Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma.

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.

Lack of claudin-7 is a strong predictor of regional recurrence in oral and oropharyngeal squamous cell carcinoma.

OBJECTIVES: Adequate treatment of oral and oropharyngeal squamous cell carcinoma (OSCC) is dependent on correctly predicting the presence of lymph node metastases. Current methods to diagnose nodal metastases partly result in overtreatment with associated morbidity and undertreatment with decreased disease-free survival. E-cadherin has been studied extensively as potential marker for lymph node metastases. EpCAM and claudin-7 have a functional relationship with E-cadherin, forming a complex that promotes tumourigenicity in vitro. We hypothesize that the co-expression patterns of these related molecules is a better prognostic marker for nodal status and regional recurrences. MATERIALS AND METHODS: We constructed separate tissue microarrays of tumour centre and tumour invasive front of 227 OSCC with complete clinicopathological and follow-up data, including HPV status, and performed immunohistochemistry for these molecules. RESULTS: Lack of E-cadherin and presence of cytoplasmic EpCAM expression in the tumour front were predictive for nodal metastasis, but no co-expression pattern was found clinically relevant. Lack of claudin-7 in the tumour centre was highly and independently predictive for shorter regional disease-free survival (HR=0.19; 95%CI: 0.06-0.62) and disease-specific survival (HR=0.43; 95%CI: 0.21-0.87). High-risk HPV was not associated with any marker. CONCLUSIONS: The expression of E-cadherin and EpCAM, depending on the specific tumour sublocalization, is predictive for nodal status. However, co-expression did not improve the prediction of nodal status, indicating that the proposed in vitro complex is not functional in clinical samples. Additionally, lack of claudin-7 expression in the tumour centre may be used to identify patients with increased risk for regional recurrence.

FADD expression is associated with regional and distant metastasis in squamous cell carcinoma of the head and neck.

AIMS: The Fas-associated death domain gene (FADD) is often overexpressed in squamous cell carcinoma of the head and neck (HNSCC), and is considered to be a driver gene in amplification of the chromosomal 11q13.3 region. Amplification of 11q13.3 is associated with increased metastasis in HNSCC and breast cancer. The aim of this study was to investigate the association between FADD protein expression in advanced-stage HNSCC and clinicopathological features and outcome. METHODS AND RESULTS: Tumour tissues of 177 HNSCC patients uniformly treated with primary surgery and postoperative radiotherapy were collected. FADD expression was assessed on pretreatment tumour biopsies using immunohistochemistry. High FADD expression was detected in 44% of the HNSCC patients. High expression was associated with an increased rate of lymph node metastasis (P = 0.001) and with a shorter distant metastasis-free interval (DMFI) (HR 2.6, 95% CI 1.0-6.7, P = 0.046) when lymph node metastases were present. CONCLUSIONS: Our data show that an increase in FADD expression is associated with a higher incidence of lymph node metastasis at presentation, and with shorter DMFI when lymph node metastases are present. High FADD expression in the primary tumour could be a useful marker to select patients for systemic treatment strategies that reduce the risk of distant metastases.

[A local gingival swelling which was based on a peripheral giant cell lesion]. / Lokale zwelling van de gingiva die berustte op een perifere reuscellaesie.

A 29-year-old woman consulted her dentist about a localgingival swelling which bled during tooth-brushing and had been present for 1 year. In the differential diagnosis, epulis gravidarum, a peripheral giant cell lesion, and peripheral ossifying granuloma were considered. After surgical removal, histopathological examination revealed a peripheral giant cell lesion. The presence of a peripheral giant cell lesion requires thorough excochleation of the underlying bone. If the bone is cleaned thoroughly, recurrence is rare.

Scandinavian Fellowship for Oral Pathology and Oral Medicine: guidelines for oral pathology and oral medicine in the dental curriculum.

