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Background:Trismus, a restricted mouth opening in head and neck cancer patients may be caused by tumor infiltration in masticatory muscles, radiation-induced fibrosis or scarring after surgery. It may impede oral functioning severely. The aims of our study were to determine: (1) the incidence of trismus at various time points; and (2) the patient, tumor, and treatment characteristics that predict the development of trismus after radiotherapy in head and neck cancer patients using a large database (n = 641). Methods:Maximal mouth opening was measured prior to and 6, 12, 18, 24, 36, and 48 months after radiotherapy. Patient, tumor, and treatment characteristics were analyzed as potential predictors for trismus using a multivariable logistic regression analysis. Results:At six months after radiotherapy, 28.1% of the patients without trismus prior to radiotherapy developed trismus for the first time. At subsequent time points the incidence declined. Over a total period of 48 months after radiotherapy, the incidence of trismus was 3.6 per 10 person years at risk. Patients who had tumors located in the oral cavity, oropharynx or nasopharynx, and the salivary glands or ear, and who had a longer overall treatment time of radiotherapy, were more likely to develop trismus in the first six months after radiotherapy. Maximal mouth opening was a predictor for developing trismus at all time points. Conclusion:Incidence of trismus is 3.6 per 10 person years at risk. Tumor localization and overall treatment time of radiotherapy are predictors for developing trismus the first six months after radiotherapy. Maximal mouth opening is a significant predictor for developing trismus at all time points. Regular measurements of maximal mouth opening are needed to predict trismus. [ABSTRACT FROM PUBLISHER]

Autofluorescence spectroscopy is a tool for detecting tissue alterations in vivo. In a previous study, we found spectral differences between clinically normal mucosa of different patient groups. These are possibly caused by associated patient characteristics. In the present study, we explore the influences of volunteer characteristics on healthy oral mucosa autofluorescence.Autofluorescence spectra were recorded in 96 volunteers with no clinically observable oral lesions. We applied principal components analysis to extract the relevant information. We used multivariate linear regression techniques to estimate the effect of volunteer characteristics on principal component scores.Statistically significant differences were found for all factors but age. Skin color strongly affected autofluorescence intensity. Gender differences were found in blood absorption. Alcohol consumption was associated with porphyrin-like peaks. However, all differences but those associated with skin color were of the same order of magnitude as standard deviations within categories.The effects of volunteer characteristics on autofluorescence spectra of the oral mucosa are measurable. Only the effects of skin color were large. Therefore, in lesion classification, skin color should be taken into account. [Copyright &y& Elsevier]

Objective: In line with screening guidelines, cancer survivors were consecutively screened on depressive symptoms (as part of standard care), with those reporting elevated levels of symptoms offered psychological care as part of a trial. Because of the low uptake, no conclusions could be drawn about the interventions' efficacy. Given the trial set-up (following screening guidelines and strict methodological quality criteria), we believe that this observational study reporting the flow of participation, reasons for and characteristics associated with nonparticipation, adds to the debate about the feasibility and efficiency of screening guidelines.Methods: Two thousand six hundred eight medium- to long-term cancer survivors were consecutively screened on depressive symptoms using the Patient Health Questionnaire-9 (PHQ-9). Those with moderate depressive symptoms (PHQ-9 ≥ 10) were contacted and informed about the trial. Patient flow and reasons for nonparticipation were carefully monitored.Results: One thousand thirty seven survivors (74.3%) returned the questionnaire, with 147 (7.6%) reporting moderate depressive symptoms. Of this group, 49 survivors (33.3%) were ineligible, including 26 survivors (17.7%) already receiving treatment and another 44 survivors (30.0%) reporting no need for treatment. Only 25 survivors (1.0%) participated in the trial.Conclusion: Of the approached survivors for screening, only 1% was eligible and interested in receiving psychological care as part of our trial. Four reasons for nonparticipation were: nonresponse to screening, low levels of depressive symptoms, no need, or already receiving care. Our findings question whether to spend the limited resources in psycho-oncological care on following screening guidelines and the efficiency of using consecutive screening for trial recruitment in cancer survivors. [ABSTRACT FROM AUTHOR]

