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Background: Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas., Methods: Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation., Findings: A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC., Conclusions: Organoids hold potential as a diagnostic tool in personalized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation., Funding: This work was funded by Oncode PoC 2018-P0003., Competing Interests: Declaration of interests H.C.’s full disclosure is given at https://www.uu.nl/staff/JCClevers/. H.C. is inventor of several patents related to organoid technology, is a co-founder of Xilis, Inc., and is currently an employee of Roche, Basel. E.D. is inventor on a patent related to head and neck organoid technology. R.M. is currently an employee of Roche Products, Welwyn. S.M.W. holds unrestricted research grants from Bayer, Pfizer, Roche, MSD, Amgen, and AstraZeneca, all paid to the UMCG., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study aimed to assess the prognostic value of intratumoral CD57+ cells in head and neck squamous cell carcinoma (HNSCC) and to examine the reproducibility of these analyses using QuPath. Pretreatment biopsies of 159 patients with HPV-negative, stage III/IV HNSCC treated with chemoradiotherapy were immunohistochemically stained for CD57. The number of CD57+ cells per mm 2 tumor epithelium was quantified by two independent observers and by QuPath, software for digital pathology image analysis. Concordance between the observers and QuPath was assessed by intraclass correlation coefficients (ICC). The correlation between CD57 and clinicopathological characteristics was assessed; associations with clinical outcome were estimated using Cox proportional hazard analysis and visualized using Kaplan-Meier curves. The patient cohort had a 3-year OS of 65.8% with a median follow-up of 54 months. The number of CD57+ cells/mm 2 tumor tissue did not correlate to OS, DFS, or LRC. N stage predicted prognosis (OS: HR 0.43, p = 0.008; DFS: HR 0.41, p = 0.003; LRC: HR 0.24, p = 0.007), as did WHO performance state (OS: HR 0.48, p = 0.028; LRC: 0.33, p = 0.039). Quantification by QuPath showed moderate to good concordance with two human observers (ICCs 0.836, CI 0.805-0.863, and 0.741, CI 0.692-0.783, respectively). In conclusion, the presence of CD57+ TILs did not correlate to prognosis in advanced stage, HPV-negative HNSCC patients treated with chemoradiotherapy. Substantial concordance between human observers and QuPath was found, confirming a promising future role for digital, algorithm driven image analysis., (© 2022. The Author(s).)

Background: Recurrences remain an important problem in laryngeal squamous cell carcinoma. Little has been described about histological characteristics of the primary laryngeal tumor that may be associated with recurrences. Identifying risk factors for recurrences might help in adapting treatment or follow-up. Using real-life population-based data, we aimed to identify histological features of the primary tumor associated with recurrences and overall survival., Material and Methods: Demographic, clinical and treatment information on all first primary invasive laryngeal tumors diagnosed in 2010-2014 (N = 3705) were extracted from the population-based nationwide Netherlands cancer registry (NCR) and linked to PALGA, the nationwide Dutch pathology registry, to obtain data on histological factors and recurrences. For a random 1502 patients histological information i.e., keratinization, perineural invasion (PNI+), vascular invasion (VI+), growth pattern, degree of differentiation, extracapsular spread (ECS+), cartilage- and bone invasion and extralaryngeal extension, was manually extracted from narrative pathology reports and analyzed for locoregional recurrence and overall survival using cox regression analysis., Results: In total, 299 patients developed a locoregional recurrence and 555 patients died. Keratinization (HR = 0.96 (95%CI: 0.68-1.34) p = 0.79), two or three adverse characteristics (PNI+, VI+, non-cohesive growth) (HR = 1.38 (95% CI: 0.63-3.01) p = 0.42), and ECS+ (HR = 1.38 (95% CI: 0.48-4.02) p = 0.55) were not associated to recurrence. For death, also no significant association was found., Conclusion: In this population-based real-life dataset on laryngeal carcinoma in the Netherlands, histological factors were not associated with locoregional recurrences or overall survival, but future studies should investigate the role of these features in treatment decisions., (© 2022. The Author(s).)

Human papilloma virus (HPV) is a well-established causative factor in a subset of squamous cell carcinomas of the head and neck (HNSCC). Although HPV can be detected in various anatomical subsites, HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) is the most common HPV-related malignancy of the head and neck, and its worldwide incidence is constantly rising. Patients with OPSCC are generally younger, have less co-morbidities and generally have better prognosis due to different biological mechanisms of carcinogenesis. These facts have generated hypotheses on potential treatment modifications, aiming to minimize treatment-related toxicities without compromising therapy efficacy. Numerous randomized clinical trials have been designed to verify this strategy and increasingly real-world evidence data from retrospective, observational studies is becoming available. Until now, the data do not support any modification in contemporary treatment protocols. In this narrative review, we outline recent data provided by both randomized controlled trials and real-world evidence of HPV-positive OPSCC in terms of clinical value. We critically analyze the potential value and drawbacks of the available data and highlight future research directions. This article was written by members and invitees of the International Head and Neck Scientific Group.(www.IHNSG.com)., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: Squamous cell carcinoma of the tongue (SCCT) is preferably treated by surgery. Free resection margins (≥5 mm) provide local control and disease-free survival. However, close (1-5 mm) and positive margins (<1 mm) are frequently encountered. We present our first experience of in-vivo ultrasound (US) guided SCCT resections followed by ex-vivo US control on the resection specimen to obtain free margins. We compare the results with those from a hisorical cohort of 91 conventionally treated SCCT patients., Materials and Methods: Ten patients with SCCT were included in a consecutive US-cohort. We aimed for a 5-10 mm margin during surgery, while we visualized the resection plane on US. Ex-vivo US measurements on the resection specimen determined whether there was any need for an immediate re-resection. US measurements were then compared with histopathology. Histopathological margins were compared with a consecutive cohort of 91 patients who had undergone conventional surgery for a SCCT., Results: In the US cohort, 70% of the margins were free. In the conventional cohort, this figure was 17% (P = 0.005). US predicted minimal histopathological margin distance with a mean ± SD error of 1.9 ± 1.8 mm. The mean ± SD of the histopathological overall submucosal/deep margin distance was 7.9 ± 2.1 mm in the US cohort and 7.0 ± 2.2 mm in the conventional cohort (P = 0.188). Ex-vivo examination through use of US indicated an immediate re-resection, which prevented local adjuvant treatment., Conclusion: Use of US-guided SCCT resection is feasible and improves margin control., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Hypoxia is a negative prognostic factor in head and neck squamous cell carcinomas. Under hypoxia, the hypoxia-inducible factor (HIF)-1a transcription factor is overexpressed. We investigated whether there were site differences in HIF-1a expression and its effect on patient outcomes per subsite., Design/method: A total of 941 patients with HNSCC in the squamous cell carcinoma of the oropharynx (OPSCC, n = 302), oral cavity (OSCC, n = 391), or larynx (LSCC, n = 248) were included. Expression of HIF-1a in tissue samples was investigated using immunohistochemistry. Overall survival (OS), disease-free survival (DFS), and locoregional control (LRC) were analyzed., Results: HIF-1a expression was higher in OSCC than in LSCC and OPSCC. High HIF-1a expression led to worse prognosis in OPSCC (OS P = .029, DFS P = .085) and LSCC (OS P = .041, DFS P = .011) and better in OSCC (OS P = .055, DFS P = .012). There was no association between HIF-1a and LRC., Conclusions: High HIF-1a expression is related to poor outcome in OPSCC and LSCC and better outcome in OSCC., (© 2020 Wiley Periodicals LLC.)

