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Objective identification of the miRNA expression profile in sinonasal inverted papilloma (SNIP) as a tool to evaluate the risk of transformation into sinonasal squamous cell carcinoma (SNSCC). Materials and Methods paired tumour tissues and adjacent normal tissues were obtained from SNIP and SNSCC patients who had undergone surgical resection and used for next-generation sequencing (NGS)-based miRNome analysis. SNIP tissues with concomitant dysplasia (SNIP-DISP) were used as malignant transition samples. By comparing the deregulated miRNAs in SNIP and SNSCC, an miRNA cluster was identified and its physio- and clinical-pathological value was predicted. Results NGS identified 54 miRNAs significantly down- and upregulated in SNIP. Among them, the miR-449 cluster was upregulated in SNIP and could differentiate the benign tumour from normal tissue. Notably, the miR-449 cluster was found to be significantly underexpressed in SNSCC, and the cluster markedly changed in SNIP during the malignant transition into SNSCC. miRNA enrichment analysis and GO analysis revealed that miR-449 is involved in apoptotic and cell proliferation pathways. Conclusions Our findings suggest that miR-449 may be involved in the molecular pathogenesis of SNIP and its malignant transformation into SNSCC. miR-449 might therefore be a useful tumour biomarker in patients with SNIP and may also have the potential to be used as a tool for detecting and monitoring the course of the possible malignant transformation.

Cell-cell and cell-matrix interactions regulate important cellular functions; they involve a number of specialised molecules and the corresponding receptors, among which a key role is played by cadherins and the associated catenins. Deregulation of these molecules has been associated with tumour progression in many human malignancies. While catenins expression has been extensively studied in many human cancers, including oral carcinoma (OSCC), less is known about their expression in oral epithelial dysplasia. The objective of this study was to evaluate the expression of these proteins in a large group of displastic lesions of the oral mucosa and their relation with subsequent malignant transformation. Expression of beta- and gamma-catenin was investigated by immunohistochemistry using specific monoclonal antibodies in 49 cases of oral epithelial dysplasia and 10 samples of normal oral mucosa. The presence and absence of beta- and gamma-catenin staining was expressed differently in relation to dysplasia grade; while the degree of dysplasia became more severe, we observed a statistically significant loss and/or reduction of catenins expression, the loss of the exclusive membranous expression and a cytoplasmic delocalisation. Progression to OSCC occurred in 10 out of our 49 cases (20.4%); all of them, except one, showed a concurrent and concordantly located beta- and gamma-catenin staining even, if no statistically significant differences were found between cases progressed to invasive OSCC or not. Catenins physiology alterations may be involved in the transformation process; however, the role of catenins expression as possible prognostic markers in precancerous oral lesions seems to be limited. Nonetheless, further studies on larger series of samples are necessary in order to clarify the role of catenins expression in oral carcinogenesis from both a biological and clinical point of view.

Granular cell odontogenic tumours (GCOT) are rare neoplasms that usually manifest a benign clinical behaviour. We document the first case of GCOT exhibiting clinico-pathological features of malignancy that occurred in the maxilla of a 40-year-old male. The lesion appeared as an intra-oral polypoid mass and, at CT scan, as a poorly demarcated radiolucency eroding the cortical plate. Histologically, the tumour consisted of clusters of granular cells, exhibiting nuclear pleomorphism, prominent nucleoli and mitotic figures, and spindle cells in a collagenous stroma containing cementicles and strands of odontogenic epithelium. Morphologic transition from fibroblast-like to granular cells was frequently detected. The tumour cells extensively invaded the oral and respiratory mucosae and the adjacent soft tissues and exhibited vimentin and CD 68 immunoreactivity and high (21%) Ki 67 immunolabeling but not cytokeratins, E.M.A. actin, desmin, myosin or S-100 protein positivity. The patient experienced tumour recurrence 16 months after radical surgery. While the histogenesis of GCOT remains to be clarified, we document the existence of a malignant counterpart of this tumour and propose the name of malignant GCOT or granular cell odontogenic sarcoma for such entity.