Background: For patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) palliative treatment options that improve overall survival are limited. The prognosis in this group remains poor and there is an unmet need for new therapeutic options. An emerging class of therapeutics, targeting tumor-specific antigens, are antibodies bound to a cytotoxic agent, known as antibody-drug conjugates (ADCs). The aim of this study was to prioritize ADC targets in HNSCC.Methods: With a systematic search, we identified 55 different ADC targets currently targeted by registered ADCs and ADCs under clinical evaluation. For these 55 ADC targets, protein overexpression was predicted in a dataset containing 344 HNSCC mRNA expression profiles by using a method called functional genomic mRNA profiling. The ADC target with the highest predicted overexpression was validated by performing immunohistochemistry (IHC) on an independent tissue microarray containing 414 HNSCC tumors.Results: The predicted top 5 overexpressed ADC targets in HNSCC were: glycoprotein nmb (GPNMB), SLIT and NTRK-like family member 6, epidermal growth factor receptor, CD74 and CD44. IHC validation showed combined cytoplasmic and membranous GPNMB protein expression in 92.0% of the cases. Strong expression was seen in 65.9% of the cases. In addition, 86.5% and 67.7% of cases showed ≥5% and >25% GPNMB positive tumor cells, respectively.Conclusions: This study provides a data-driven prioritization of ADCs targets that will facilitate clinicians and drug developers in deciding which ADC should be taken for further clinical evaluation in HNSCC. This might help to improve disease outcome of HNSCC patients. [ABSTRACT FROM AUTHOR]

Purpose: We sought to assess the effect of age on overall survival (OS), cancer-specific survival (CSS), and non-cancer-related death (NCRD) in elderly (aged ≥70 years) head and neck squamous cell carcinoma (HNSCC) patients treated with definitive radiation therapy. The results were compared with those of younger patients, and the most important prognostic factors for survival endpoints were determined. Treatments may be better justified based on identification of the main differences in survival between young and elderly patients.Methods and Materials: Data were analyzed from all consecutive HNSCC patients treated with definitive radiation therapy (66-70 Gy) in our department between April 2007 and December 2014. A total of 674 patients, including 168 elderly patients (24.9%), were included in the study. Multivariate association models were constructed to assess the effect of age on survival endpoints. Multivariate analysis was performed to identify potential prognostic factors for survival in elderly patients.Results: A total of 674 consecutive patients, including 168 elderly patients, were analyzed. The 5-year OS and NCRD rates were significantly worse for elderly patients than for young patients: 45.5% versus 58.2% (P=.007) and 39.0% versus 20.7% (P<.001), respectively. In the multivariate association analysis on the relationship between age and OS, lymph node involvement and worse World Health Organization (WHO) performance status were identified as significant confounders. Multivariate association analysis between age and NCRD identified Union for International Cancer Control stage as a significant confounder. After correction for confounders, the effect of age on OS and NCRD increased. Worse WHO performance status, lymph node involvement, and specific tumor site were independent prognostic factors for OS and CSS in the elderly patient group. Of the elderly patients, 80 (47%) died during follow-up; 45% of these deaths were ascribed to the index tumor. For elderly patients, radiation therapy combined with systemic forms of treatment was significantly associated with adverse NCRD rate (hazard ratio, 8.02; 95% confidence interval, 2.36-27.2; P=.001) after we performed a multivariate association analysis.Conclusions: Elderly HNSCC patients have worse survival outcomes than young HNSCC patients. Age is an independent prognostic factor for OS, mainly due to an increase in non-cancer-related mortality and comorbid diseases. The differences in CSS between young and elderly patients are negligible. [ABSTRACT FROM AUTHOR]