Background : Despite the improvement in therapeutic interventions, 5-year survival rates in Head and Neck Squamous Cell Carcinoma (HNSCC) are limited. HNSCC is an immunogenic cancer type for which molecular stratification markers are lacking. Tumor-infiltrating lymphocytes (TILs) have shown a favorable prognostic role in different cancer types. This study focused on the prognostic role of NK cells in HNSCC. Methods : A systematic search was conducted in Pubmed/Medline and Embase. Articles that correlated the presence of intratumoral NK cells, activating/inhibiting receptors, death receptors, or their ligands with clinicopathologic characteristics or survival were included. A meta-analysis was performed that assessed the association between CD56+ and CD57+ and overall survival (OS), disease-free survival (DFS), and progression-free survival (PFS). Results : A pooled analysis indicated a favorable prognostic role of CD56+ and CD57+ NK cells for OS (HR 0.19 CI 0.11-0.35). NK cell markers NKp46 and Granzyme B (GrB) also have a favorable prognostic role. NK cell ligand Fas correlated with better survival and better characteristics. NK cell marker Fas-L, NK cell ligands CEACAM1, RCAS1, CD70 and TRAIL-R, and effector molecules of these ligands, FADD and FAP1, correlated to features of worse prognosis. Conclusion : A favorable prognostic role of NK cells in HNSCC was found in this review. Some studies implied the opposite, indicating the fine balance between pro- and anti-tumor functions of NK cells. Future studies using homogeneous patient cohorts regarding tumor subsite and treatment modality, are necessary to further provide insight into the prognostic role of NK cells., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Aim: This study aimed to evaluate the presence and prognostic value of tumor-infiltrating T cells in the tumor epithelium in advanced stage, HPV-negative head and neck squamous cell carcinoma (HNSCC) patients treated with primary chemoradiotherapy using digital pathology., Methods: Pre-treatment biopsies from 80 oropharyngeal, 52 hypopharyngeal, and 29 laryngeal cancer patients were collected in a tissue microarray (TMA) and immunohistochemically stained for T-cell markers CD3, CD4, CD8, FoxP3, and PD1, and for immune checkpoint PD-L1. For each marker, the number of positive tumor-infiltrating lymphocytes (TILs) per mm 2 tumor epithelium was digitally quantified and correlated to overall survival (OS), disease-free survival (DFS), and locoregional control (LRC), as well as to clinicopathological characteristics. Differences in clinical outcome were estimated using Cox proportional hazard analysis and visualized using Kaplan-Meier curves., Results: The patient cohort had a 3-year OS of 58%, with a median follow-up of 53 months. None of the T-cell markers showed a correlation with OS, DFS or LRC. A low N stage was correlated to a better prognosis (OS: HR 0.39, p = 0.0028, DFS: HR 0.34, p =  < 0.001, LRC: HR 0.24, p = 0.008). High TIL counts were more often observed in PD-L1-positive tumors (p < 0.05)., Conclusion: This study showed an objective, digital pathology-aided method to assess TILs in the tumor epithelium. However, it did not provide evidence for a prognostic role of the presence of CD3 + , CD4 + , CD8 + , FoxP3 + , and PD1 + TILs in the tumor epithelium of advanced stage, HPV-negative HNSCC patients treated with primary chemoradiotherapy.

Background: To investigate if depth of invasion (DOI) can predict occult nodal disease in patients with cT1-2N0 (7th TNM) oral squamous cell carcinoma (OSCC) staged by sentinel lymph node biopsy (SLNB)., Methods: In 199 OSCC patients, DOI measurements and SLNB were performed., Results: Metastases were found in 64 of 199 patients (32%). Of these 64 patients, the mean DOI was 6.6 mm compared to 4.7 mm in patients without metastases (P = .003). The ROC-curve showed an area under the curve of 0.65 with a most optimal cutoff point of 3.4 mm DOI (sensitivity 83% and specificity 47%). Regional metastases were found in 15% of patients with DOI ≤ 3.4 mm., Conclusion: DOI seems to be a poor predictor for regional metastasis in patients with cT1-2N0 OSCC. Therefore, staging of the neck using SLNB in patients with early stage oral cancer should also be performed in tumors with limited DOI and probably in T3 (8th TNM) OSCC ≤4 cm diameter., (© 2019 The Authors. Head & Neck published by Wiley Periodicals, Inc.)

