The protective effect of p16(INK4a) in oral cavity carcinomas: p16(Ink4A) dampens tumor invasion-integrated analysis of expression and kinomics pathways.

A large body of evidence shows that p16(INK4a) overexpression predicts improved survival and increased radiosensitivity in HPV-mediated oropharyngeal squamous cell carcinomas.(OPSCC). Here we demonstrate that the presence of transcriptionally active HPV16 in oral cavity squamous cell carcinomas does not correlate with p16(INK4a) overexpression, enhanced local tumor immunity, or improved outcome. It is interesting that HPV-mediated oropharyngeal squamous cell carcinomas can be categorized as having a 'nonaggressive' invasion phenotype, whereas aggressive invasion phenotypes are more common in HPV-negative squamous cell carcinomas. We have developed primary cancer cell lines from resections with known pattern of invasion as determined by our validated risk model. Given that cell lines derived from HPV-mediated oropharyngeal squamous cell carcinomas are less invasive than their HPV-negative counterparts, we tested the hypothesis that viral oncoproteins E6, E7, and p16(INK4a) can affect tumor invasion. Here we demonstrate that p16(INK4a) overexpression in two cancer cell lines (UAB-3 and UAB-4), derived from oral cavity squamous cell carcinomas with the most aggressive invasive phenotype (worst pattern of invasion type 5 (WPOI-5)), dramatically decreases tumor invasiveness by altering expression of extracellular matrix remodeling genes. Pathway analysis integrating changes in RNA expression and kinase activities reveals different potential p16(INK4a)-sensitive pathways. Overexpressing p16(INK4a) in UAB-3 increases EGFR activity and increases MMP1 and MMP3 expression, possibly through STAT3 activation. Overexpressing p16(INK4a) in UAB-4 decreases PDGFR gene expression and reduces MMP1 and MMP3, possibly through STAT3 inactivation. Alternatively, ZAP70/Syk might increase MUC1 phosphorylation, leading to the observed decreased MMP1 expression.

Caveolin-1 accumulation in the tongue cancer tumor microenvironment is significantly associated with poor prognosis: an in-vivo and in-vitro study.

BACKGROUND: Caveolin-1 (CAV1) may be upregulated by hypoxia and acts in a tumor-dependent manner. We investigated CAV1 in tongue squamous cell carcinoma (TSCC) and its association with clinical outcomes, and studied in vitro possible ways for CAV1 accumulation in the tumor microenvironment (TME). METHODS: TSCC cases (N = 64) were immunohistochemically stained for CAV1. Scores were separately assessed in the tumor and TME and plotted for association with recurrence and survival (univariate analysis with log-rank test). In vitro studies were performed on a 3D myoma organotypic model, a mimicker of TME. Prior to monoculturing HSC-3 tongue cancer cells, the model underwent modifications in oxygenation level (1%O2 hypoxia to upregulate CAV1) and/or in the amount of natural soluble factors [deleted by 14-day rinsing (rinsed myoma, RM), to allow only HSC-3-derived factors to act]. Controls included normoxia (21%O2) and naturally occurring soluble factors (intact myoma, IM). HSC-3 cells were also co-cultured with CaDEC12 cells (fibroblasts exposed to human tongue cancer). CAV1 expression and cellular distribution were examined in different cellular components in hypoxic and rinsed myoma assays. Twist served as a marker for the process of epithelial-mesenchymal transition (EMT). Exosomes isolated from HSC-3 media were investigated for containing CAV1. RESULTS: Expression of CAV1 in TSCC had a higher score in TME than in the tumor cells and a negative impact on recurrence (p = 0.01) and survival (p = 0.003). Monocultures of HSC-3 revealed expression of CAV1 mainly in the TME-like myoma assay, similar to TSCC. CAV1+, alpha-smooth muscle actin (αSMA) + and Twist + CAF-like cells were observed surrounding the invading HSC-3, possibly reflecting EMT. RM findings were similar to IM, inferring action of HSC-3 derived factors, and no differences were seen when hypoxia was induced. HSC-3-CaDEC12 co-cultures revealed CAV1+, αSMA+ and cytokeratin-negative CAF-like cells, raising the possibility of CaDEC12 cells gaining a CAF phenotype. HSC-3-derived exosomes were loaded with CAV1. CONCLUSIONS: Accumulation of CAV1-TME in TSCC had a negative prognostic value. In vitro studies showed the presence of CAV1 in cancer cells undergoing EMT and in fibroblasts undergoing trans-differentiation to CAFs. CAV1 delivery to the TME involved cancer cell-derived exosomes.