In Scandinavia, as in many European countries, most patients consult their general dentist once a year or more. This gives the dentist a unique opportunity and an obligation to make an early diagnosis of oral diseases, which is beneficial for both the patient and the society. Thus, the dentist must have knowledge of clinical symptoms, local and systemic signs and clinical differential diagnoses to make an accurate diagnosis. The dentist must be competent in selecting appropriate diagnostic tests, for example, tissue biopsy and microbiological samples, and conducting them correctly, as well as in interpreting test results and taking appropriate action accordingly. Furthermore, the dentist must be aware of diseases demanding multidisciplinary cooperation and be able to recognise his/her professional limitation, and to refer to other specialists when required. The dental curriculum changes over time as new approaches, treatments and diagnostic possibilities develop. Likewise, the role of the dentist in the community changes and may vary in different countries. As members of the Scandinavian Fellowship for Oral Pathology and Oral Medicine and subject representatives of oral pathology and oral medicine, we feel obliged to contribute to the discussion of how the guidelines of the dental curriculum support the highest possible standards of dental education. This article is meant to delineate a reasonable standard of oral pathology and oral medicine in the European dental curriculum and to guide subject representatives in curriculum development and planning. We have created an advisory topic list in oral pathology and oral medicine.

Tumour infiltration depth ≥4 mm is an indication for an elective neck dissection in pT1cN0 oral squamous cell carcinoma.

Patients with pT1cN0 oral squamous cell carcinomas (OSCC) are generally not treated with a neck dissection (ND). However, in 25% of cN0 patients, nodal metastases become apparent during follow-up. Infiltration depth of the primary tumour has been consistently associated with the presence of nodal metastasis, but proposed cut-off depths for performing a ND vary considerably. The aim of this study was to explore the infiltration depth as predictor for the nodal status and to recommend a cut-off depth for performing a ND. From our database of 351 primary oral carcinomas, we selected all pT1-2 tumours (n=246). Infiltration depth was measured in 212 cases. Neck status was determined by histopathological examination of the dissection specimen, or by at least two years of follow-up. Mean infiltration depth was 5.49 mm (95% CI: 4.86-6.12) in the N0 and 8.40 mm (95% CI: 7.38-9.43) in the N+ group (p<0.001). cN status, lymphovascular invasion and infiltration depth were the only independent predictors for nodal status in multiple logistic regression. ROC-analysis on pT1cN0 tumours resulted in an optimal cut-off for the prediction of the nodal status at a depth of 4.59 mm. This cut-off identified a subgroup of patients at increased risk for nodal metastasis (OR=8.3) and with significantly shorter survival. Tumour infiltration depth is an independent predictor for nodal status in pT1-2 OSCC. In pT1cN0 tumours, a cut-off at 4.59 mm results in the best predictive value. We recommend an infiltration depth of ≥4 mm as an indication to perform a neck dissection in pT1cN0 OSCC.

Applicability of equine hydroxyapatite collagen (eHAC) bone blocks for lateral augmentation of the alveolar crest. A histological and histomorphometric analysis in rats.

This study assessed the mechanical characteristics, biocompatibility and osteoconductive properties of an equine hydroxyapatite collagen (eHAC) bone block when applied as a bone substitute for lateral augmentation of rat mandible. 96 rats underwent lateral augmentation of the mandible, using two substitute bone blocks (eHAC or Bio-Oss spongiosa) or autologous bone grafts. Signs of inflammation, amount of bone formation and ingrowth of bone into the bone blocks were assessed at 1 and 3 months. eHAC blocks were mechanically rigid and could be fixed firmly and easily. Bio-Oss spongiosa blocks were brittle and fixation was difficult. eHAC and Bio-Oss spongiosa blocks were biocompatible and induced few or no signs of inflammation. Inflammation prevalence between the groups was not statistically different. Bone formation and bone growth into the blocks was significantly higher in eHAC than Bio-Oss spongiosa blocks, but lower than in autologous bone grafts (after 1 and 3 months). Regression analysis showed that the autologous bone graft predicted new bone formation at both time points. The eHAC block was only a predictor at 1 month; a trend was found at 3 months. The application of biodegradable membranes was not related to more bone ingrowth.

Scandinavian Fellowship for Oral Pathology and Oral Medicine: statement on oral pathology and oral medicine in the European Dental Curriculum.