With mass spectrometry imaging (MSI) on tissue microarrays (TMAs) a large number of biomolecules can be studied for many patients at the same time, making it an attractive tool for biomarker discovery. Here we investigate whether lymph node metastasis can be predicted from MALDI-MSI data. Measurements are performed on TMAs and then filtered based on spectral intensity and the percentage of tumor cells, after which the resulting data for 122 patients is further preprocessed. We assume differences between patients with and without metastasis are expressed in a limited number of features. Two univariate feature selection methods are applied to reduce the dimensionality of the MALDI-MSI data. The selected features are then used in combination with three classifiers. The best classification scores are obtained with a decision tree classifier, which classifies about 72% of patients correctly. Almost all the predictive power comes from a single peak (m/z 718.4). The sensitivity of our classification approach, which can be generically used to search for biomarkers, is investigated using artificially modified data., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: In oral squamous cell carcinoma (OSCC) the differentiation grade of the tumor is determined on the biopsy and the resection specimen. The relation between tumor grade, nodal metastasis and survival is debatable. The aims of this study were to determine the correlation between differentiation grade of the biopsy and the resection specimen. Furthermore, we wanted to correlate tumor differentiation grade with nodal stage and survival., Patients and Methods: One-hundred and forty-five patients with OSCC staged as T1-2, N0 of the tongue, floor of mouth or cheek with primary resection of the tumor were examined. Biopsy and resection specimen were histologically re-assessed with regard to differentiation grade, as well as infiltrative, peri-neural and vascular invasive growth., Results: This study showed a poor correlation between differentiation grade in the incisional biopsy and the resection specimen of the same tumor. No significant relation between differentiation grade of the resection specimen and nodal involvement, as well as overall and disease-specific survival was found., Conclusion: In early OSCC the differentiation grade determined by biopsy is of little predictive value for the grading of the resection specimen. Poor differentiation grade could not be related to the presence of nodal metastasis or survival and seems not to have any prognostic value concerning outcome. Treatment planning must be related to these findings., (Copyright © 2018 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Background: Lymph node metastasis (LNM) is a major determinant of prognosis and treatment planning of oral squamous cell carcinoma (OSCC). Cysteine cathepsins constitute a family of proteolytic enzymes with known role in the degradation of the extracellular matrix. Involvement in pathological processes, such as inflammation and cancer progression, has been proved. The aim of the study was to discover the clinicopathological and prognostic implications of cathepsin K (CTSK) expression in oral squamous cell carcinoma., Methods: Eighty-three patients with primary OSCC, treated surgically between 1996 and 2000, were included. Gene expression data were acquired from a previously reported study. Human papilloma virus (HPV) status was previously determined by an algorithm for HPV-16. CTSK Protein expression was semi-quantitatively determined by immunohistochemistry in tumor and stromal cells. Expression data were correlated with various clinicopathological variables., Results: Elevated gene and protein expression of CTSK were strongly associated to LNM and perineural invasion (p < 0.01). Logistic regression analysis highlighted increased CTSK protein expression in tumor cells as the most significant independent factor of lymphatic metastasis (OR = 7.65, CI:2.31-23.31, p = 0.001). Survival analysis demonstrated CTSK protein expression in both stromal and tumor cells as significant indicators of poor 5-year disease specific survival (HR = 2.40, CI:1.05-5.50, p = 0.038 for stromal cells; HR = 2.79, CI:1.02-7.64, p = 0.045 for tumor cells)., Conclusion: Upregulation of CTSK seems to be associated with high incidence of lymphatic spread and poor survival in OSCC. CTSK could therefore serve as a predictive biomarker for OSCC.

Aims: To assess differences in methylation profiles, and thus pathogenesis, between Epstein-Barr virus (EBV)-positive and negative nasopharyngeal carcinomas (NPCs). Also, promoter hypermethylation is a common phenomenon in early carcinogenesis to inactivate tumour suppressor genes. Since epigenetic changes are reversible, the therapeutic application of methylation inhibitors could provide treatment options., Methods: We evaluated promoter hypermethylation profiles of 22 common tumour suppressor genes in 108 NPCs using methylation-specific multiplex ligation-dependent probe amplification. Correlation between methylation, clinicopathological features (including EBV) and survival was examined. Cluster analysis was also performed., Results: Hypermethylation of RASSF1A and ESR1 was significantly more frequent in EBV-positive NPC, while hypermethylation of DAPK1 was more frequent in EBV-negative NPC. In logistic regression, age, with EBV-positive NPC occurring at earlier age, and RASSF1, with RASSF1 hypermethylation being more frequent in EBV-positive NPC, remained significant. In EBV-positive NPC, hypermethylation of RASSF1A predicted worse overall survival (OS) (HR 3.058,95% CI 1.027 to 9.107). In EBV-negative NPC, hypermethylated adenomatous polyposis coli (APC) was a predictor of poor disease-free survival (DFS) (HR 6.868, 95% CI 2.142 to 22.022)., Conclusion: There are important epigenetic differences between EBV-negative and EBV-positive NPCs, with EBV-negative NPC having a more similar hypermethylation profile to other head and neck squamous cell carcinomas than EBV-positive NPC. Hypermethylation of RASSF1A might contribute to worse OS in EBV-positive NPC, and may be an important event in the pathogenesis of EBV-infected NPC. Hypermethylation of APC might contribute to worse DFS in EBV-negative NPC., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Early oral cancer is preferably treated by surgery. Its complete removal is essential for locoregional control and disease-free survival. Inadequate resection margins require adjuvant therapy such as re-resection or (chemo)radiation, that causes extra morbidity and oral discomfort. Intraoral ultrasonography (US) is reported to be of value in determining tumor thickness. Intraoperative visualization of the tumor may facilitate the resection and ensure adequate surgical margins. Furthermore, accurate prediction of tumor thickness could help determine the treatment strategy of the clinically node-negative neck, as thickness and depth of invasion are predictors of cervical metastasis as well as prognosticators of survival. The 8th edition of the American Joint Committee on Cancer staging system for oral squamous cell carcinoma has included depth of invasion as parameter for cT-stage. The aim of this review is to analyze the accuracy of intraoral US in determining tumor thickness in oral cancer. A systematic search was conducted, and the quality of the included papers was assessed using the QUADAS-2 tool for diagnostic accuracy studies. Subsequently, a meta-analysis was performed on the available individual participant data of 240 patients. Most of the twelve included studies focused on T1-2 tongue cancer (n = 129). Meta-analysis showed a high correlation in tumor thickness within this subgroup as measured by intraoral US and histopathology (r = 0.82, p < .001), with minor overestimation of 0.5 mm on US. It is concluded that intraoral US is very accurate in determining tumor thickness in early oral tongue cancer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Liquid biopsy is a minimally invasive detection method for molecular biomarkers in body fluids which may serve as a novel tool in management of head and neck cancer. The purpose of this systematic review is to outline the current status of liquid biopsy in head and neck squamous cell carcinoma (HNSCC) patients by systematically identifying and qualifying all published studies on the diagnostic or prognostic value of cell-free nucleic acids detection for posttreatment disease monitoring and/or disease outcome. A search was performed in PubMed, EMBASE, and Cochrane Library. Thirty articles met the inclusion criteria for further analysis. Study and patient characteristics, molecular analysis method and treatment or prognostic outcomes were extracted. Seventeen studies investigated circulating miRNAs in blood. Of these studies, 16 found statistically significant results for a total of 24 different candidate miRNAs for prognostication or treatment monitoring. The remaining studies investigated circulating tumor DNA by targeting somatic mutations, allelic imbalances, hypermethylation, or HPV-DNA. Of these studies, 2 found a statistically significant association between nucleic acid levels (tumor DNA targeted by allelic imbalances and HPV-DNA) in blood and/or saliva and prognostic outcome. One study found significantly different pre- and posttreatment levels of mitochondrial DNA in serum. Despite large differences among these studies in both design and results, individual results are promising and provide ground for more large-scale studies with standardized serial assessment of patient samples in the future., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Intraoperative analysis of lymph nodes during a sentinel lymph node biopsy (SLNB) procedure could result in one-step surgery for early oral squamous cell carcinoma (OSCC) with an occult nodal metastasis. One-step nucleic acid amplification rapidly detects cytokeratin 19 (CK19) RNA with high accuracy. Sensitivity and specificity of CK19 expression in OSCC was evaluated., Methods: Immunohistochemical CK19 expression was done in 207 patients with OSCC with 65 cases of paired nodal metastases., Results: CK19 was expressed in 65% of all OSCC and even less in early OSCC (56%), with poor correlation between primary tumor and (occult) nodal metastasis. Furthermore, ectopic glandular tissue in close proximity of lymph nodes showed strong CK19 expression., Conclusion: CK19 lacks both sensitivity and specificity as a biomarker for nodal metastasis in OSCC, which questions the suitability for CK19-based one-step nucleic acid amplification in SLNB procedures. Therefore, future studies should focus on other biomarkers, because the concept of fast intraoperative diagnostics during SLNB remains attractive., (© 2017 Wiley Periodicals, Inc.)

