E-cadherin as a potential biomarker of malignant transformation in oral leukoplakia: a retrospective cohort study.

BACKGROUND: Numerous attempts have been made to establish and develop tumor markers that could determine the susceptibility of normal tissues to transform into cancerous ones. To determine whether altered expression patterns of E-cadherin could be an early event in the progression of potentially malignant disorders to oral squamous cell carcinoma, this study aimed to assess the relationship between the immunoexpression of E-cadherin and the different degrees of epithelial dysplasia in oral leukoplakia. METHODS: Surgically excised specimens from patients with oral leukoplakia (n=31), oral cavity squamous cell carcinoma with cervical lymph node metastasis (n=12) and normal oral mucosa (n=9) were immunostained for E-cadherin. Oral leukoplakia samples were distributed into low and high risk group according to a binary system for grading oral epithelial dysplasia. Comparative analyses between E-cadherin expression and microscopic features (WHO histological grading and epithelial dysplasia) were performed by Pearson Chi-square test (P<0.05). RESULTS: Differences in E-cadherin expression were observed between normal oral mucosa and low risk oral leukoplakia (P=0.006), low and high risk oral leukoplakia (P=0.019), and high risk oral leukoplakia and oral cavity squamous cell carcinoma with cervical lymph node metastasis (P=0.0001). In addition, as epithelia undergo dysplastic changes, the risk of malignant transformation increases, and there is a reduction or loss of E-cadherin expression by keratinocytes. Reduced E-cadherin expression was an early phenomenon and it was observed in moderate-severe dysplasia, showing that the loss of epithelial cohesion may be an indicator of progression to oral cavity squamous cell carcinoma. CONCLUSIONS: E-cadherin could be used as a novel biomarker to identify lesions with potential risk for malignant transformation, which may provide opportunities for prophylactic interventions in high risk patient groups.

The participation of tumor residing pericytes in oral squamous cell carcinoma.

Pericytes are perivascular cells related to vessel structure and angiogenesis that can interact with neoplastic cells, interfering with cancer progression and outcomes. This study focused on the characterization of pericytes in oral squamous cell carcinoma (OSCC) using clinical samples and a transgenic mouse model of oral carcinogenesis. Nestin-/NG2+ (type-1) and nestin+/NG2+ (type-2) pericytes were analyzed by direct fluorescence after induction of oral carcinogenesis (4-nitroquinoline-1-oxide). Gene expression of neuron glial antigen-2 (NG2), platelet-derived growth factor receptor beta (PDGFR-ß), and cluster of differentiation 31 (CD31) was examined in human OSCC tissues. The protein expression of von Willebrand factor and NG2 was assessed in oral leukoplakia (i.e., oral potentially malignant disorders) and OSCC samples. Additionally, clinicopathological aspects and survival data were correlated and validated by bioinformatics using The Cancer Genome Atlas (TCGA). Induction of carcinogenesis in mice produced an increase in both NG2+ pericyte subsets. In human OSCC, advanced-stage tumors showed a significant reduction in CD31 mRNA and von Willebrand factor-positive vessels. Low PDGFR-ß expression was related to a shorter disease-free survival time, while NG2 mRNA overexpression was associated with a reduction in overall survival, consistent with the TCGA data. Herein, oral carcinogenesis resulted in an increase in NG2+ pericytes, which negatively affected survival outcomes.

Promoter hypermethylation in primary squamous cell carcinoma of the oral cavity and oropharynx: a study of a Brazilian cohort.

Epigenetic silencing of cancer-related genes plays an important role in oral/oropharyngeal squamous cell carcinoma (OSCC). We evaluated promoter hypermethylation of 4 cancer-related genes in OSCCs of a Brazilian cohort and determined its relationship with exposure to alcohol, tobacco, HPV infection and clinicopathological parameters. CDKN2A (cyclin-dependent kinase inhibitor 2A or p16), SFN (stratifin or 14-3-3 σ), EDNRB (endothelin receptor B) and RUNX3 (runt-related transcript factor-3) had their methylation patterns evaluated by MSP analysis in OSCC tumors (n = 45). HPV detection was carried out by PCR/RFLP. Aberrant methylation was detected in 44/45 (97.8 %) OSCC; 24.4 % at CDKN2A, 77.8 % at EDNRB, 17.8 % at RUNX3 and 97.8 % at SFN gene. There was no significant association between methylation patterns and clinical parameters. HPV (subtype 16) was detected in 3 out of 45 patients (6 %). Our findings indicate that HPV infection is uncommon and methylation is frequent in Brazilian OSCCs, however, EDNRB and SFN gene methylation are not suitable OSCC biomarkers due to indistinct methylation in tumoral and normal samples. In contrast, CDKN2A and RUNX3 genes could be considered differentially methylated genes and potential tumor markers in OSCCs.