Insights into the role of components of the tumor microenvironment in oral carcinoma call for new therapeutic approaches.

The research on oral cancer has focused mainly on the cancer cells, their genetic changes and consequent phenotypic modifications. However, it is increasingly clear that the tumor microenvironment (TME) has been shown to be in a dynamic state of inter-relations with the cancer cells. The TME contains a variety of components including the non-cancerous cells (i.e., immune cells, resident fibroblasts and angiogenic vascular cells) and the ECM milieu [including fibers (mainly collagen and fibronectin) and soluble factors (i.e., enzymes, growth factors, cytokines and chemokines)]. Thus, it is currently assumed that TME is considered a part of the cancerous tissue and the functionality of its key components constitutes the setting on which the hallmarks of the cancer cells can evolve. Therefore, in terms of controlling a malignancy, one should control the growth, invasion and spread of the cancer cells through modifications in the TME components. This mini review focuses on the TME as a diagnostic approach and reports the recent insights into the role of different TME key components [such as carcinoma-associated fibroblasts (CAFs) and inflammation (CAI) cells, angiogenesis, stromal matrix molecules and proteases] in the molecular biology of oral carcinoma. Furthermore, the impact of TME components on clinical outcomes and the concomitant need for development of new therapeutic approaches will be discussed.

The hypoxic tumor microenvironment regulates invasion of aggressive oral carcinoma cells.

Invasion is an important hallmark of cancer involving interactions between the tumor microenvironment and the cancer cells. Hypoxia, low oxygen level, is related to increased invasion and metastasis in many cancers. The aim was to elucidate the effect of hypoxia on invasion of oral squamous cell carcinoma cells (OSCCs), and the applicability of a novel 3-dimentional myoma organotypic invasion model in hypoxia experiments. OSCC cell lines (primary oral carcinoma derived cells UT-SCC-43A, recurrent oral carcinoma cells UT-SCC-43B and aggressive tongue carcinoma cells HSC-3) were studied for their migration and invasion capabilities under normoxia, hypoxia, and in the presence a hypoxia-mimicker cobalt chloride. As expected, the recurrent UT-SCC-43B cells were significantly more aggressive than the primary tumor derived cells. In contrast to tongue carcinoma HSC-3 cells, they only mildly responded to hypoxia in the migration or invasion assays, indicating a cell line specific response of hypoxia on the invasive potential. The modification of the organotypic human tissue-derived matrix via the removal of various yet unidentified soluble factors by rinsing the tissue resulting in stripped matrix substantially changed the invasion pattern of HSC-3 cells and the outcomes of hypoxic treatments. Only in the stripped tissue hypoxia significantly increased invasion, whereas in native intact tissue the induced invasion was not observed. This demonstrates the importance of the soluble factors to the invasion pattern and to the hypoxia response. A metastasis and poor prognosis marker, hypoxia-regulated lysyl oxidase (LOX), was present in the myoma tissue, but could be removed by rinsing. The inhibition of LOX resulted in a decrease in invasion area, but only very mildly in invasion depth. Thus, it may have a role in the modulation of the invasion pattern. Another hypoxia-related poor prognosis marker carbonic anhydrase 9 (CAIX) was induced in HSC-3 cells both by the hypoxic exposure and interestingly in invading HSC-3 cells inside the tissue even in normoxic conditions. In conclusion, this suggests that the intact myoma organotypic model offers optimally hypoxic surroundings, thus being an excellent human tumor microenvironment mimicker.

Inflammatory cells of immunosuppressive phenotypes in oral lichen planus have a proinflammatory pattern of expression and are associated with clinical parameters.