The Nucleosome Remodeling and Deacetylase (NURD) complex is a key regulator of cell differentiation that has also been implicated in tumorigenesis. Loss of the NURD subunit Deleted in Oral Cancer 1 (DOC1) is associated with human oral squamous cell carcinomas (OSCCs). Here, we show that restoration of DOC1 expression in OSCC cells leads to a reversal of epithelial-mesenchymal transition (EMT). This is caused by the DOC1-dependent targeting of NURD to repress key transcriptional regulators of EMT. NURD recruitment drives extensive epigenetic reprogramming, including eviction of the SWI/SNF remodeler, formation of inaccessible chromatin, H3K27 deacetylation, and binding of PRC2 and KDM1A, followed by H3K27 methylation and H3K4 demethylation. Strikingly, depletion of SWI/SNF mimics the effects of DOC1 re-expression. Our results suggest that SWI/SNF and NURD function antagonistically to control chromatin state and transcription. We propose that disturbance of this dynamic equilibrium may lead to defects in gene expression that promote oncogenesis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Accurate nodal staging is pivotal for treatment planning in early (stage I-II) oral cancer. Unfortunately, current imaging modalities lack sensitivity to detect occult nodal metastases. Chromosomal region 11q13, including genes CCND1, Fas-associated death domain (FADD), and CTTN, is often amplified in oral cancer with nodal metastases. However, evidence in predicting occult nodal metastases is limited., Methods: In 158 patients with early tongue and floor of mouth (FOM) squamous cell carcinomas, both CCND1 amplification and cyclin D1, FADD, and cortactin protein expression were correlated with occult nodal metastases., Results: CCND1 amplification and cyclin D1 expression correlated with occult nodal metastases. Cyclin D1 expression was validated in an independent multicenter cohort, confirming the correlation with occult nodal metastases in early FOM cancers., Conclusion: Cyclin D1 is a predictive biomarker for occult nodal metastases in early FOM cancers. Prospective research on biopsy material should confirm these results before implementing its use in routine clinical practice. © 2016 Wiley Periodicals, Inc. Head Neck 39: 326-333, 2017., (© 2016 Wiley Periodicals, Inc.)

The prognosis of head and neck squamous cell carcinoma (HNSCC) is largely based on disease stage. Despite improvements in treatment, recurrence rates are still considered high. Currently, disease progression or regression after curative treatment is monitored by clinical evaluation combined with flexible endoscopy and/or imaging. However, specificity of imaging is low due to the posttreatment effects. Detection of circulating tumor DNA (ctDNA) from blood samples of HNSCC patients is a minimally invasive technique that could lead to an earlier detection of recurrence. In addition, digital droplet PCR (ddPCR) could be used to sensitively detect these mutational targets. Future study on ctDNA using ddPCR in blood samples of HNSCC patients is recommended during the follow-up stage to detect recurrences in a timely manner., (© 2016 S. Karger AG, Basel.)

Head and neck squamous cell carcinomas (HNSCC) form a large heterogeneous group of tumors and have a relatively poor outcome in advanced cases. Revealing the underlying genetic mutations in HNSCC facilitates the development of diagnostic biomarkers, which might lead to improved diagnosis and post treatment surveillance. We retrospectively analyzed mutational hotspots using targeted next-generation sequencing (NGS) of 239 HNSCC tumor samples in order to examine the mutational profile of HNSCC. Furthermore, we assessed prevalence, co-occurrence, and synonymy of gene mutations in (matched) tumor samples. TP53 was found mutated the most frequent with mutation rates of up to 83% in all tumors, compared to mutation rates of between 0 and 21% of CDKN2A, PIK3CA, HRAS, CDK4, FBXW7 and RB1. Mutational co-occurrence predominantly existed between TP53 and PIK3CA, TP53 and CDKN2A, and HRAS and PIK3CA. Mutational synonymy between primary tumor and associated metastasis and recurrence was present in respectively 88% and 89%. TP53 mutations were concordantly mutated in 95% of metastases and in 91% of recurrences. This indicates TP53 mutations to be highly prevalent and concordant in primary tumors and associated locoregional metastases and recurrences. In turn, this provides ground for further investigating the use of TP53 mutations as diagnostic biomarkers in HNSCC patients., Competing Interests: The authors declare no conflicts of interest.

