPV) is firmly established as an important cause of oropharyngeal carcinoma. Recent studies have also implicated HPV as a cause of mucoepidermoid carcinoma (MEC)-a tumor of salivary gland origin that frequently harbors MAML2 translocations. The purpose of this study was to determine the prevalence of transcriptionally active HPV in a large group of MECs and to determine whether HPV infection and the MAML2 translocation are mutually exclusive events. Break-apart fluorescence in situ hybridization for MAML2 was performed on a tissue microarray containing 92 MECs. HPV testing was performed using RNA in situ hybridization targeting high risk HPV mRNA E6/E7 transcripts. Of the 71 MECs that could be evaluated by FISH, 57 (80 %) harbored the MAML2 rearrangement. HPV was not detected in any of the 57 MECs that contained a MAML2 rearrangement, in any of the 14 MECs that did not contain the rearrangement, or in any of the 21 MECs where MAML2 status was unknown. High risk HPV does not appear to play any significant role in the development of MEC. It neither complements nor replaces MAML2 translocation in the tumorigenesis of MEC.

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.

Salivary mucoepidermoid carcinoma: demonstration of transcriptionally active human papillomavirus 16/18.

Herein we test the following hypotheses: (1) High-risk Human Papillomavirus (HR-HPV) may be involved in the etiology of mucoepidermoid carcinoma (MEC), and (2) The detection rate of HR-HPV in MEC has been increasing over time. Ninety-eight archival MEC specimens from three institutions spanning three decades were studied for HPV16/18 E6/E7 transcripts. RNA was extracted from formalin-fixed paraffin embedded specimens and HPV16/18 E6/E7 expression assessed by nested reverse transcription polymerase chain reaction (RT-PCR). A subset of MEC were also studied for MECT1-MAML2 fusion transcripts by nested RT-PCR and amplicon sequencing. The HPV expression data was validated by immunofluorescence (IF) with monoclonal HPV16/18 E6 antibody, PCR with the GP5+/6+ consensus primers, and sequencing of RT-PCR amplicons. HPV genome was localized by in-situ hybridization with the Ventana Inform HPVIII Family 16 probe. P16(INK4a) overexpression and aberrant p53 expression were assessed by immunohistochemistry. HPV16 E6/E7 transcripts were demonstrated in (29/98) 30% of MEC by RT-PCR. HPV18 E6/E7 transcripts were demonstrated in 13/98 (13%) of MEC by RT-PCR. Seven of 98 tumors (7%) demonstrated both HPV16/18. No significant association was found between HPV status and gender, age, and tumor site. All 13 HPV18+ MEC were diagnosed between 2001 and 2010, whereas 45 MEC diagnosed from 1977 to 2000 were negative for HPV18 (p = 0.002). By contrast, there was no significant difference with respect to HPV16 detection and date of diagnosis. All MEC that were positive for E6 protein were also HPV16/18 positive by RT-PCR. Sequencing a subset of RT-PCR amplicons confirmed HPV type- and region-specific sequences. PCR using GP5+/6+ consensus primers demonstrated HPV status concordance in 9 of 10 cases. DNA degradation was present in the last case; the RT-PCR amplicons were sequenced from this case which confirmed the presence of HPV type- and region-specific sequences. Strong (+4/+4) and diffuse (>50%) nuclear and cytoplasmic p16 expression was seen in 64% of MEC in the glandular regions, and 18% of MEC in the solid, squamoid regions. No correlation was seen between p16 expression and HPV status. Twenty-nine MEC (22 HPV+ and 7 HPV-negative) were selected for further evaluation for p53 expression. Strong aberrant nuclear p53 expression was present in only 2/22 HPV + MEC (9%, both Grade 3); no HPV-negative MEC demonstrated aberrant p53 expression. MECT1-MAML2 fusion transcripts were demonstrated in 23/37 (62%) MEC. No significant association was found between the presence of the MECT1-MAML2 fusion transcripts and tumor grade, HPV status, gender, era of diagnosis (2000 and earlier vs. 2001-2010) or tumor site. We demonstrate for the first time that transcriptionally active HPV16/18 is common to MEC. These findings were validated by demonstrating concordant results by separate PCR with consensus primers, and/or confirming the presence of HPV type- and region-specific sequences in the RT-PCR amplicons. We also visualized E6 viral oncoprotein and HPV genome within tumor cells. HR-HPV is thus potentially implicated in the pathogenesis of MEC. The frequency of HPV18 detection is significantly increased in MEC diagnosed after 2001, whereas we found no differences in the HPV16 detection rates per era of diagnosis.