Purpose The aim of this study was to clinically validate a multivariable normal tissue complication probability (NTCP) model for grade 2–4 swallowing dysfunction at 6 months after radiotherapy or chemoradiation (SWAL M6 ) in head and neck cancer patients treated with swallowing sparing intensity modulated radiotherapy (SW-IMRT) and to test if SW-IMRT resulted in a reduction of the prevalence of SWAL M6 . Materials and methods The primary endpoint was SWAL M6 . For all 186 patients, a standard IMRT (parotid sparing) and a SW-IMRT plan (additional constraints for swallowing organs at risk) was created. The difference in NTCP for SWAL M6 (ΔNTCP SWALM6 = NTCP standard − NTCP SW-IMRT ) was calculated. Patients were treated with SW-IMRT. The external validation of the NTCP model was analyzed by comparing performance measures. Results The mean ΔNTCP SWALM6 was 4.9% (range 0.01–17.3%), with a significant lower mean predicted NTCP SW-IMRT of 22.6% (95% CI 20.2–24.9%), compared to NTCP standard of 27.5% (95% CI 24.9–29.9%) ( p < 0.001). There was a perfect correspondence of NTCP SW-IMRT with the observed prevalence of SWAL M6 (22.6%). The overall model performance, discrimination and ‘goodness of fit’ were good. Conclusion We externally validated the multivariable NTCP model for SWAL M6 in SW-IMRT treated patients, showing reduced swallowing dysfunction by reducing the dose parameters included in this NTCP model. [ABSTRACT FROM AUTHOR]

Objectives To identify patterns of long-term, radiation-induced swallowing dysfunction after definitive radiotherapy with or without chemotherapy (RT or CHRT) and to determine which factors may explain these patterns over time. Material and methods The study population consisted of 238 consecutive head and neck cancer patients treated with RT or CHRT. The primary endpoint was ⩾grade 2 swallowing dysfunction at 6, 12, 18 and 24 months after treatment. Cluster analysis was used to identify different patterns over time. The differences between the mean dose to the swallowing organs at risk for each pattern were determined by using dose maps. Results The cluster analysis revealed five patterns of swallowing dysfunction: low persistent, intermediate persistent, severe persistent, transient and progressive. Patients with high dose to the upper pharyngeal, laryngeal and lower pharyngeal region had the highest risk of severe persistent swallowing dysfunction. Transient problems mainly occurred after high dose to the laryngeal and lower pharyngeal regions, combined with moderate dose to the upper pharyngeal region. The progressive pattern was mainly seen after moderate dose to the upper pharyngeal region. Conclusions Various patterns of swallowing dysfunction after definitive RT or CHRT can be identified over time. This could reflect different underlying biological processes. [ABSTRACT FROM AUTHOR]

Purpose To determine if acute symptoms during definitive radiotherapy (RT) or chemoradiation (CHRT) are prognostic factors for late dysphagia in head and neck cancer (HNC). Material and methods This prospective cohort study consisted of 260 HNC patients who received definitive RT or CHRT. The primary endpoint was grade 2–4 swallowing dysfunction at 6 months after completing RT (SWALM6). During treatment, acute symptoms, including oral mucositis, xerostomia and dysphagia, were scored, and the scores were accumulated weekly and entered into an existing reference model for SWALM6 that consisted of dose–volume variables only. Results Both acute xerostomia and dysphagia were strong prognostic factors for SWALM6. When acute scores were added as variables to the reference model, model performance increased as the course of treatment progressed: the AUC rose from 0.78 at the baseline to 0.85 in week 6. New models built for weeks 3–6 were significantly better able to identify patients with and without late dysphagia. Conclusion Acute xerostomia and dysphagia during the course of RT are strong prognostic factors for late dysphagia. Including accumulated acute symptom scores on a weekly basis in prediction models for late dysphagia significantly improves the identification of high-risk and low-risk patients at an early stage during treatment and might facilitate individualized treatment adaptation. [ABSTRACT FROM AUTHOR]