Nestin and Neuron-glial antigen 2 transgenes unveil progenitor units in murine salivary glands.

OBJECTIVE: Undifferentiated cells play pivotal roles in sustaining tissue homeostasis during physiological turnovers and after tissue impairment. Nestin and Neuron-glial antigen 2 (NG2) are markers frequently deployed to distinguish progenitor populations. In the salivary gland scenario, these markers remain largely unknown. Particularly for a double-labeled group of progenitor cells (NG2+Nestin+), their phenotype and distribution have never been explored in freshly isolated tissues. Herein, we analyzed a subset of plastic cells that express Nestin and NG2 near the ducts and in the periacinar region of the major salivary glands of murine samples. DESIGN: The major salivary glands tissues of Nestin-GFP/NG2-DsRed mice were analyzed under a fluorescence microscope. The cells marked by GFP and DsRed were counted in the merged image component of random representative images obtained for each gland sample at × 20 magnification. RESULTS: In the parotid, submandibular, and sublingual glands, the population of cells exclusively expressing Nestin was more abundant. There was a predominance of Nestin, NG2, and double-labeled cells in the submandibular gland compared to the parotid gland, mainly near the ductal system. Of note, the sublingual and parotid glands had similar populations of Nestin+ and NG2+, especially in acini, and some positive cells were observed surrounding ducts. CONCLUSIONS: Collectively, our study revealed differential expression patterns of Nestin and NG2, alone or in combination, in the salivary gland subset during homeostasis.

Methylation analysis of cancer-related genes in non-neoplastic cells from patients with oral squamous cell carcinoma.

Early detection of Oral Squamous Cell Carcinoma (OSCC) is important to reduce mortality rates and to help provide successful cancer treatment. Hypermethylation of CpG islands is a common epigenetic mechanism that leads to gene silencing in tumors and could be a useful biomarker in OSCC. Abnormal DNA hypermethylation can occur very early in cancer development and may be induced by exposure to environmental carcinogens. We set out to investigate the methylation status of cancer-related genes in normal oral exfoliated cells from OSCC patients and healthy volunteers, as well as possible associations with alcohol/tobacco exposure or specific tumor characteristics. The methylation status of CDKN2A (cyclin-dependent kinase inhibitor 2A or p16), SFN (stratifin or 14-3-3 σ), EDNRB (endothelin receptor B) and RUNX3 (runt-related transcript factor-3) was evaluated by MSP (Methylation-Specific Polymerase Chain Reaction) analysis in non-neoplastic oral epithelial cells from OSCC patients (n = 70) and cancer-free subjects (n = 41). Hypermethylation was observed in CDKN2A, EDNRB and SFN genes, whereas no methylation was found in the RUNX3 gene. CDKN2A hypermethylation occurred only in the OSCC group (5.7%) while SFN and EDNRB hypermethylation occurred in both groups. There was no association between hypermethylation and smoking, drinking habits or specific tumor characteristics.

Central giant cell granuloma of the maxilla: Long-term follow-up of a patient treated with an adjuvant corticosteroid.