Is human papilloma virus associated with salivary gland neoplasms? An in situ-hybridization study.

OBJECTIVE: HPV can infect cells of epithelial origin and is closely associated with carcinomas. Studies investigating its presence in salivary gland neoplasms are few and conflicting. METHODS: Detection of HPV types 16 & 18 was done on 34 formalin-fixed, paraffin-embedded archival material of different salivary gland neoplasms using Digene HPV types 16 & 18 probe using in situ hybridization technique. RESULTS: Eight of neoplastic salivary gland specimens were positively infected by HPV types 16 & 18. Seven of them were benign (4 Warthin's tumour, 2 pleomorphic adenoma and one myoepithelioma), in addition to one malignant specimen (lymphoma). Correlation was found between the incidence of HPV infection and histological differentiation of salivary gland neoplasms. CONCLUSIONS: An association exists between HPV infections and salivary gland neoplasms. However, given the sparse pattern of reactive cells, it cannot be confirmed that this virus is implicated in the aetiology of this group of tumours.

Human cytomegalovirus and mucoepidermoid carcinoma of salivary glands: cell-specific localization of active viral and oncogenic signaling proteins is confirmatory of a causal relationship.

Human cytomegalovirus (hCMV) infection is common. Although still controversial, there is growing evidence that active hCMV infection is associated with a variety of malignancies, including brain, breast, lung, colon, and prostate. Given that hCMV is frequently resident in salivary gland (SG) ductal epithelium, we hypothesized that hCMV would be important to the pathogenesis of SG mucoepidermoid carcinoma (MEC). This was initially supported by our finding that purified CMV induces malignant transformation in SG cells in an in vitro mouse model, and utilizes a pathogenic pathway previously reported for human MEC. Here we present the histologic and molecular characterizations of 39 human SG MECs selected randomly from a repository of cases spanning 2004-2011. Serial sections were obtained from formalin-fixed, paraffin embedded, tissue blocks from previous incisional or excisional biopsies. Immunohistochemical assays were performed for active hCMV proteins (IE1 and pp65) and the activated COX/AREG/EGFR/ERK signaling pathway. All four prospective causal criteria for viruses and cancer are fully satisfied: (1) protein markers for active hCMV are present in 97% of MECs; (2) markers of active hCMV are absent in non-neoplastic SG tissues; (3) hCMV-specific proteins (IE1, pp65) are in specific cell types and expression is positively correlated with severity; (4) hCMV correlates and colocalizes with an upregulation and activation of an established oncogenic signaling pathway (COX/AREG/EGFR/ERK). Thus, the evidential support reported here and previously in a mouse model is strongly confirmatory of a causal relationship between hCMV and SG mucoepidermoid carcinoma. To our knowledge, this is the first demonstration of hCMV's role in human oncogenesis that fully responds to all of Koch's Postulates as revised for viruses and cancer. In the absence of any contrary evidence, hCMV can reasonably be designated an "oncovirus."

Herpes oncolytic therapy of salivary gland carcinomas.