Background and purpose Curative radiotherapy/chemo-radiotherapy for head and neck cancer (HNC) may result in severe acute and late side effects, including tube feeding dependence. The purpose of this prospective cohort study was to develop a multivariable normal tissue complication probability (NTCP) model for tube feeding dependence 6 months (TUBE M6 ) after definitive radiotherapy, radiotherapy plus cetuximab or concurrent chemoradiation based on pre-treatment and treatment characteristics. Materials and methods The study included 355 patients with HNC. TUBE M6 was scored prospectively in a standard follow-up program. To design the prediction model, the penalized learning method LASSO was used, with TUBE M6 as the endpoint. Results The prevalence of TUBE M6 was 10.7%. The multivariable model with the best performance consisted of the variables: advanced T-stage, moderate to severe weight loss at baseline, accelerated radiotherapy, chemoradiation, radiotherapy plus cetuximab, the mean dose to the superior and inferior pharyngeal constrictor muscle, to the contralateral parotid gland and to the cricopharyngeal muscle. Conclusions We developed a multivariable NTCP model for TUBE M6 to identify patients at risk for tube feeding dependence. The dosimetric variables can be used to optimize radiotherapy treatment planning aiming at prevention of tube feeding dependence and to estimate the benefit of new radiation technologies. [ABSTRACT FROM AUTHOR]

Purpose To assess the prevalence of EGFRvIII, a specific variant of EGFR (epidermal growth factor receptor), in 3 well-defined cohorts of head and neck squamous cell carcinoma (HNSCC). Methods and Materials Immunohistochemistry for the specific detection of EGFRvIII using the L8A4 antibody was optimized on formalin-fixed, paraffin-embedded tissue using glioblastoma tissue. It was compared with EGFR and EGFRvIII RNA expression using a specific reverse transcription–polymerase chain reaction also optimized for formalin-fixed, paraffin-embedded tissue. Tissue microarrays including 531 HNSCCs of various stages with complete clinicopathologic and follow-up data were tested for the presence of EGFRvIII. Results None of the 531 cases showed EGFRvIII protein expression. Using an immunohistochemistry protocol reported by others revealed cytoplasmic staining in 8% of cases. Reverse transcription–polymerase chain reaction for the EGFRvIII transcript of the 28 highest cytoplasmic staining cases, as well as 69 negative cases, did not show expression in any of the tested cases, suggesting aspecific staining by a nonoptimal protocol. Conclusions The EGFRvIII mutation is not present in HNSCC. Therefore, EGFRvIII does not influence treatment response in HNSCC and is not a usable clinical prognostic marker. [ABSTRACT FROM AUTHOR]

Purpose: The objective of this study was to evaluate the treatment outcomes (implant survival, denture satisfaction, subjective chewing ability, oral function and quality of life) of oral cancer patients with implant-retained mandibular overdentures, in whom the implants were installed during ablative tumor surgery, up to 14years after placement of the implants. Methods and materials: Edentulous oral cancer patients with the need for surgery and in whom prosthetic problems were expected after oncological treatment, were offered implant-based treatment in the mandibula. All implants were installed during ablative tumor surgery in native bone in the interforaminal area. The patients and implants were evaluated by clinical assessments and standardized questionnaires. Results: Of the 181 patients that fulfilled the inclusion criteria, 165 patients were included. One hundred patients received postoperative radiotherapy. In total 138 patients were rehabilitated with an implant-based prosthesis (84%). Of the 528 placed implants, 35 implants were lost during the follow-up (overall survival rate=93.4%). Implant loss was higher in irradiated patients. Rehabilitation not depend on radiotherapy, type of reconstruction used during surgery or the number of implants placed. Irradiated patients reported lower scores on several items of the quality of life and oral functioning questionnaires. Patients not wearing the prosthesis had lower scores on oral functioning questionnaires and reported less chewing ability. The type of reconstruction used or the number of implants placed did not give different outcome of the questionnaires. Conclusions: It was concluded that oral cancer patients can benefit from implants installed during ablative surgery, with a high survival rate of the implants, a high percentage of rehabilitated patients and short time between surgery and prosthetic rehabilitation. Patients not receiving post-operative radiotherapy report a better oral function and more prosthesis-related satisfaction. When possible from a prosthetic point of view, two mandibular implants should be placed. [Copyright &y& Elsevier]