BACKGROUND: Central giant cell granuloma (CGCG) is one of the most intriguing lesions of the jaws and its nature has not yet been fully elucidated. Clinically, some CGCG behave more aggressively, while others have an indolent course. In cases of aggressive CGCG of the maxilla, effective personalized therapies are worth understanding. CASE REPORT: We report here a challenging case of aggressive CGCG in a 15-year-old girl which was misdiagnosed as an endodontic lesion. Radiographically, a large osteolytic lesion involving the hard palate from the central incisor to the second premolar, extending into the nasal cavity, with loss of the lamina dura and cortical resorption was observed. The lesion expanded aggressively after extensive curettage. With possible mutilation and defects due to a more radical approach to the lesion, treatment with systemic prednisone and intralesional triamcinolone hexacetonide associated with a calcitonin nasal spray was instituted. The decision in favor of this therapeutic strategy was made after careful immunohistochemical analysis of calcitonin and glucocorticoid receptors. The H-score for the staining of glucocorticoid and calcitonin receptors in multinucleated giant cells was 222 and 153.6, respectively. The lesion reduced in size, and no adverse effects associated with medications were observed. Another curettage was performed, and only fibrous connective tissue was found. The patient is in follow-up for 11 years without evidence of recurrence. CONCLUSION: Pharmacological agents hold clinical promise in cases of aggressive CGCG affecting the maxilla of pediatric patients. Investigating the expression of calcitonin and glucocorticoid receptors in order to plan treatment is very helpful in the decision to manage aggressive CGCG.

Human Papillomavirus DNA Detection by Droplet Digital PCR in Formalin-Fixed Paraffin-Embedded Tumor Tissue from Oropharyngeal Squamous Cell Carcinoma Patients.

INTRODUCTION: High-risk human papillomavirus infection impacts staging and prognosis of oropharyngeal squamous cell carcinomas (OPSCCs). Determination of HPV status in tumor tissue by p16-immunohistochemistry (p16-IHC) can be challenging; therefore, complementary methodologies could be useful in a clinical setting. OBJECTIVE: To test for accuracy and clinical relevance of HPV-DNA detection in formalin-fixed and paraffin-embedded (FFPE) tumor samples by droplet digital PCR (ddPCR). MATERIALS AND METHODS: Fifty OPSCCs were tested for p16-IHC status followed by HPV-16/18 DNA detection/quantification in FFPE-recovered DNA using ddPCR. Accuracy for HPV status determination and association with patient information were also evaluated. RESULTS: 32.0% (16/50) of the cases were p16-IHC positive (p16 +), 42.0% (21/50) had detectable levels of HPV-16 DNA, and none were positive for HPV-18 DNA. A higher median viral load of HPV-16 DNA was observed in p16 + cases (p < 0.0001). Concordance between p16-IHC and HPV-16 DNA ranged from 78.0 to 86.0% and accuracy rates were between 78.0 and 86.0%. P16-IHC and HPV-16 DNA detection was associated with gender, smoking status, and tumor subsite, while only HPV-16 DNA was associated with cT stage. The combination of HPV positivity by p16-IHC and ddPCR showed higher overall survival rates in comparison with p16 + /HPV-DNA- and p16 - /HPV-DNA- results. CONCLUSIONS: Type-specific HPV-DNA detection by ddPCR is highly specific but moderately sensitive for the determination of HPV status and showed clinical relevance, mainly when associated with p16-IHC status. Results highlight the importance of performing HPV-DNA testing in combination with p16-IHC for proper identification of HPV-associated OPSCC and to improve clinical management of OPSCC patients.

Differential Expression of Potential Biomarkers of Oral Squamous Cell Carcinoma Development.

To evaluate molecular epithelial changes, we investigated whether a profile of survivin, cyclin dependent kinase inhibitor 2A (CDKN2A), epidermal growth factor receptor (EGFR), polo like kinase 1 (PLK1), p63, p40 (Δnp63 isoform), cyclin D1 (CCND1) and BCL2 apoptosis regulator (BCL2) proteins could predict malignant transformation. Different tissue segments (tumor adjacent epithelium; dysplasia and tumor) from a total of 109 patients were analyzed by immunohistochemistry. An increased expression of survivin (p < 0.001), PLK1 (p = 0.001), and p63 (p < 0.001) in parallel to reduced immunostaining of p40 (p < 0.001) and BCL2 (p = 0.029) was observed among the tissue segments analyzed. Our study revealed that survivin, PLK1, p63, p40 and BCL2 play a role in oral tumorigenesis and represent promising biomarkers able to recognize mesenchymal phenotype induction in the transition from nonmalignant cells to tumor cells. These results reveals critical interaction between survivin, PLK1, p63, p40 promising proteins during invasive carcinoma development.