Clear cell tumours, in the head and neck region, are usually derived from salivary or odontogenic tissues, or may be metastatic. A few clear cells may be present in odontogenic cysts, while, odontogenic neoplasms composed predominantly of clear cells are quite rare. They include calcifying epithelial odontogenic tumours (CEOT), ameloblastoma and odontogenic carcinoma. Clear cell odontogenic tumour (CCOT) has been classified in the last WHO classification as a benign tumour, but it is now recognized as a more sinister lesion and current opinion is that CCOT should be designated as a carcinoma. These tumours are characterized by aggressive growth, recurrences, and metastatic disease. A recent review of the literature has yielded 30 cases of tumours with similar characteristics. These tumours have a peak incidence in the 5th-7th decades, with a female predilection. The anterior portions of the jaws, especially the mandible, are most frequently affected. The aggressive potential of these neoplasms is well documented by the extensive invasion of adjacent tissues, multiple recurrences and regional or distant metastases.

The identification of the proliferative activity in tumours may be useful to predict the biological behaviour of different lesions. Proliferating cell nuclear antigen (PCNA) has been used for the evaluation of the proliferative ability of many lesions. In this study 22 ameloblastomas (4 follicular, 5 plexiform, 4 acanthomatous, 5 unicystic, 4 recurrent), 12 odontogenic keratocysts (OKC), 8 dentigerous cysts (DC), and 12 radicular cysts (RC) were analysed. PCNA+ cells were present in all cyst types but the OKC contained the highest number of PCNA+ cells. In OKC the location of PCNA+ cells was mainly suprabasal. In ameloblastoma PCNA+ cells were located mainly in the peripheral portion of the tumour islands. Statistical analysis showed that ameloblastoma had higher PCNA+ cell counts than OKC (P < 0.0001); OKC had higher values than DC and RC (P < 0.0001). Recurrent ameloblastoma presented higher PCNA+ cell counts than other types of ameloblastoma, while unicystic ameloblastoma showed lower values than acanthomatous, plexiform and follicular ameloblastomas (in this latter case the difference was not statistically significant). These data could help to explain the different biological behaviour of these lesions.

Summary Squamous cell carcinoma is the most frequent malignant tumor of the oral cavity and often arises from premalignant lesions. Traditional methods used by the pathologist are subjective and lack the sensitivity to predict accurately which precancers may progress with time. Therefore, it is important to search for markers that may identify progression of premalignant lesions. Microarray technology can be use with this aim. Here, we define the genetic expression profile of lingual dysplasia (DS) progression. By using cDNA microarray containing 19.2 K clones and a baseline of 11 normal tissues, we compared 5 mild and 4 severe DS. We identified 270 genes differentially expressed in normal tissue vs. mild DS (i.e. 161 up- and 109 down-regulated) and 181 genes differentially expressed in mild vs. severe DS (i.e. 63 up- and 118 down-regulated). The described genes cover a broad range of functional activities: (a) anti-oxidative, (b) DNA-repair, (c) inflammatory response, (d) cell-adhesion/mobility, (e) extracellular matrix depolimerization, and (f) cell-cycle regulation. The data reported better define DS progression and can help in classifying premalignant lesions.

Sebaceous adenoma is a tumour only rarely located in the oral cavity. Less than 10 cases have been reported. Sebaceous adenoma represents 0.5-0.7% of all monomorphic adenomas. Sebaceous adenoma is mainly constituted by two types of cells, undifferentiated peripheral basaloid cells and cells showing different degrees of sebaceous differentiation located in the center of the lesion. The differential diagnosis must be made with sebaceous hyperplasia. Sebaceous adenomas are benign, and they do not recur after a conservative excision.

Lipomas are common, benign tumours located in any part of the body in which fat is normally present. Some variants of lipoma have been described according to the type of tissue present. A rare variant consists of a lipoma with osseous or cartilaginous metaplasia. These lesions have been called chondrolipoma, osteolipoma, lipoma with chondroid or osseous metaplasia, lipoma with cartilaginous or osseous change, or ossifying lipoma. We present the case of an osteolipoma of the tongue in a 49-year-old female who was referred for a painless mass on the left lateral margin of the tongue, and present for about 8 years. Osteolipomas have been reported in middle-aged or elderly patients with a very long clinical history. These tumours tend to be large and to arise from the deep soft or subcutaneous tissues. The cartilage and bone is probably produced by metaplasia of fibroblasts in chondroblasts or osteoblasts. These lesions are benign and do not recur.

Angiomyolipoma (AML) is a tumour or an hamartomatous growth that usually affects the kidney. Only rarely has AML been described in the oral cavity. The authors report a case of AML located in the palate in a 43-year-old patient. AML is composed of smooth muscle cells, blood vessels and mature fat cells. In 50% of cases, AML presents with symptoms of tuberous sclerosis. Renal AML are often invasive, may involve regional nodes and may recur, while, on the contrary, AML are most often well circumscribed and easily resected. AML seems to follow an entire benign course.