Oncolytic herpes simplex viruses (HSV) have demonstrated potent antitumoral effects against a variety of human malignancies in preclinical studies and are in early clinical trials. We explored the activity of an attenuated, replication-competent, oncolytic HSV (NV1023) for the treatment of human salivary gland carcinomas. NV1023 was able to successfully enter into 4 mucoepidermoid carcinoma (H292, H3118, HTB-41, UT-MUC-1) and 2 adenocarcinoma (HSY, HSG) cell lines, as measured by lacZ assays after exposure to 5 viral particles per cell (MOI 5). Viral plaque assays showed variation of viral replication within these cell lines, ranging from a 268-fold increase (H292) to a 3-fold increase (HSG) in viral titer. At MOI 5, all cell lines showed >95% cytotoxicity from NV1023 by Day 7, except for HSY (73%). At MOI 0.1, H3118 and UT-MUC-1 remained highly sensitive to NV1023, both showing >95% cytotoxicity by Day 7. The mucoepidermoid carcinomas were more sensitive to NV1023 at low viral concentrations compared with the adenocarcinomas. Flank tumors of H3118, HTB-41 and HSY in nude mice showed significant tumor volume reductions after a single intratumoral injection of NV1023 (2 x 10(7) plaque-forming units). These data suggest that oncolytic herpes viruses have significant efficacy entering, replicating within, and lysing human salivary gland carcinomas. These promising biologic agents should be further investigated as novel therapy for patients with salivary carcinomas failing conventional treatment.

[Mucoepidermoid carcinoma of nasopharynx: a report of twelve cases].

BACKGROUND & OBJECTIVE: Mucoepidermoid carcinoma of nasopharynx, a kind of primary adenocarcinoma of nasopharynx, is rare, and has seldom been reported. This article was to summarize the pathogenesis, clinical features, and treatment outcomes of this disease according to our experiences. METHODS: From Jan. 1975 to Dec. 2003, 12 patients with pathologically confirmed primary mucoepidermoid carcinoma of nasopharynx were treated in our hospital. We analyzed retrospectively their clinical data with literature review. RESULTS: The 12 patients with mucoepidermoid carcinoma of nasopharynx accounted for only 0.026% of all nasopharyngeal cancer patients diagnosed simultaneously in our hospital. Age of getting disease was 20 to 60 years old with male to female ratio of 2:1. Positive rates of Epstein-Barr virus serological tests (VCA-IgA, EA-IgA, and DNA enzyme ratio) were very low [50.0% (6/12), 11.1% (1/9), and 20.0% (1/5)]. Of the 11 patients with follow-up data, 4 received chemotherapy, 4 received surgery plus radiotherapy, and 3 received radiochemotherapy. The 5-, and 10-year survival rates of the 12 patients were 27.3%, and 9.0%. CONCLUSIONS: Mucoepidermoid carcinoma of nasopharynx is a special type of nasopharyngeal carcinoma with specific pathogenesis features. Surgery-predominant of complete lumpectomy combined treatment strategy is possibly accommodating.

Epstein-Barr virus infection in salivary gland tumors in children and young adults.

BACKGROUND: Epstein-Barr virus (EBV) infection has been implicated in the pathogenesis of certain subtypes of salivary gland tumors in the adult population. However, to the authors' knowledge its role in pediatric salivary gland tumors, a rare disease, has not been explored previously. METHODS: Thirteen cases of primary tumors of the salivary gland occurring in children were retrieved from the tumor registry at the St. Jude Children's Research Hospital in Memphis, Tennessee. Clinical data were analyzed from the medical records and formalin fixed, paraffin embedded tumor tissues were examined by the in situ hybridization (ISH) technique for the presence of latent EBV infection. RESULTS: Twelve of 13 tumors originated from the parotid gland and 1 originated from the submandibular gland. Mucoepidermoid carcinoma was the predominant tumor type; it was observed in seven patients, rhabdomyosarcoma was the diagnosis in three patients, acinic cell carcinoma was noted in two patients, and malignant fibrous histiocytoma was diagnosed in one patient. The ages of the patients ranged from 4.1-29.2 years, with a median age of 11 years. The outcome was excellent with all patients alive and free of disease at the time of last follow-up. The ISH tested negative in all tumor samples. CONCLUSIONS: Based on the results of the current study, EBV infection does not appear to play a major role in the pathogenesis of pediatric salivary gland tumors.