OBJECTIVES: We sought to investigate the expression of cells with immunosuppressive/protumorigenic phenotypes in oral lichen planus (OLP), such as M2-tumor-associated macrophages (TAM2), myeloid-derived suppressive cells (MDSCs), and regulatory T cells (Tregs) in association with clinical parameters. MATERIALS AND METHODS: Cases of hyperkeratotic (HK)-OLP (n = 23) and erosive (E)-OLP (n = 26) were immunohistochemically stained to determine the percentages of CD163-TAM2, CD80-MDSCs, and FOXP3-Tregs of proinflammatory CD121a-Th17, CD4 and CD8 lymphocytes, and of cells positive for nuclear factor kappa B (NF-κB) and transforming growth factor beta. Clinical parameters included symptoms, treatment approach, treatment response, and others. RESULTS: The inflammatory infiltrate in HK-OLP and E-OLP contained immunosuppressive cells; however, their pattern of expression was compatible with a proinflammatory response [membranous CD163-TAM2 staining (not extracellular), CD80+ lymphocytes (not macrophages), and a few Tregs]. The presence of CD4+, CD8+, and CD121a+ T lymphocytes was extensive. TAM2 were more frequent in E-OLP than in HK-OLP (P = 0.017). A higher frequency of CD80+ lymphocytes was associated with partial to no response to treatment (P = 0.028). Nuclear expression of NF-κB in the inflammatory cells was absent. CONCLUSIONS: The pattern of expression of the immunosuppressive cells, together with numerous CD4+, CD8+, and Th17-CD121a+ lymphocytes, suggest an extensive proinflammatory response rather than an immunosuppressive/protumorigenic response. CLINICAL RELEVANCE: The frequency of selective types of inflammatory cells calls for individual profile analyses of inflammatory infiltrates and individually adjusted treatment.

[Tongue cancer: an ongoing problem which needs new therapeutic approaches].

Oral cancer together with oropharyngeal cancer constitutes the tenth most common malignancy in men worldwide. In spite of a general trend of mild decreases in the frequency of oral and pharyngeal cancers, there appears to be an increasing trend in the occurrence of oral tongue cancer (Localized in the anterior two-thirds of the tongue). Furthermore, in recent years, tongue cancer is on the rise in young patients (fifth decade and less). Tongue cancer is the most frequent type of malignancy in the oral cavity and is characterized by remarkable aggressive biological behavior. The 5-year survival rate in patients with tongue cancer has steadily remained lower than 50% during the last 40 years. This is in contrast to the considerable improvement in the survival rates that have been achieved in patients with solid tumors in other parts of the body, due to the introduction of advanced diagnostic means and new treatment modalities. Our recent studies have shown an important role that components of the tumor microenvironment (TME) play in the proliferation, invasion and local and distant spread of tongue cancer, which is not less than that of the cancer cells. The results of these studies suggest the development of new therapeutic modalities in tongue cancer, which should combine anti-cancer and anti-TME agents.

Immunohistochemical features of 3,3',4,4'-tetrachloroazobenzene-induced rat gingival lesions.

Gingival lesions of squamous hyperplasia, cystic keratinizing hyperplasia (CKH), and squamous cell carcinoma (SCC) can be induced in rats treated by chronic gavage with 10-100 mg/kg 3,3',4,4'-tetrachloroazobenzene. We evaluated gingival squamous hyperplasia (GSH), CKH, and SCC for the immunohistochemical pattern of expression of carcinogenesis-associated markers. The 3 types of lesions and controls were stained with proliferation markers (proliferating cell nuclear antigen [PCNA] and cyclin-D1), tumor-suppressor markers (ß-catenin and mammary serine protease inhibitor [maspin]) and stroma-related markers (α-smooth muscle actin [SMA] and osteonectin/SPARC). The lesions had common immunohistochemical characteristics that differed in their expression patterns among the various diagnoses. PCNA and cyclin-D1 expression was higher in GSH, CKH, and SCC than in controls. The normal membranous expression of ß-catenin was lower in GSH, and almost absent in CKH and SCC. Maspin expression was similar in GSH and controls, whereas both CKH and SCC showed decreased expression. SMA and/or osteonectin/SPARC were seen in stromal cells in CKH and SCC. Collectively, there appears to be a progression from hyperplastic and cystic lesions toward malignancy based on the morphological changes, supported by the expression of carcinogenesis-associated proteins. The exact sequence of events leading to SCC remains to be defined in a time-dependent manner.