Purpose: FGFR1 is a promising therapeutic target in multiple types of solid tumors, including head and neck squamous cell carcinoma (HNSCC). FGFR inhibitors have shown great therapeutic value in preclinical models. However, resistance remains a major setback. In this study, we have investigated the prognostic value of FGFR1 expression in HNSCC, the therapeutic relevance of targeting FGFR with AZD4547, and potential resistant mechanisms., Experimental Design: IHC and FISH were applied on tissue microarrays to investigate FGFR1 protein expression and FGFR1 gene copy numbers in 452 HNSCCs. The sensitivity of HNSCC cell lines to AZD4547, either as single or combination treatment with the EGFR inhibitor gefitinib, was assessed using long-term colony formation assays, short-term viability assays, and biochemical analysis., Results: FGFR1 protein overexpression occurred in 82% (36/44) of human papillomavirus (HPV)-positive HNSCC and 75% (294/392) of HPV-negative HNSCC and relates with poor overall survival and disease-free survival in HPV-negative HNSCC [HR, 3.07; 95% confidence interval (CI), 1.74-6.90; P = 0.001 and HR, 1.53; 95% CI, 1.04-2.39; P = 0.033]. Moreover, the FGFR1 gene was amplified in 3% (3/110) of HPV-negative HNSCC. Treatment of the high FGFR1-expressing cell line CCL30 with AZD4547 reduced cell proliferation and FGFR signaling. Two FGFR-amplified cell lines, SCC147 and BICR16, were resistant to AZD4547 treatment due to EGFR signaling. Combined AZD4547 and gefitinib treatment synergistically inhibited the proliferation of resistant cell lines., Conclusions: Here, we identify high FGFR1 expression as a candidate prognostic biomarker in HPV-negative HNSCC. Furthermore, we provide a rationale for treating FGFR1-expressing HNSCC with the FGFR inhibitor AZD4547 and for combining AZD4547 and gefitinib in FGFR inhibitor-resistant HNSCC patients. Clin Cancer Res; 22(15); 3884-93. ©2016 AACR., (©2016 American Association for Cancer Research.)

Introduction: Fibroblast growth factor receptor family member proteins (FGFR1-4) have been identified as promising novel therapeutic targets and prognostic markers in a wide spectrum of solid tumors. The present study investigates the expression and prognostic value of four FGFR family member proteins in a large multicenter oral cavity squamous cell carcinoma (OCSCC) and oropharyngeal squamous cell carcinoma (OPSCC) cohort., Methods: Protein expression of FGFR1-4 was determined by immunohistochemistry on tissue microarrays containing 951 formalin-fixed paraffin embedded OCSCC and OPSCC tissues from the University Medical Center Utrecht and University Medical Center Groningen. Protein expression was correlated to overall survival using Cox regression models, and bootstrapping was performed as internal validation., Results: FGFR proteins were highly expressed in 39-64 % of OCSCC and 63-79 % of OPSCC. Seventy-three percent (299/412) of OCSCC and 85 % (305/357) of OPSCC highly co-expressed two or more FGFR family member proteins. FGFR1 protein was more frequently highly expressed in human papillomavirus (HPV)-negative OPSCC than HPV-positive OPSCC (82 vs. 65 %; p = 0.008). Furthermore, protein expression of FGFR family members was not related to overall survival in OCSCC or OPSCC (p > 0.05)., Conclusion: FGFR family members are frequently highly expressed in OCSCC and OPSCC. These FGFR family member proteins are therefore potential targets for novel therapies that are urgently required to improve survival of OCSCC and OPSCC patients.

Objectives: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs) have an improved prognosis compared to HPV-negative OPSCCs. Several theories have been proposed to explain this relatively good prognosis. One hypothesis is a difference in immune response. In this study, we compared tumor-infiltrating CD3+, CD4+, CD8+ T-cells, and granzyme inhibitors (SERPINB1, SERPINB4, and SERPINB9) between HPV-positive and HPV-negative tumors and the relation with survival., Methods: Protein expression of tumor-infiltrating lymphocytes (TILs) (CD3, CD4, and CD8) and granzyme inhibitors was analyzed in 262 OPSCCs by immunohistochemistry (IHC). Most patients (67%) received primary radiotherapy with or without chemotherapy. Cox regression analysis was carried out to compare overall survival (OS) of patients with low and high TIL infiltration and expression of granzyme inhibitors., Results: HPV-positive OPSCCs were significantly more heavily infiltrated by TILs (p < 0.001) compared to HPV-negative OPSCCs. A high level of CD3+ TILs was correlated with a favorable outcome in the total cohort and in HPV-positive OPSCCs, while it reached no significance in HPV-negative OPSCCs. There was expression of all three granzyme inhibitors in OPSCCs. No differences in expression were found between HPV-positive and HPV-negative OPSCCs. Within the group of HPV-positive tumors, a high expression of SERPINB1 was associated with a significantly worse overall survival., Conclusion: HPV-positive OPSCCs with a low count of CD3+ TILs or high expression of SERPINB1 have a worse OS, comparable with HPV-negative OPSCCs. This suggests that the immune system plays an important role in the carcinogenesis of the virally induced oropharynx tumors.

Background: Identification of prognostic patient characteristics in head and neck squamous cell carcinoma (HNSCC) is of great importance. Human papillomavirus (HPV)-positive HNSCCs have favorable response to (chemo)radiotherapy. Apparent diffusion coefficient, derived from diffusion-weighted MRI, has also shown to predict treatment response. The purpose of this study was to evaluate the correlation between HPV status and apparent diffusion coefficient., Methods: Seventy-three patients with histologically proven HNSCC were retrospectively analyzed. Mean pretreatment apparent diffusion coefficient was calculated by delineation of total tumor volume on diffusion-weighted MRI. HPV status was analyzed and correlated to apparent diffusion coefficient., Results: Six HNSCCs were HPV-positive. HPV-positive HNSCC showed significantly lower apparent diffusion coefficient compared to HPV-negative. This correlation was independent of other patient characteristics., Conclusion: In HNSCC, positive HPV status correlates with low mean apparent diffusion coefficient. The favorable prognostic value of low pretreatment apparent diffusion coefficient might be partially attributed to patients with a positive HPV status. © 2015 Wiley Periodicals, Inc. Head Neck 38: E613-E618, 2016., (© 2015 Wiley Periodicals, Inc.)