Odontogenic tumours are rare neoplasms whose classification is sometime controversial. Among these entities, granular cell odontogenic tumour (GCOT) is extremely rare and usually has a benign clinical behaviour. While the histogenesis of GCOT remains to be clarified, we documented the existence of a malignant counterpart of this neoplasm and proposed the name of malignant GCOT. Expression profiling by cDNA microarrays is a molecular technology that enables a global gene expression analysis. By using cDNA microarrays, we identified in malignant GCOT several genes with significantly differentially regulated genes when compared to non neoplastic tissues. These cancer specific genes include a range of functional activities: (1) transcription, (2) signaling transduction, (3) cell-cycle regulation, (4) apoptosis, (5) differentiation and (6) angiogenesis. In conclusion, we show that cDNA microarrays is a useful approach to investigate the biology of tumours. Moreover, this technology might lead to identification of gene targets for cancer therapy and to molecular classification of odontogenic tumours.

Salivary gland papillomas are rare tumours arising from ductal epithelium. Intraduct papillomas are the most rare of all duct papillomas. Only four intraduct papillomas have been described in a review of nearly 3100 epithelial salivary tumours. Intraduct papillomas are located almost exclusively in the excretory ducts of the minor salivary glands. However, also the major salivary glands may be affected. Microscopically, the tumour consists of fibrovascular papillae covered by a columnar or cuboidal epithelium. The authors describe an intraduct papilloma of the palate in a 74-year-old woman. The excision of the lesion was curative.

The authors present the case of a 36-year-old HIV+ male patient with a 1-cm diameter papillary exophytic lesion of the right cheek. Microscopic examination showed a papillary epithelial neoplasm with invasion of the stroma in the peripheral part. Cellular and nuclear atypia were present in the superficial and in the deep layers of the neoplasm. An in situ hybridization for human papillomavirus (HPV) 6, 11, 16, 18, 31, 35 and 51 was performed. A focal positivity only for HPV 16 and 18 was present in koilocytotic cells of the most peripheral portion of the lesion. The microscopic definitive diagnosis was warty carcinoma of the cheek. No recurrence was observed at a 3-year follow-up.

Spindle-cell lipoma (SCL) is a distinct histological variant of lipoma. Clinically, it appears as a solitary, subcutaneous, circumscribed lesion. SCL accounts for about 1.5% of all adipocytic tumours. Only nine cases of intraoral SCL were found in the literature. Microscopically, mature adipocytes and spindle cells are immersed in a myxoid stroma. SCL needs only local excision, and it does not recur.

In a double-blind study 10 patients with oral leukoplakia were treated daily (three topical applications) with 0.1% isotretinoin gel or a placebo for 4 months. Nine patients completed the treatment, while one patient was lost to follow-up. Subsequently, the patients who had received the placebo used the active medication for an additional 4 months. All patients treated with the active medication showed a significant improvement of the oral lesions while, in the patients receiving only the placebo, the size of the lesions remained the same. Also the group of patients who received the active medication after the placebo showed an improvement in the size and clinical appearance of the lesions. A complete response was defined as the complete disappearance of the lesion as assessed by visual inspection, while a partial response was defined as a 50% or more reduction in the size of the lesions. In total we had a complete response and eight partial responses. No side-effects from the use of the gel were ever observed. The percentage of bcl-2-positive cells was evaluated in the basal layer from a minimum of 1000 cells in each case and the bcl-2 immunostaining was scored using three groups: - (or = 10% cells); + (or = 50% cells); +2 (or = 50% cells). The presence of apoptotic bodies was evaluated in a random fashion in the parabasal layer in 20 HPF. Immunohistochemical analysis for bcl-2 protein showed that before treatment a weak positivity of the basal layers, with a focal positivity of some parabasal cells, was present: five out of nine specimens were positive. Only a few apoptotic bodies were observed. After treatment in almost all specimens it was possible to observe a complete bcl-2 negativity with a positivity in only one specimen out of nine. An increase in apoptotic bodies was observed. Statistical analysis showed that the difference between the bcl-2 positivity in the two groups was not statistically significant (P = 0.134) while, on the contrary, the difference in the count of the apoptotic bodies between the same two groups was statistically significant (P = 0.0193). In conclusion, the data obtained from this pilot study show that good results can be obtained with the topical use of 13-cis-retinoic acid.