Cancer-associated fibroblasts and epithelial-mesenchymal transition in metastatic oral tongue squamous cell carcinoma.

We examined cancer-associated fibroblasts (CAFs) and a panel of immunohistochemical markers of epithelial-mesenchymal transition (EMT) in 19 pair-matched oral tongue squamous cell carcinoma (SCC) and metastatic tumors to regional lymph nodes (RLNs). alpha-Smooth muscle actin (alpha-SMA) was studied to identify CAFs. EMT was studied with syndecan-1, Cadherin-11, fibroblast-specific protein (FSP)-1, secreted protein acidic and rich in cysteine (SPARC) and Twist. Triple immunostaining in RLNs was used to highlight the carcinoma cells (E-cadherin and Ki-67) and their relationship to the CAFs (alpha-SMA). We found that metastatic RLNs hosted CAFs similarly as in pair-matched primary tumors. Expression of EMT markers is common in both primary and metastatic tumors. We demonstrate that metastatic carcinoma cells (Ki-67 positive) downregulate E-cadherin expression at the periphery of cancer islands, where they are in direct contact with CAFs. The supporting connective tissue microenvironment also commonly expresses syndecan-1, Cadherin-11, FSP-1, and SPARC. In conclusion, CAFs are common to both primary and metastatic SCC. We hypothesize that CAFs not only promote tumor invasion but also facilitate metastases, either by cometastasizing and/or being recruited to lymph nodes. Evidence of EMT is common within primary tumors and metastatic SCC and may be further modulated by CAFs.

Tumor-host histopathologic variables, stromal myofibroblasts and risk score, are significantly associated with recurrent disease in tongue cancer.

Margin status, a major prognostic parameter in oral cancer, was analyzed vis-à-vis the histopathologic parameters of risk scores and stromal myofibroblasts. Specimens of tongue carcinoma (n = 50) were submitted to a risk score assignment consisting of the worst pattern of invasion, lymphocytic infiltration, and perineural invasion. Frequency of stromal myofibroblasts (alpha-smooth muscle actin stain) was assessed. A triple immunostaining assay with E-cadherin, Ki-67 and alpha-smooth muscle actin was used to identify carcinoma cells undergoing epithelial-mesenchymal transition. Margins were considered 'clean' if the tumor was >or=5 mm away from them. Patients

The effect of desalivation on the malignant transformation of the tongue epithelium and associated stromal myofibroblasts in a rat 4-nitroquinoline 1-oxide-induced carcinogenesis model.

The aim of our study was to analyse desalivated rat tongue epithelium for histopathological changes, proliferating cell nuclear antigen (PCNA), and epithelium-associated stromal myofibroblasts [SMF; alpha-smooth muscle actin (alphaSMA)] following 0.001% 4-nitroquinoline 1-oxide (4NQO) administration in drinking water. Results were compared with those of identically treated but salivated specimens. 4NQO was administered for 7, 14, 22 and 28 weeks. Tongue length was divided into anterior, middle and posterior 'thirds'. The histopathological changes per 'third' were scored as normal epithelium, hyperplasia, dysplasia, carcinoma-in-situ, and superficial and invasive carcinoma. The PCNA and alphaSMA stains were assessed by a point-counting method. At all time points, the histopathological changes in the anterior and middle thirds were higher in the desalivated than in the salivated group (P < 0.05) but almost identical in the posterior third (P > 0.05). PCNA scores were significantly lower in the desalivated vs. the salivated group at almost all time points and tongue thirds (P < 0.05). SMF were usually scarce in both groups, but there was a significant surge in the posterior third at 28 weeks: the score in the desalivated group was only about one-half that of the salivated group (P < 0.05). The absence of saliva seems to promote malignant transformation of the tongue epithelium in the early stages. PCNA cannot be regarded as a marker of proliferation and probably contributes to this process by other mechanisms. Emergence of SMF seems to be highly dependent on growth factors from saliva in addition to factors from cancerous cells.

Maspin, p53, p63, and Ki-67 in epithelial lesions of the tongue: from hyperplasia through dysplasia to carcinoma.