Although the receptor tyrosine kinase (RTK) MET is widely expressed in head and neck squamous cell carcinoma (HNSCC), its prognostic value remains unclear. This might be due to the use of a variety of antibodies and scoring systems. Here, the reliability of five commercial C-terminal MET antibodies (D1C2, CVD13, SP44, C-12 and C-28) was evaluated before examining the prognostic value of MET immunoreactivity in HNSCC. Using cancer cell lines, it was shown that D1C2 and CVD13 specifically detect MET under reducing, native and formalin-fixed paraffin-embedded (FFPE) conditions. Immunohistochemical staining of routinely FFPE oral SCC with D1C2 and CVD13 demonstrated that D1C2 is most sensitive in the detection of membranous MET. Examination of membranous D1C2 immunoreactivity with 179 FFPE oral and oropharyngeal SCC - represented in a tissue microarray - illustrated that staining is either uniform (negative or positive) across tumors or differs between a tumor's center and periphery. Ultimately, statistical analysis revealed that D1C2 uniform staining is significantly associated with poor 5-year overall and disease free survival of patients lacking vasoinvasive growth (HR = 3.019, p < 0.001; HR = 2.559, p < 0.001). These findings might contribute to reliable stratification of patients eligible for treatment with biologicals directed against MET.

Fibroblast growth factor receptor 3 (FGFR3) is a member of the fibroblast growth factor receptor tyrosine kinase family. It has been identified as a promising therapeutic target in multiple types of cancer. We have investigated FGFR3 protein expression and FGFR3 gene copy-numbers in a single well-documented cohort of oral and oropharyngeal squamous cell carcinoma. Tissue microarray sets containing 452 formalin-fixed paraffin-embedded tissues were immunohistochemically stained with an anti-FGFR3 antibody and hybridized with a FGFR3 fluorescence in situ hybridization probe. FGFR3 protein expression was correlated with clinicopathological and survival data, which were retrieved from electronic medical records. FGFR3 mRNA data of 522 head and neck squamous cell carcinoma (HNSCC) were retrieved from The Cancer Genome Atlas (TCGA). Fibroblast growth factor receptor 3 (FGFR3) protein was overexpressed in 48% (89/185) of oral and 59% (124/211) of oropharyngeal squamous cell carcinoma. Overexpression of FGFR3 protein was not related to overall survival or disease-free survival in oral (HR[hazard ratio]: 0.94; 95% CI: 0.64-1.39; P = 0.77, HR: 0.94; 95% CI: 0.65-1.36; P = 0.75) and oropharyngeal squamous cell carcinoma (HR: 1.21; 95% CI: 0.81-1.80; P = 0.36, HR: 0.42; 95% CI: 0.79-1.77; P = 0.42). FGFR3 mRNA was upregulated in 3% (18/522) of HNSCC from the TCGA. The FGFR3 gene was gained in 0.6% (1/179) of oral squamous cell carcinoma but no amplification was found in oral and oropharyngeal squamous cell carcinoma. In conclusion, FGFR3 protein is frequently overexpressed in oral and oropharyngeal squamous cell carcinoma. Therefore, it may serve as a potential therapeutic target for FGFR3-directed therapies in oral and oropharyngeal squamous cell carcinoma., (© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Since head and neck cancer is characterized by poor survival rates, there is a demand for novel therapeutic targets and prognostic biomarkers. An upcoming therapeutic target is the fibroblast growth factor receptor (FGFR) family. However, their prognostic role in head and neck cancer remains unclear., Objective: To systematically review current evidence on the prognostic value of FGFR family members in head and neck squamous cell carcinoma (HNSCC)., Methods: A systematic search of PubMed, Embase, and the Cochrane Library was performed for publications up to 14 May 2014. Two reviewers screened all articles and included prognostic studies on the molecular biomarkers FGFR1-5 in any type of HNSCC. Relevant studies were assessed on risk of bias using the Quality in Prognostic Studies (QUIPS) tool. Data on FGFR aberrations and survival outcome were extracted from relevant studies. The prognostic value of FGFR aberrations was compared among studies., Results: The initial search yielded 1568 publications of which 12 fulfilled the inclusion criteria. Four studies reported FGFR1 gene amplification (9.3-17.4 %) and FGFR1 protein overexpression (11.8 %) in HNSCC. FGFR1 protein expression by cancer-associated fibroblasts correlated with poor survival outcome in one study (p < 0.01). Eight studies reported high rates of FGFR4 Gly388Arg polymorphisms (32.5-54.2 %) and FGFR4 protein overexpression (16-35 %), with varying correlations with survival. So far, no studies assessed the prognostic role of FGFR2, FGFR3, or FGFR5 in HNSCC., Limitations: Significant risk of bias has been identified among included studies. Therefore, cautious interpretation of the results is recommended., Conclusion: In conclusion, evidence was found for prognostic value of FGFR1 expression in cancer-associated fibroblasts in HNSCC. Prognostic evidence on the other FGFR family members in HNSCC is limited and conflicting. This emphasizes the need for future well-conducted prognostic studies.

Current conventional treatment modalities in head and neck squamous cell carcinoma (HNSCC) are nonselective and have shown to cause serious side effects. Unraveling the molecular profiles of head and neck cancer may enable promising clinical applications that pave the road for personalized cancer treatment. We examined copy number status in 36 common oncogenes and tumor suppressor genes in a cohort of 191 oropharyngeal squamous cell carcinomas (OPSCC) and 164 oral cavity squamous cell carcinomas (OSCC) using multiplex ligation probe amplification. Copy number status was correlated with human papillomavirus (HPV) status in OPSCC, with occult lymph node status in OSCC and with patient survival. The 11q13 region showed gain or amplifications in 59% of HPV-negative OPSCC, whereas this amplification was almost absent in HPV-positive OPSCC. Additionally, in clinically lymph node-negative OSCC (Stage I-II), gain of the 11q13 region was significantly correlated with occult lymph node metastases with a negative predictive value of 81%. Multivariate survival analysis revealed a significantly decreased disease-free survival in both HPV-negative and HPV-positive OPSCC with a gain of Wnt-induced secreted protein-1. Gain of CCND1 showed to be an independent predictor for worse survival in OSCC. These results show that copy number aberrations, mainly of the 11q13 region, may be important predictors and prognosticators which allow for stratifying patients for personalized treatment of HNSCC., (© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Objectives: Infiltration depth, perineural growth (PG), vascular invasive growth (VG), and infiltrative growth (IG) are associated with regional metastases in oral squamous cell carcinomas (OSCCs). Preoperative knowledge of these parameters could facilitate the treatment planning of the neck. The aim of this study was to evaluate if the biopsy specimen correlates with the resection specimen., Methods: In total, 149 patients with a pT1-2cN0 OSCC were included. Biopsy thickness and tumor thickness were analyzed. Occurrence of PG, VG, and IG was determined on biopsy and resection specimens and correlated with the N status and survival. Sensitivity, specificity, positive and negative predictive value, and diagnostic gain of the biopsy specimen were calculated., Results: N+ patients showed PG, VG, and IG significantly more often in the resection specimen compared with N- patients (P = .02, P = .001, and P = .001, respectively). Histologic parameters in the biopsy specimens did not correlate with N status or survival. The positive diagnostic gain for biopsy specimens with PG, VG, and IG was 57%, 40%, and 19%, respectively. The negative diagnostic gain was 2%, 0%, and 22%, respectively., Conclusions: Histologic parameters in biopsy specimens do not represent the resection specimen. Determination of histologic parameters in routinely taken biopsy specimens of OSCC is not helpful in deciding whether to treat the neck., (Copyright© by the American Society for Clinical Pathology.)