Human Papillomavirus E6/E7 mRNA detection by in situ hybridization in oral cavity squamous cell carcinoma.

OBJECTIVE: The aim of this study was to evaluate the application of in situ hybridization using E6/E7 mRNA probes to identify the frequency of high-risk HPV transcriptionally active and the use of HPV status as a prognostic biomarker in oral cavity squamous cell carcinoma (OCSCC). METHODS: Ninety-nine OCSCC samples were evaluated from Hospital Santa Rita de Cassia, Hospital Universitário Cassiano Antônio de Moraes and University Hospitals Coventry and Warwickshire NHS Trust. After tissue microarray construction, the slides were submitted to an in situ hybridization detection method for HPV E6/E7 mRNA. HPV status was designated a binary classification. Multiple logistic regression examined the association of HPV with clinical features and other risk factors, using SPSS® software. For all hypothesis tests, a significance level of p ≤ 0.05 was considered. RESULTS: HPV frequency in oral squamous cell carcinoma was 8%. There was no association between HPV and clinical variables and between the main prognostic features and known risk factors. There was no difference in the prevalence of HPV for oral cavity squamous cell carcinoma by geography (Brazil vs UK). CONCLUSIONS: A low frequency of E6/E7 mRNA by RNA in situ hybridization was found in oral cavity squamous cell carcinoma, which supports the evidence that HPV-driven cancer of the oral cavity is uncommon.

Polymorphisms in methylenetetrahydrofolate reductase and cystathionine beta-synthase in oral cancer - a case-control study in southeastern Brazilians.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a serious public health problem, due to its high mortality rate and worldwide rising incidence. OSCC susceptibility is mediated by interactions between genetic and environmental factors. Studies suggest that genetic variants encoding enzymes involved in folate metabolism may modulate OSCC risk by altering DNA synthesis/repair and methylation process. OBJECTIVE: The goals of this study were to evaluate the association of three genotypic polymorphism (MTHFR C677T, MTHFR A1298C and CBS 844ins68) and oral cancer risk in southeastern Brazilians and evaluate the interactions between polymorphisms and clinical histopathological parameters. METHODS: This case-control study included 101 cases and 102 controls in the state of Espírito Santo, Brazil. MTHFR genotyping was done by PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) and CBS genotyping by PCR (polymerase chain reaction) analysis. RESULTS: MTHFR C677T polymorphism was associated with lymph node involvement. Genotype CT+TT acted as a protective factor. MTHFR A1298C AC+CC genotype was associated with tumor differentiation, and possibly with a better prognosis. In risk analysis, no correlation was observed between genotypes and OSCC. CONCLUSION: We concluded that MTHFR C677T, MTHFR A1298C and CBS 844ins68 polymorphisms were not associated with OSCC risk in southeastern Brazilians; however, we suggest a prognosis effect associated with MTHFR C677T and A1298C polymorphisms in OSCC.

Atypical esthesioneuroblastoma invading oral cavity: a case report and review of the literature.

Esthesioneuroblastoma is an uncommon tumour of neuroectodermal origin. The authors describe a rare presentation of an atypical esthesioneuroblastoma invading oral cavity. The clinical presentation, aetiology, diagnosis, and management of this condition are discussed. The patient developed significant swelling in the right anterosuperior alveolar mucosa and had moderate tooth mobility. Conventional x-rays and computed tomography revealed a large osteolytic lesion, with imprecise limits. Histological findings along with immunohistochemical staining results and clinical features led to the diagnosis of high-grade esthesioneuroblastoma. Local recurrences and neck metastasis were detected. The rare oral findings produced delayed in diagnosis which may lead to a compromise in planning and execution of further radical management and thus a poor prognosis. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1168853011139286.