BACKGROUND: The pattern of changes in the expression of mammary serine protease inhibitor (maspin) tumor suppressor protein in tongue epithelial lesions [hyperplasia (HP), mild dysplasia (MD), moderate-to-severe dysplasia (MSD) and squamous cell carcinoma (SCC)] was investigated and correlated to the expression of maspin-regulating factors p53 and p63, and the proliferation marker Ki-67. METHODS: Cases of HP (n = 16), MD (n = 12), MSD (n = 11), and SCC (n = 22) were immunostained for maspin, p53, p63, and Ki-67. Maspin expression was scored separately for the basal, middle, and upper thirds of the epithelial width, and as the total sum of all 'thirds' (maspin-total). p53, p63, and Ki-67 were immuno-morphometrically assessed for the entire epithelial width. RESULTS: Maspin expression was differential and progressive extending to higher epithelial layers as dysplastic changes aggravated and culminated in carcinoma. Strong expression was related to MSD in the middle third and to carcinoma in the upper third. It was frequently lost at the invasion front, where the tumor was less differentiated. The changes in mean scores of maspin-total in the different study groups were positively correlated to the mean scores of p63 (r = 0.5, P < 0.001), p53 (r = 0.4, P = 0.004), and Ki-67 (r = 0.5, P < 0.001). CONCLUSIONS: Strong expression of maspin in the middle third of the epithelium may be considered a diagnostic sign of mild-to-moderate dysplasia and an indication of carcinoma in the upper third. The correlations between maspin and controlling factors (e.g. p63 and p53) may be events with key roles in the development of tongue carcinoma.

Foreign body granulomas after injection of Bio-alcamid for lip augmentation.

Bio-alcamid is one of the newest agents on the market for soft tissue augmentation. Seven studies were documented in the medical literature that examined the safety of Bio-alcamid (Polymekon, Brindisy, Italy); all reported no cases of tissue migration, foreign body granulomas, allergenicity, or interference with the control of cell proliferation. On 2 separate occasions, a woman who had recently undergone lip augmentation presented at our hospital with submucosal nodules of the lip. Histologic examination revealed multiple foreign body-type granulomas composed of giant cells, epithelioid cells, and chronic inflammation of the lip. Efforts to produce a cosmetic material that fulfills all the criteria as an "ideal" agent has not yet been found because all injectable foreign agents have the potential to induce adverse reactions. Caution must be exercised in all cases and the risks explained to the patient before its use.

4NQO-induced rat tongue carcinoma: an ultrastructural study.

The 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinoma, in which the carcinogen is administered systemically in drinking water, is the most comparable animal model to the development of human oral carcinoma. This is the first study to report the ultrastructural changes in this model. The most significant changes were observed in the carcinoma cells at the invasion front and included unique modifications in the basal lamina, presence of micropinocytotic vesicles (plasmalemmal caveolae), and emergence of cytoplasmic microfilaments featuring a parallel arrangement. The microfilaments, in both appearance and organization, were consistent with contractile microfilaments. These observations may be the morphological reflection of the phenotypic modifications occurring within the carcinoma cells, approaching smooth muscle differentiation.

Stromal myofibroblasts and malignant transformation in a 4NQO rat tongue carcinogenesis model.

This study assesses the correlation of changes in the density of stromal myofibroblasts to propagation of histopathologic alterations and proliferative activity of the epithelium in a rat 4NQO-induced tongue rat carcinogenesis model.Forty-three male Wistar rats were administered 0.001% 4NQO in drinking water for: 0 (n=7, control), 7 (n=4), 8 (n=8), 14 (n=6), 22 (n=9), and 28 (n=9) weeks, after which they were euthanized. Tongue sections were divided equally into anterior, middle and posterior thirds. Each third was given a histopathologic score (hematoxylin and eosin), ranging from normal, hyperplasia/hyperkeratosis, and escalating degrees of dysplasia to carcinoma, and analyzed by the point-counting method for density of epithelium-associated stromal myofibroblasts (alphaSMA) and of proliferating epithelial cells (PCNA). Histopathologic changes significantly increased in severity (carcinoma) with duration of 4NQO administration (p<0.001) in the posterior third of the tongue. The density of stromal myofibroblasts and proliferating epithelial cells was significantly higher in the posterior third of the tongue at 28 weeks compared to the other time points and locations (p<0.001, p=0.01, respectively). Significant correlations were found between occurrence of carcinoma and the increase in density of stromal myofibroblasts (p<0.001) and of proliferating epithelial cells (p=0.001) in the posterior third of the tongue. Increased density of stromal myofibroblasts was distinctively associated with the development of carcinoma but not with pre-malignant changes. Defining the mechanism of evolvement of carcinoma-associated stromal myofibroblasts is expected to further broaden our knowledge on the micro-environmental events occurring during the malignant transformation.