BACKGROUND: For many years, dentists have migrated between the Scandinavian countries without an intentionally harmonized dental education. The free movement of the workforce in the European Union has clarified that a certain degree of standardization or harmonization of the European higher education acts, including the dental education, is required. As a result of the Bologna process, the Association for Dental Education in Europe and the thematic network DentEd have generated guidelines in the document 'Profile and Competences for the European Dentist' (PCD). This document is meant to act as the leading source in revisions of dental curricula throughout Europe converging towards a European Dental Curriculum. In order to render the best conditions for future curriculum revisions providing the best quality dentist we feel obliged to analyse and comment the outlines of oral pathology and oral medicine in the PCD. METHODS: The representatives agreed upon definitions of oral pathology and oral medicine, and competences in oral pathology and oral medicine that a contemporary European dentist should master. The competences directly related to oral pathology and oral medicine were identified, within the PCD. RESULTS: The subject representatives suggested eighteen additions and two rewordings of the PCD, which all were substantiated by thorough argumentation. PERSPECTIVES: Hopefully, this contribution will find support in future revisions of the PCD in order to secure the best quality dental education.

The phosphatase and tensin homologue deleted on chromosome 10 mediates radiosensitivity in head and neck cancer.

BACKGROUND: For locally advanced squamous cell carcinoma of the head and neck (HNSCC), the recurrence rate after surgery and postoperative radiotherapy is between 20 and 40%, and the 5-year overall survival rate is approximately 50%. Presently, no markers exist to accurately predict treatment outcome. Expression of proteins in the human epidermal growth factor receptor (EGFR) pathway has been reported as a prognostic marker in several types of cancer. METHODS: The aim of this study was to investigate the prognostic value of proteins in the EGFR pathway in HNSCC. For this purpose, we collected surgically resected tissue of 140 locally advanced head and neck cancer patients, all treated with surgery and postoperative radiotherapy. RESULTS: In a multivariate analysis, expression of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was significantly related to worse locoregional control (LRC; HR: 2.2, 95% CI: 1.1-4.6; P=0.03), independent of lymph node metastases (HR: 5.6, 95% CI: 1.2-27.4; P=0.03) and extranodal spread (HR: 2.7; 95% CI: 1.2-6.5; P=0.02). In vitro clonogenic radiosensitivity assays confirmed that overexpression of PTEN resulted in increased radioresistance. CONCLUSION: Our study is the first report showing that expression of PTEN mediates radiosensitivity in vitro and that increased expression in advanced HNSCC predicts worse LRC.

[An obscure anomaly: regional odontodysplasia]. / Een spookachtige afwijking: regionale odontodysplasie.

In a 3-year-old child, acute dental abscesses in combination with clinical and radiographic impressions of a number of deciduous teeth indicated regional odontodysplasia as probable diagnosis. Histological examination of the removed deciduous teeth confirmed the diagnosis. Early determination of this regional developmental anomaly in the odontogenesis is of great importance for optimal guidance of the dental care of a patient with regional odontodysplasia.

Clinical and histologic evidence of salivary gland restoration supports the efficacy of rituximab treatment in Sjögren's syndrome.

OBJECTIVE: To assess the effect of rituximab (anti-CD20 antibody) therapy on the (immuno)histopathology of parotid tissue in patients with primary Sjögren's syndrome (SS) and the correlation of histologic findings with the flow rate and composition of parotid saliva. METHODS: In a phase II study, an incisional parotid biopsy specimen was obtained from 5 patients with primary SS before and 12 weeks after rituximab treatment (4 infusions of 375 mg/m(2)). The relative amount of parotid parenchyma, lymphocytic infiltrate, and fat, and the presence/quantity of germinal centers and lymphoepithelial duct lesions were evaluated. Immunohistochemical characterization was performed to analyze the B:T cell ratio of the lymphocytic infiltrate (CD20, CD79a, CD3) and cellular proliferation in the acinar parenchyma (by double immunohistologic labeling for cytokeratin 14 and Ki-67). Histologic data were assessed for correlations with the parotid flow rate and saliva composition. RESULTS: Four patients showed an increased salivary flow rate and normalization of the initially increased salivary sodium concentration. Following rituximab treatment, the lymphocytic infiltrate was reduced, with a decreased B:T cell ratio and (partial) disappearance of germinal centers. The amount and extent of lymphoepithelial lesions decreased in 3 patients and was completely absent in 2 patients. The initially increased proliferation of acinar parenchyma in response to inflammation was reduced in all patients. CONCLUSION: Sequential parotid biopsy specimens obtained from patients with primary SS before and after rituximab treatment demonstrated histopathologic evidence of reduced glandular inflammation and redifferentiation of lymphoepithelial duct lesions to regular striated ducts as a putative morphologic correlate of increased parotid flow and normalization of the salivary sodium content. These histopathologic findings in a few patients underline the efficacy of B cell depletion and indicate the potential for glandular restoration in SS.