Abstract: Background and purpose: The purpose of this large multicentre prospective cohort study was to identify which dose volume histogram parameters and pre-treatment factors are most important to predict physician-rated and patient-rated radiation-induced swallowing dysfunction (RISD) in order to develop predictive models for RISD after curative (chemo) radiotherapy ((CH) RT). Material and methods: The study population consisted of 354 consecutive head and neck cancer patients treated with (CH) RT. The primary endpoint was grade 2 or more swallowing dysfunction according to the RTOG/EORTC late radiation morbidity scoring criteria at 6months after (CH) RT. The secondary endpoints were patient-rated swallowing complaints as assessed with the EORTC QLQ-H&N35 questionnaire. To select the most predictive variables a multivariate logistic regression analysis with bootstrapping was used. Results: At 6months after (CH) RT the bootstrapping procedure revealed that a model based on the mean dose to the superior pharyngeal constrictor muscle (PCM) and mean dose to the supraglottic larynx was most predictive. For the secondary endpoints different predictive models were found: for problems with swallowing liquids the most predictive factors were the mean dose to the supraglottic larynx and radiation technique (3D-CRT versus IMRT). For problems with swallowing soft food the mean dose to the middle PCM, age (18–65 versus >65years), tumour site (naso/oropharynx versus other sites) and radiation technique (3D-CRT versus IMRT) were the most predictive factors. For problems with swallowing solid food the most predictive factors were the mean dose to the superior PCM, the mean dose to the supraglottic larynx and age (18–65 versus >65years). And for choking when swallowing the V60 of the oesophageal inlet muscle and the mean dose to the supraglottic larynx were the most predictive factors. Conclusions: Physician-rated and patient-rated RISD in head and neck cancer patients treated with (CH) RT cannot be predicted with univariate relationships between the dose distribution in a single organ at risk and an endpoint. Separate predictive models are needed for different endpoints and factors other than dose volume histogram parameters are important as well. [Copyright &y& Elsevier]

Summary: Treatment choice in elderly pharyngeal cancer patient is disputed. This study was aimed to asses association of co-morbidity, complications and survival in different treatment modalities of pharyngeal cancer patients. Retrospective analysis of pharyngeal cancer patients, diagnosed between 1997 and 2007 in a tertiary referral hospital was performed. Patients 75years and older (n =42), were matched with two control patients 64years and younger (n =84). Co-morbidity (ACE-27), treatment related complications and survival data were assessed and analyzed. Frequency of co-morbidity was similar in both age groups, although discarding alcohol abuse resulted in higher incidence of co-morbidity in the elderly group. Complication rate was not significantly different. In a multivariate analysis only stage found to be a significant predictor of complications. Survival estimates adjusted to sex, age and birth cohort revealed co-morbidity to be a significant predictor for survival in elderly and young patients. No evidence has been found to treat elderly pharyngeal cancer patients differently than younger ones. Treatment related complications are not predicted by co-morbidities in young and elderly patients; however survival is predicted by comorbidity. Therefore thorough pre-treatment evaluation and care necessary in the elderly population. [Copyright &y& Elsevier]

Summary: Detection of distant metastases and second primary tumors in patients with head and neck squamous cell carcinoma (HNSCC) is of importance because of the impact on treatment and prognosis. The aim of this study was to assess the value of whole-body fluorodeoxyglucose-positron emission tomography (FDG-PET) in detecting distant metastases and second primary tumors below clavicular level in HNSCC compared to chest CT and chest radiography (CXR). Patient records of 149 consecutive patients with a primary HNSCC in whom a whole-body FDG-PET for initial staging was performed were reviewed. FDG-PET showed a sensitivity and specificity for detecting distant disease of 92% and 93%, respectively. Chest CT (n =82) showed a sensitivity and a specificity of 74% and 61% respectively. CXR (n =106) showed a sensitivity and a specificity of 41% and 91%, respectively. FDG-PET was shown to be able to detect distant metastases and second primary tumors in HNSCC with a high sensitivity and specificity. The higher sensitivity of PET compared to chest CT is mainly due to the detection of extrapulmonary malignancy. Sensitivity of PET and CT were similar in detecting intrapulmonary malignancy, but PET specificity was significantly higher. FDG-PET helps to determine the nature of pulmonary lesions and might be considered as a first diagnostic modality for detecting distant disease in advanced HNSCC. [Copyright &y& Elsevier]