Stromal differences in salivary gland tumors of a common histopathogenesis but with different biological behavior: a study with picrosirius red and polarizing microscopy.

Salivary gland neoplasms - pleomorphic adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma - share a common histogenetic trait, but differ markedly in their biological properties. The objective of the study was to assess the polarization colors of picrosirius red-stained stromal collagen fibers in these salivary gland neoplasms to evaluate their possible role in the histopathogenesis of the tumors and to evaluate the potential usefulness of this approach as a diagnostic tool. Ten cases of each tumor type and 10 cases of mucous extravasation phenomenon (control) were examined using picrosirius red staining and polarizing microscopy. In each case, at least 50 thin ( approximately 0.8 microm) and 50 thick (1.6-2.4 microm) collagen fibers were counted and classified as green-yellow or yellow-orange, the mean percentage was calculated and statistical differences analyzed by one-way ANOVA. Results showed a similar thin fiber distribution in all tumor types and controls (82-88% green-yellow, 12-18% yellow-orange, p>0.05). Thick fibers showed a different distribution in polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (approximately 50% green-yellow) compared to pleomorphic adenoma and mucous extravasation phenomenon (approximately 13% green-yellow) (p=0.001). Thick fiber distribution was similar in polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (p>0.05). We conclude that with picrosirius red staining and polarizing microscopy, stromal collagen fibers differ significantly in pleomorphic adenoma from those in polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma, but not from mucous extravasation phenomenon. Similarity between polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma may indicate that these tumor types represent a single entity with a broad spectrum of biological behavior.

Morphological and molecular features of oral fluid-derived exosomes: oral cancer patients versus healthy individuals.

PURPOSE: Oral cancer (OC) patients are at high risk to develop recurrent disease or secondary primary cancers with no available biomarkers to detect these events until a visible lesion is readily present and diagnosed by biopsy. Exosomes secreted by cancer cells are involved in tumor growth, invasion and metastasis. We aimed to determine morphological and molecular differences between oral fluid (OF)-derived exosomes of OC patients and those isolated from healthy individuals (HI). METHODS: OF from OC patients (n = 36) and HI (n = 25) was initially assessed by nanoparticle tracking analysis (NTA). Following ultracentrifugation, exosomal pellets of OC patients and HI were morphologically examined by transmission electron microscopy and atomic force microscopy (AFM). Enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) were used to analyze the expression of exosomal markers--CD9, CD81 and CD63. RESULTS: NTA showed that OC samples of OF had a significantly higher concentration of nanoparticles/ml (p = 0.01) and modal nanoparticle size (p = 0.002) compared to HI. The difference in size was structurally highlighted by AFM three-dimensional images applied on exosomal pellets. ELISA and WB showed differential expression of exosomal markers in OC exosomes compared to HI: lower expression of CD81 and CD9 in contrast to a higher expression of CD63 (~53 kDa). CONCLUSIONS: OF-derived exosomes from OC patients differ both morphologically and molecularly from exosomes present in HI. This study is a baseline that provides a starting point for finding exosomal biomarkers for early detection of malignant changes in high-risk patients without overt clinical signs/lesions.

Liposarcoma of the tongue: clinico-pathologic correlations of a possible underdiagnosed entity.