Autogenous femoral head as grafting material for mandibular augmentation.

Bone grafting is commonly used for augmentation of the atrophic edentulous maxilla and mandible. Although bone substitutes and allogeneic frozen bone grafts have been applied successfully, fresh autogenous bone grafts remain the 'gold standard' in maxillofacial reconstructive surgery. A disadvantage of harvesting autogenous bone is the resulting donor-site morbidity. The authors present a case in which an autogenous femoral head, which was removed because of a prosthetic hip replacement, was used for augmentation of the extreme atrophic mandible. Using this procedure avoids donor-site morbidity.

Overexpression of intrinsic hypoxia markers HIF1alpha and CA-IX predict for local recurrence in stage T1-T2 glottic laryngeal carcinoma treated with radiotherapy.

PURPOSE: To examine the prognostic value of three endogenous hypoxia markers (hypoxia inducible factor 1 alpha subunit [HIF1 alpha], carbonic anhydrase IX [CA-IX], and glucose transporter type 1 [GLUT-1]) on the clinical outcome in patients with early-stage glottic carcinoma primarily treated with radiotherapy (RT) and to determine the predictive hypoxic profile to choose the optimal treatment of early-stage laryngeal carcinoma. METHODS AND MATERIALS: Immunohistochemistry for HIF1 alpha, CA-IX, and GLUT-1 was performed on formalin-fixed, paraffin-embedded, pretreatment tissue samples of 91 glottic squamous cell carcinoma specimens. The patient group consisted only of those with early-stage (T1-T2) glottic carcinoma, and all patients were treated with RT only. Relative tumor staining was scored on the tissue samples. Receiver operating curve analysis was performed to determine the optimal cutoff value for each tumor marker. Cox regression analyses for the variables HIF1 alpha, CA-IX, GLUT-1, gender, age, hemoglobin level, T category, N category, tobacco use, and alcohol use were performed with local control and overall survival as endpoints. RESULTS: HIF1 alpha overexpression in early-stage glottic carcinoma correlated significantly with worse local control (hazard ratio [HR], 3.05; p = 0.021) and overall survival (HR, 2.92; p = 0.016). CA-IX overexpression correlated significantly with worse local control (HR, 2.93; p = 0.020). GLUT-1 overexpression did not show any correlation with the clinical outcome parameters. Tumors with a nonhypoxic profile (defined as low HIF1 alpha and low CA-IX expression) had significantly better local control (HR, 6.32; p = 0.013). CONCLUSION: The results of our study have shown that early-stage glottic laryngeal carcinomas with low HIF1 alpha and CA-IX expression are highly curable with RT. For this group, RT is a good treatment option. For tumors with HIF1 alpha or CA-IX overexpression, hypoxic modification before RT or primary surgical treatment should be considered.

CO2 laser evaporation of oral lichen planus.