Abstract: Background and purpose: The aim of the study was to define the added value of whole body FDG-PET in screening for distant metastases in patients with head and neck squamous cell carcinoma and risk factors. Materials and methods: In a multi-center prospective study between 1998 and 2003, 145 consecutive HNSCC patients with risk factors for distant metastases underwent chest CT and whole body FDG-PET for screening of distant metastases. The data of 92 evaluable patients who developed distant metastases or who had a follow-up of at least 12 months were analyzed. Besides their performance in clinical practice, the operational characteristics of PET and CT using ROC analyses were investigated. Results: Pretreatment screening identified distant metastases in 19 patients (21%). FDG-PET had a higher sensitivity (53% vs. 37%) and positive predictive value (80% vs. 75%) than CT. The combination of CT and FDG-PET had the highest sensitivity (63%). The ROC analyses of the five point ordinal scales revealed that the “area under the curve” (AUC) of FDG-PET was significantly higher as compared to CT. Conclusion: In HNSCC patients with risk factors, pretreatment screening for distant metastases by chest CT is improved by FDG-PET. [Copyright &y& Elsevier]

Summary: Treatment of the clinical N0 neck in squamous cell carcinoma (SCC) of oral cavity and oropharynx remains a dilemma. None of the current imaging modalities are able to detect the presence of micrometastases in the lymph nodes of clinical N0 necks reliably. The aim of this study was to determine the diagnostic properties of fluorine-18 fluorodeoxyglucose positron emission tomography (FDG PET) in patients clinically staged as N0. FDG PET results of 38 patients were compared to histologic specimens obtained with neck dissections or to follow-up. FDG PET performance was compared to computed tomography (CT), magnetic resonance imaging (MRI) or ultrasonography-guided fine needle aspiration cytology (USgFNAC). Sensitivity and specificity of FDG PET in detecting occult cervical metastases were 50% and 97% respectively. Although FDG PET performed better than conventional imaging modalities, sensitivity was lower than desired. As a consequence, clinical application of FDG PET in the patient staged as N0 is limited. [Copyright &y& Elsevier]

Summary: Surgical treatment of malignancies in the oral cavity and subsequent radiotherapy often result in an anatomic and physiological oral condition unfavorable for prosthodontic rehabilitation. The objective of this prospective study was to assess the effect of hyperbaric oxygen therapy on treatment outcome (condition of peri-implant tissues, implant survival, oral functioning and quality of life) of prosthodontic rehabilitation with implant-retained lower dentures in radiated head and neck cancer patients 6weeks and 1year after placing the new dentures. The treatment outcome was assessed in a group of 26 head neck cancer patients who were subjected to radiotherapy after tumour surgery. Standardized questionnaires were completed and clinical and radiographic assessments were performed. After randomization, endosseous Brånemark implants were placed in the anterior part of the mandible either under antibiotic prophylaxis (13 patients) or under antibiotic prophylaxis combined with pre and postsurgery hyperbaric oxygen (HBO) treatment (13 patients). In the HBO and non-HBO group eight implants (implant survival 85.2%) and three implants (implant survival 93.9%) were lost, respectively. Peri-implant tissues had a healthy appearance in both groups. Osteoradionecrosis developed in one patient in the HBO group. All patients functioned well with their implant-retained lower denture. The quality of life related to oral functioning and denture satisfaction were improved to a comparable extent in the HBO and non-HBO group. Implant-retained lower dentures can improve the quality of life related to oral functioning and denture satisfaction in head and neck cancer patients. Adjuvant hyperbaric oxygen therapy could not be shown to enhance implant survival in radiated mandibular jaw bone. [Copyright &y& Elsevier]