A dentinogenic ghost cell tumour in an 80-year-old male patient is presented. It is an extremely rare tumour and only 10 cases have been reported in the English literature. The lesion showed odontogenic epithelium, ghost cells, dentinoid, giant cells. The immunohistochemical analysis for Mib-1 and bel-2 showed a strong positivity of the cells of the odontogenic epithelium, while with p53 only a rare positivity was observed. Completely negative were the ghost cells, giant cells and dentinoid material. In this tumour the cells expressing Mib-1 and bcl-2 could be the cells that proliferate, and that could undergo malignant transformation.

Odontogenic keratocysts (OKC) present an aggressive course with a marked tendency to recurrence. The epithelium of OKC is thought to have an intrinsic growth potential and has been shown to present a higher rate of proliferation as compared to other types of cyst. bcl-2 has a role in the extension of cell survival. The objective of the present study was to evaluate the bcl-2 protein expression in different odontogenic cysts. A total of 19 dentigerous cysts (DC), 20 radicular cysts (RC) and 14 OKC were used in the present study. DC and RC showed an almost complete negativity for bcl-2. OKC, on the other hand, presented in all cases a strong positivity in the cells of the basal layer, with, in most cases, more than 50% of the cells positive. This bcl-2 positivity of the basal layer of OKC could point to an abnormal control of the cell cycle. The bcl-2 protein overexpression could then produce an increase in the survival of the epithelial cells, and this increased lifespan could, in turn, lead to the peculiar aggressive growth pattern of OKC. Moreover the bcl-2 staining can be useful to differentiate OKC from other types of odontogenic cysts.

Purpose Although deregulation of the Wnt pathway via β -catenin is a frequent event in several human cancers, its potential implication in oral cancer is largely unexplored. Aim of the work was to define both the pathogenetic and the prognostic role of beta-catenin in a large series of oral and oropharyngeal squamous cell carcinomas (OSCC and OPSCC respectively). Materials and methods 374 OSCCs/OPSCCs selected from three different geographic areas were analysed by IHC for beta-catenin and Lef/TCF1/TCF4. All cases were stratified according to intracellular staining localization. The association with clinico-pathological parameters was assessed by statistical analysis. Survival rates were valued by Kaplan–Meier curves. Beta-catenin expression was also investigated on both normal and neoplastic oral cell lines, by Real time PCR. Results Beta-catenin alterations were statistically evident in SCCs when compared to normal mucosa ( p p Conclusions Our work has underlined the key-role of beta-catenin in oral and oropharyngeal carcinogenesis, and in the prognostic stratification of patients. To our knowledge, the present work is the largest study to highlight the existence of a statistical association between beta-catenin and classical prognostic factors in OSCC/OP-SCC.

Purpose Hyper-methylation in CpG islands of gene promoters, an epigenetic phenomenon down-regulating gene expression, is directly linked with carcinogenesis. In our laboratory we have analysed, in a series of primary OSCCs with matched normal oral mucosa under alcohol-smoking chronic exposure, the methylation status of a panel of genes, including hMLH1, CDH1, CDKN2a, MGMT, Rar-β2, SFRP-1, SFRP-2, SFRP-4, SFRP-5, Wif-1 and DKK-3 in order to define an epigenetic fingerprint for the precancer lesions and oral cancers. Materials and methods Thirty-seven FFPE OSCCs with relative controls of normal oral epithelium were analysed by methylation specific PCR (MSP). We have observed different frequencies of gene methylation. For all genes, the Wald Test and the logistic multiple regression were performed, in order to verify the association between methylation status of gene promoter (covariates) and presence of cancer (response variable). Results Interestingly also the apparently health oral mucosa shows a methylated background. The most frequently methylated gene in OSCC were WNT-related genes (SFRP2, 45%; SFRP4, 78%; SFRP5, 59%), CDKn2a (74%) and hMLH1(53%). RAR-beta-2 and MGMT promoter hyper-methylation was found in both cases only in 13.5% of OSCCs, but more frequently in healthy oral mucosa (respectively, 28.5% and 23% of studied cases). The Wald test confirmed the statistical significance for RAR-beta-2 de-methylation in cancer (p = 0.044; CI at 95%). Conclusions This work, highlighting the importance of epigenetic silencing, have shown that a panel of genes may be useful in clinical practice separating normal oral epithelia from the cancerous. All these results suggest that employing of an epigenetic fingerprint may improve the current diagnostic tools, but also contribute indirectly to therapeutics for the correct clinical management of oral neoplastic and pre-neoplastic lesions.