Aspectos clínicos e epidemiológicos do câncer bucal em um hospital oncológico: predomínio de doença localmente avançada / Clinical and epidemiological features of oral cancer in a hospital cancer center: predominance of advanced local disease

O câncer bucal é a sétima neoplasia mais frequente na populaçãobrasileira, com elevada taxa de mortalidade. O objetivo desteestudo foi realizar um levantamento epidemiológico da populaçãocom câncer bucal atendida no Programa de Prevenção eDetecção Precoce de Câncer de Boca Hospital Santa Rita, ES.Foram avaliados 152 pacientes, tendo sido diagnosticados 30,3%casos de câncer bucal. As variáveis gênero, idade, grupo étnico,localização da lesão, sinais e sintomas, tempo de evolução,estadiamento clínico, graduação histológica do tumor e históriado consumo de álcool e uso do tabaco foram avaliadas. Dentreos pacientes diagnosticados com câncer bucal, 80,4% eram dogênero masculino com média de idade de 57,7 anos. A língua foi osítio mais frequente de tumor primário. Os sinais e sintomas maiscomumente relatados foram presença de lesão em boca e dor.Dos tumores diagnosticados, 63,0% encontrava-se em estadioavançado com tempo médio de evolução de 8,7 meses. Pacientescom história de consumo de álcool e uso do tabaco apresentaramestadio avançado da doença durante o diagnóstico. Estesdados nos permitiram concluir que o consumo de álcool e usodo tabaco, bem como o tempo entre a percepção dos primeirossinais e sintomas e o diagnóstico são fatores determinantes paraa progressão da doença.

Oral cancer is the seventh most frequent neoplasia with highdeath rate in the Brazilian population. This study aims at carryingout an epidemiological investigation in the population with oralcancer being assisted by in the Program of Prevention and EarlyDetection of Oral Cancer - Santa Rita Hospital in Brazil. Onehundred fifty-two patients were evaluated, of which 30.3% werediagnosed with oral cancer. The variables gender, age, ethnicgroup, location of the tumor, signs and symptoms, evolution time,clinical staging, tumor histopathological grading, and history ofalcohol and tobacco use were assessed. Among the patients withoral cancer, 80.4% were men with average age of 57.7 years. Themost frequent site of the primary tumor was the tongue (41.3%).The most commonly reported signs and symptoms were presenceof lesions in the mouth and pain. During clinical assessment, itwas detected that 63,0% of the cases were advanced, and theaverage evolution time of the disease was 8.7 months. Patientswith concurrent history of alcohol and tobacco consumptionpresented with a more advanced stage of the disease duringdiagnosis. These data allow us to conclude that alcohol andtobacco use, as well as the time between noticing the first signsand symptoms and the diagnosis are determining factors in thedisease progression.

Prevalência de metilaçäo do gene p16 em populaçäo de risco ao câncer bucal / Prevalence of methylation of the p16 gene in population of risk to oral cancer

A tumorigênese do carcinoma epidermóide de cabeça e pescoço tem sido associada a carcinógenos exógenos, destacando-se aqueles presentes no tabaco. Estes agentes podem causar alterações genéticas que levam à inativaçäo de genes supressores de tumor e descontrole na proliferaçäo celular. O gene p16 (CDKN2/INK4a) está localizado sobre o cromossomo 9p21 e codifica uma proteína de 16kDa, que controla negativamente a progressäo do ciclo celular nas fases G1/S. Uma das formas de silenciamento deste gene é a hipermetilaçäo, inativando sua transcriçäo e contribuindo para o desenvolvimento tumoral. O objetivo deste estudo foi analisar o estado de metilaçäo do gene p16 em células provenientes da mucosa bucal de indivíduos tabagistas crônicos, sem evidência clínica de câncer bucal. Foram analisadas 516 amostras, obtidas de 258 indivíduos tabagistas crônicos. Para detectar metilaçäo em ilhas CpG na regiäo promotora do gene p16 foi utilizada a técnica de digestäo do DNA genômico com enzimas de restriçäo sensíveis à metilaçäo e amplificaçäo por PCR. Hipermetilaçäo em ilhas CpG do gene p16 foi observada em 17,4 por cento (45/258) dos casos analisados. Estes dados nos levam a considerar a importância da análise do estado de metilaçäo do gene p16 como um marcador dos estágios iniciais da tumorigênese, o que poderá, futuramente, ser utilizado como um método eficiente e rápido de detecçäo precoce de alterações moleculares associadas ao câncer bucal em populaçäo de risco