Summary: The obstructive sleep apnoea–hypopnoea syndrome (OSAHS) is a sleep-related breathing disorder characterised by repetitive pharyngeal collapse. OSAHS is associated with a reduced quality of life. A high OSAHS prevalence has been reported in patients treated for head and neck cancer (HNC). The aim of the present study was to identify the prevalence of OSAHS within a Dutch population of patients treated for HNC. Consecutive HNC patients with a follow-up of 6 months to 5 years after treatment of an oral or oropharynx carcinoma were eligible for inclusion. Two questionnaires were used to assess the presence of OSAHS-related complaints. Subsequently, polysomnography was used in patients with OSAHS-related complaints to confirm the diagnosis of OSAHS. Four out of 33 included patients were diagnosed with OSAHS, yielding a prevalence of 12%. Since recognition and treatment of OSAHS might play an important role in improving quality of life of HNC patients, we suggest screening all patients with an oral or oropharynx carcinoma for the presence of OSAHS-related complaints prior to and following HNC treatment. [Copyright &y& Elsevier]

Purpose: HPV-associated cancer in the head-neck region has been reported at high frequencies in certain countries and is considered a separate entity. Since the prevalence of HPV positivity in oropharyngeal squamous cell carcinomas is currently not known in the Northern part of the Netherlands, the aim was to analyze HPV prevalence in oropharyngeal tumours treated at our centre. Material and methods: Based on the Dutch cancer registration, we collected clinical, pathological, and follow-up data and formalin-fixed paraffin embedded samples of a complete cohort of 125 tonsillar and base-of-tongue oropharyngeal tumours treated in our centre from 1997 to 2009. HPV testing was performed using an established algorithm of p16 immunohistochemistry, followed by HPV-PCR of the positive cases. HPV-BRISH was performed as extra control of p16 negative cases. Results: p16 staining could be assessed on 109 cases. 32/109 (29%) were p16+ of these 32 cases 21/32 (66%) were HPV-GP+, 16 cases were found HPV16+ and one HPV18+, three were HPV33+ and one HPV35+. HPV-BRISH showed 83/102 (81%) negativity and 19/102 (19%) positivity. BRISH had a sensitivity of 85% and a specificity of 98%, as only two p16- cases were positive for BRISH. HPV prevalence increased from 11% between 1997 and 2002, to 27% between 2003 and 2009. Conclusions: In the period 1997–2009, 19% of the tonsillar and base-of-tongue tumours in the Northern Netherlands was HPV+. This prevalence is lower than in other regions in the Netherlands (33%) and in Europe (40%). Remarkably, a large increase in prevalence was observed in the more recent years. The relative low incidence in our region might be due to the relative high percentage of smokers, associated with non-HPV related oropharyngeal tumours. Currently HPV testing of a series of 200 oral squamous cell carcinomas from our centre is in progress. [Copyright &y& Elsevier]

About halve of all oral squamous cell carcinoma (OSCC) patients have occult nodal metastases (N-status) due to the inaccuracy of current detection methods. The aim of our study is to find DNA methylation markers in the DNA of the primary tumor that predict N-status. For biomarker discovery we used a new approach combining a global DNA methylation screen, next generation sequencing, quantitative methylation detection and microarray expression profiling. Here we report the success of this new approach and the further validation of novel methylation markers. We assessed global methylation levels on DNA extracted from 6 oral squamous cell carcinomas (OSCC) with nodal metastases (N+) and 6 OSCC without nodal metastases (N0) by MethylCap-Seq. Isolated methylated DNA fragments were sequenced by Illumina GA II and computationally mapped back to the genome. A list was composed of the 5000 most differentially methylated genes between the N+and the N0 group. The most significantly differentially methylated genes were selected for further validation by Quantitative Methylation Specific PCR and Pyrosequencing on an independent series of 20N+and 20 N0 OSCC. In addition the MethylCap-Seq methylation data was combined with microarray expression data of 696 genes for a N-status validation cohort containing 222 OSCC (Hoof et al., 2012, J. Clin. Oncol.). The effect of DNA methylation on gene expression will be measured by (Q)RT-PCR and immunohistochemistry on genes differently hypermethylated and downregulated. The most promising methylation markers will be further validated on a N-status validation cohort containing 463 cases for which complete clinicopathological and follow-up data are available. These markers might contribute to better diagnosis, improved quality of life and may be associated with increased survival. [Copyright &y& Elsevier]