Liposarcoma (LS), one of the most common malignant tumors, is considered as a rarity in the oral cavity, the tongue being the most frequent site for its occurrence. The purpose of the present study was to review and analyze the clinico-pathologic correlations of 32 cases of LS of the tongue, including four new cases from the files of our department. A total of 32 cases of LS of the tongue were collected, 29 from a MEDLINE search of the English language literature (1966-2004). Since the histopathologic features of one of the four new cases have been presented previously, this case was counted only once. LS of the tongue is a tumor of adult and old age, with a mean age of approximately 62 years and a peak incidence in the seventh and eighth decades. Clinically, it is a long-standing tumor that commonly presents as a solitary nodular mass, but can also present as a multi-nodular lesion. The most common histopathologic type is that of atypical lipomatous tumor (ALT)/well-differentiated LS (75%). LS can be easily misdiagnosed with both benign and malignant lipomatous and non-lipomatous tumors. LS of the tongue was commonly treated by local excision, however, there were cases of (multiple) recurrences and dedifferentiation. When a lipomatous lesion is encountered in the oral cavity, and especially in the tongue, LS should certainly be among the lesions that top the list of differential diagnosis. The follow-up must be on a long-term basis since this tumor can recur years after initial surgical treatment and can also undergo dedifferentiation.

Myofibroblasts in stroma of odontogenic cysts and tumors can contribute to variations in the biological behavior of lesions.

Stromal myofibroblasts (MF) have the potential to facilitate progression of neoplastic epithelial lesions that could contribute to their biological behavior. To assess immunohistochemically the frequency of stromal MF in different odontogenic cysts and tumors and correlate it to their aggressive biological behavior. The study included cases of dentigerous cyst (DC, n = 7), odontogenic keratocyst-parakeratinized type (OKC-P, n = 8), orthokeratinized type (OKC-O, n = 9), ameloblastic fibroma/fibro-odontoma (AMF/O, n = 11), unicystic ameloblastoma (UAM, n = 6), and solid ameloblastoma (SAM, n = 7). Cases of oral squamous cell carcinoma (SCC, n = 5) served as control. Myofibroblast frequency was assessed as the number of alpha smooth muscle actin (alphaSMA)-positive stromal cells in 10 high-power fields, presented as the mean number of positive cells per field. Counts showed that mean number of positive cells in OKC-P (25.7+/-11.4) was significantly higher than in DC (8.7+/-11.6) (p = 0.024) and in SAM (29+/-7) it was significantly higher than in UAM (14.9+/-4.9) and AMF/O (5.6+/-7.5) (p < 0.001). Counts in OKC-P and SAM were not significantly different from SCC (21.3+/-5.3) (p > 0.05). The high frequency of stromal MF in known aggressive odontogenic lesions, such as OKC-P and SAM, implies that MF can contribute to the biological behavior of these odontogenic lesions. Various pharmacological agents that control stromal MF can be used as an aid to reduce extensive and mutilating surgery in cases of remarkably aggressive odontogenic lesions.

Human saliva-derived exosomes: comparing methods of isolation.

ExoQuick-TC(TM) (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.

Histopathological spectrum of bone lesions associated with dental implant failure: osteomyelitis and beyond.

Early or late post-implant placement complications are usually localized infectious/inflammatory processes and treated accordingly. If the healing process does not take place within a reasonable timeframe, the possibility of a pathologic process beyond localized infection/inflammation should be suspected. We describe a radiological/histopathological spectrum of bony lesions ranging from inflammatory to malignant lesions surrounding failed dental implants. Five cases of mandibular dental implant failure that clinically, radiologically and histopathologically appeared to be inflammatory processes are presented. The failure of the dental implants was immediate in two cases and late in the remaining three. The radiological features were essentially similar for all five, and they included radiolucent or mixed radiolucent-radiopaque lesions with poorly defined borders. Three lesions were limited to the area of the failed implant, while the other two extended to a large part of the mandible. The histopathological findings ranged from acute osteomyelitis and chronic osteomyelitis with features of a fibro-osseous-like lesion and occasional rimming of atypical osteoblasts to osteogenic sarcoma that was admixed with a component of osteomyelitis (diagnosis of the latter was achieved only after a series of biopsies). In-depth investigative procedures are imperative in order to establish an accurate diagnosis whenever the histopathological diagnosis is inconsistent with persisting clinical signs and symptoms in bone lesions associated with failed dental implants.