Background: Gene expression profiling revealed a strong signature predicting lymph node metastases in oral squamous cell carcinoma (OSCC). Four of the most predictive genes are secretory leukocyte protease inhibitor (SLPI), lipocalin-2 (LCN2), thrombospondin-2 (THBS2), and tumor-associated calcium signal transducer 2 (TACSTD2). This study correlates their protein expression with lymph node metastases, overall survival (OS), and disease-specific survival (DSS)., Methods: Two hundred twelve patients with OSCC were included for protein expression analysis by immunohistochemistry., Results: SLPI expression correlates with lymph node metastases in the whole cohort, not in a subgroup of cT1 to 2N0. SLPI expression correlates with OS (hazard ratio [HR] = 0.61) and DSS (HR = 0.47) in multivariate analysis. LCN2, THBS2, and TACSTD2 show no correlation with lymph node metastases, OS, or DSS., Conclusion: Although SLPI expression correlates with lymph node metastases, it has no additional value in determining lymph node metastases in early oral cancer. However, it is an independent predictor for both OS and DSS and therefore a relevant prognostic biomarker in OSCC., (© 2014 Wiley Periodicals, Inc.)

Awareness increases that the tumor biology influences treatment outcome and prognosis in cancer. Tumor hypoxia is thought to decrease sensitivity to radiotherapy and some forms of chemotherapy. Presence of hypoxia may be assessed by investigating expression of endogenous markers of hypoxia (EMH) using immunohistochemistry (IHC). In this systematic review we investigated the effect of EMH expression on local control and survival according to treatment modality in head and neck cancer (head and neck squamous cell carcinoma [HNSCC]). A search was performed in MEDLINE and EMBASE. Studies were eligible for inclusion that described EMH expression in relation to outcome in HNSCC patients. Quality was assessed using the Quality in Prognosis Studies (QUIPS) tool. Hazard ratios for locoregional control and survival were extracted. Forty studies of adequate quality were included. HIF-1a, HIF-2a, CA-IX, GLUT-1, and OPN were identified as the best described EMHs. With exception of HIF-2a, all EMHs were significantly related to adverse outcome in multiple studies, especially in studies where patients underwent single-modality treatment. Positive expression was often correlated with adverse clinical characteristics, including disease stage and differentiation grade. In summary, EMH expression was common in HNSCC patients and negatively influenced their prognosis. Future studies should investigate the effect of hypoxia-modified treatment schedules in patients with high In summary, EMH expression. These may include ARCON, treatment with nimorazole, or novel targeted therapies directed at hypoxic tissue. Also, the feasibility of surgical removal of the hypoxic tumor volume prior to radiotherapy should be investigated., (© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Despite improvements in both diagnostic and therapeutic strategies, the prognosis of oral squamous cell carcinoma (OSCC) has not changed significantly over the last decades. Prognosis of OSCC particularly depends on the presence of nodal metastasis in the neck. Therefore, proper determination of the nodal status is pivotal for appropriate treatment. Unfortunately, current available imaging techniques (magnetic resonance imaging or ultrasound even with fine needle aspiration of suspected lymph nodes (LNs)) fail to detect occult nodal disease accurately. Clinicians in head and neck oncology urgently need new diagnostic tools to reliably determine the presence of nodal metastasis of the neck. Gain of the chromosomal region 11q13 is one of the most prominent genetic alterations in head and neck cancer and is associated with poor prognosis and metastasis. The aim of this systematic review and meta-analysis was to determine the diagnostic value of either 11q13 amplification or amplification/protein overexpression of individual genes located on 11q13 to detect nodal metastasis in OSCC. A search was conducted in Pubmed, EMBASE, and Cochrane, and 947 unique citations were retrieved. Two researchers independently screened all articles and only 18 were found to meet our inclusion criteria and were considered of sufficient quality for meta-analysis. Pooled results of those show that both amplification of CCND1 and protein overexpression of cyclin D1 significantly correlate with lymph node metastasis (LNM) in OSCC. In addition, amplification of CCND1 shows a negative predictive value of 80 % in the detection of LNM in early stage OSCCs which are clinically lymph node negative although this evidence is sparse and should be validated in a larger homogeneous cohort of T1-2 OSCC.

Objective: The aim of this study was to investigate whether fibroblast growth factor receptor 4 (FGFR4) could serve as a potential therapeutic target, prognostic biomarker or biomarker predicting radiotherapy sensitivity in oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC)., Methods: FGFR4 immunohistochemistry and FGFR4/CEN5q FISH were performed on tissue microarrays from 212 OSCC and 238 OPSCC patients. FGFR4 genotypes were determined by PCR and DNA sequencing in 76 random OPSCC samples. The response to radiotherapy was evaluated 3 months after the last radiotherapy treatment session by a head and neck radiation oncologist and/or surgeon during clinic visits. The results were correlated to overall survival and response to radiotherapy., Results: The FGFR4 protein was overexpressed in 64% (153/238) of OPSCCs and 41% (87/212) of OSCCs. The FGFR4 gene was amplified in 0.47% (1/212) of OSCCs and 0.42% (1/238) of OPSCCs, and the FGFR4 Gly388Arg polymorphism was detected in 62% (47/76) of OPSCCs. FGFR4 protein expression, FGFR4 gene copy numbers and FGFR4 genotypes were not related to overall survival or response to radiotherapy in OSCC or OPSCC., Conclusion: FGFR4 is frequently overexpressed in OSCC and OPSCC in the absence of gene amplification, and may serve as a potential predictive marker for FGFR4-directed targeted therapy in OSCC and OPSCC., (© 2015 S. Karger AG, Basel.)