Oral lichen planus is a relatively common disease of the oral mucosa. The buccal mucosa and lateral border of the tongue are mostly involved, although the condition can occur anywhere in the oral cavity. The erosive type in particular can cause spontaneous pain during eating. In the period from 1975 to 2003, a group of 21 patients with 39 lesions of oral lichen planus which caused pain, even after conservative therapy, were treated with CO2 laser evaporation. During a follow-up period of 1-18 years (mean 8 years) 21 patients were pain free (85%) and 6 patients (15%) experienced painful recurrence after treatment. After retreatment with CO2 laser evaporation there were no complaints of pain. Among the many treatments available, high-potency topical corticosteroids remain the most consistent and effective. In patients whose condition is unresponsive to topical corticosteroids, CO2 laser evaporation can cause long-term remission of symptoms, and may even be the treatment of first choice in patients suffering from painful oral lichen planus.

Cortactin expression predicts poor survival in laryngeal carcinoma.

Amplification of the 11q13 region is one of the most frequent aberrations in squamous cell carcinomas of the head and neck region (HNSCC). Amplification of 11q13 has been shown to correlate with the presence of lymph node metastases and decreased survival. The 11q13.3 amplicon carries numerous genes including cyclin D1 and cortactin. Recently, we reported that FADD becomes overexpressed upon amplification and that FADD protein expression predicts for lymph node positivity and disease-specific mortality. However, the gene within the 11q13.3 amplicon responsible for this correlation is yet to be identified. In this paper, we compared, using immunohistochemical analysis for cyclin D1, FADD and cortactin in a series of 106 laryngeal carcinomas which gene correlates best with lymph node metastases and increased disease-specific mortality. Univariate Cox regression analysis revealed that high expression of cyclin D1 (P=0.016), FADD (P=0.003) and cortactin (P=0.0006) predict for increased risk to disease-specific mortality. Multivariate Cox analysis revealed that only high cortactin expression correlates with disease-specific mortality independent of cyclin D1 and/or FADD. Of genes located in the 11q13 amplicon, cortactin expression is the best predictor for shorter disease-specific survival in late stage laryngeal carcinomas.

Non-invasive measurement of the morphology and physiology of oral mucosa by use of optical spectroscopy.

Differential path-length spectroscopy (DPS) was used to non-invasively determine the superficial optical properties of oral mucosa in vivo. DPS yields information on physiological parameters such as the mucosal blood content, the microvascular blood oxygenation and the average micro-vessel diameter as well as on morphological parameters such as the scattering slope and scattering amplitude. DPS measurements were made on normal and cancerous oral mucosa using a novel fiber-optic probe, and were correlated to the histological outcome of punch biopsies taken from the same location. Our data shows that the mucosa of oral squamous cell carcinoma is characterised by a significant decrease in microvascular oxygenation and increase in mucosal blood content compared to normal oral mucosa as well as a significant decrease in scattering amplitude and increase in scattering slope.

Parotid gland biopsy compared with labial biopsy in the diagnosis of patients with primary Sjogren's syndrome.

OBJECTIVE: To assess the value of the parotid biopsy as a diagnostic tool for primary Sjögren's syndrome (pSS), and to compare the parotid biopsy and the labial biopsy with regard to diagnostic value and biopsy-related morbidity. METHODS: In 15 consecutive patients with pSS and 20 controls, the parotid biopsy was assessed as a diagnostic tool based on the presence of lymphocytic foci, benign lymphoepithelial lesions and lymphoid follicles. These new histological criteria were compared with established diagnostic criteria for the labial biopsy in 35 consecutive patients suspected for pSS who underwent simultaneous biopsies from both sites. In addition, both biopsies were compared for morbidity. RESULTS: The first analysis revealed a focus score of >or=1 or lymphocytic infiltrates (not fulfilling the criterion of a focus score of 1) combined with benign lymphoepithelial lesions as diagnostic criteria for pSS. When comparing the parotid biopsy with the labial biopsy sensitivity and specificity were comparable (sensitivity 78%, specificity 86%). Level of pain was comparable and no loss of motor function was observed. No permanent sensory loss was observed after parotid biopsy, while labial biopsy led to permanent sensory loss in 6% of the patients. Malignant lymphoma was detected in one parotid biopsy by chance, without involvement of the labial salivary gland. CONCLUSION: A parotid biopsy has a diagnostic potential comparable with that of a labial biopsy in the diagnosis of pSS, and may be associated with less morbidity.