Objective: Oral squamous-cell carcinoma (OSCC) still has a poor prognosis. Lymph node metastasis (LNM) is a major determinant of treatment decisions and prognosis. Serine protease inhibitor Kazal-type 5 (SPINK5) is the inhibitor of kallikrein 5 (KLK5) and KLK7. SPINK5, KLK5 and KLK7 are three of the genes of a recently validated LNM-predicting gene expression profile in OSCC. This study evaluates their clinicopathological role and value as biomarkers in OSCC., Methods: Eighty-three patients with primary OSCC, treated surgically between 1996 and 2000, were included. Gene expression data were acquired from a previously reported study. Human papillomavirus (HPV) status was determined by an algorithm for HPV-16. Protein expression for KLK5, KLK7 and SPINK5 was semi-quantitatively determined in all 83 tumours by immunohistochemistry. All expression data were correlated with clinicopathological parameters., Results: Concurrent loss of KLK5 and KLK7 correlates with worse disease-specific and overall survival (DSS and OS). Multivariate analysis proved that co-expression is an independent prognostic factor for DSS (p = 0.029) and OS (p = 0.001)., Conclusion: This report demonstrates that concurrent loss of KLK5 and KLK7 associates with a poor clinical outcome in OSCC and could therefore serve as prognostic marker in this disease.

Background and Objective: Current resection modalities for oral squamous cell carcinomas (OSCC) vary from cold steel over CO2 laser to monopolar electro-surgery (MO). We compared thulium laser (TL) as a new modality with MO with regards to pathological assessment., Study Design/materials and Methods: Forty-two patients who were treated for OSCC by either TL or MO were included. All resected specimens were assessed with special attention to margin interpretation and thermal damage., Results: Depth of thermal damage ranged from 1.0 to 3.5 mm in the TL group compared to 1.0-4.0 mm in the MO group without interfering with the pathological assessment. The percentage of positive margin resections was three times higher in the MO group compared with the TL group., Conclusions: This study shows resections done by TL do not impair pathological assessment when compared to MO resections., (© 2014 Wiley Periodicals, Inc.)

Oropharyngeal squamous cell carcinoma (OPSCC) is associated with human papillomavirus (HPV) in a proportion of tumors. HPV-positive OPSCC is considered a distinct molecular entity with a prognostic advantage compared to HPV-negative cases. Silencing of cancer-related genes by DNA promoter hypermethylation may play an important role in the development of OPSCC. Hence, we examined promoter methylation status in 24 common tumor suppressor genes in a group of 200 OPSCCs to determine differentially methylated genes in HPV-positive versus HPV-negative primary OPSCC. Methylation status was correlated with HPV status, clinical features, and patient survival using multivariate methods. Additionally, methylation status of 16 cervical squamous cell carcinomas (SCC) was compared with HPV-positive OPSCC. Using methylation-specific probe amplification, HPV-positive OPSCC showed a significantly higher cumulative methylation index (CMI) compared to HPV-negative OPSCC (P=0.008). For the genes CDH13, DAPK1, and RARB, both HPV-positive and HPV-negative OPSCC showed promoter hypermethylation in at least 20% of the tumors. HPV status was found to be an independent predictor of promoter hypermethylation of CADM1 (P < 0.001), CHFR (P = 0.027), and TIMP3 (P < 0.001). CADM1 and CHFR showed similar methylation patterns in OPSCC and cervical SCC, but TIMP3 showed no methylation in cervical SCC in contrast to OPSCC. Methylation status of neither individual gene nor CMI was associated with survival. These results suggest that HPV-positive tumors are to a greater extent driven by promotor hypermethylation in these tumor suppressor genes. Especially CADM1 and TIMP3 are significantly more frequently hypermethylated in HPV-positive OPSCC and CHFR in HPV-negative tumors., (© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Silencing of tumor suppressor genes (TSGs) by DNA promoter hypermethylation is an early event in carcinogenesis and a potential target for personalized cancer treatment. In head and neck cancer, little is known about the role of promoter hypermethylation in survival. Using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) we investigated the role of promoter hypermethylation of 24 well-described genes (some of which are classic TSGs), which are frequently methylated in different cancer types, in 166 HPV-negative early oral squamous cell carcinomas (OSCC), and 51 HPV-negative early oropharyngeal squamous cell carcinomas (OPSCC) in relation to clinicopathological features and survival. Early OSCC showed frequent promoter hypermethylation in RARB (31% of cases), CHFR (20%), CDH13 (13%), DAPK1 (12%), and APC (10%). More hypermethylation (≥ 2 genes) independently correlated with improved disease specific survival (hazard ratio 0.17, P = 0.014) in early OSCC and could therefore be used as prognostic biomarker. Early OPSCCs showed more hypermethylation of CDH13 (58%), TP73 (14%), and total hypermethylated genes. Hypermethylation of two or more genes has a significantly different effect on survival in OPSCC compared with OSCC, with a trend toward worse instead of better survival. This could have a biological explanation, which deserves further investigation and could possibly lead to more stratified treatment in the future.

Objectives: The treatment strategy of early stage oral squamous cell carcinoma's (OSCC) resected with close or involved margins is a returning point of discussion. In this study we reviewed the consequences of re-resection (RR), postoperative radiotherapy (PORT) or watchful waiting (WW)., Patients and Methods: Two-hundred patients with a primary resected Stage 1-2 OSCC of the tongue, floor of the mouth and cheek were included and retrospectively analysed. Local recurrence ratio was related to margin status, unfavourable histological parameters (spidery infiltrative, peri-neural and vascular-invasive growth) and postoperative treatment modality. 3-year overall survival (OS) and disease-specific survival (DSS) was calculated in relation to margin status., Results: Twenty-two of 200 (11%) patients had pathological positive margins (PM), 126 (63%) close margins (CM), and 52 (26%) free margins (FM). OS and DSS were not significantly different between these groups. Nine of 200 (4.5%) patients developed local recurrent disease. Two (9.1%) had a PM, five (4.0%) a CM and two (3.8%) a FM. Of the nine recurrences, five patients had undergone PORT, one a RR, and three follow-up. Watchful waiting for CM ⩾3 mm with ⩽2 unfavourable histological parameters showed, besides margin status no significant differences with the FM group., Conclusion: With this treatment strategy, the local recurrence rate was 4.5%. No evidence was found for local adjuvant treatment in case of close margins ⩾3 mm with ⩽2 unfavourable histological parameters. Current data do not support the use of one treatment modality above any other., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Oropharyngeal squamous cell carcinoma (OPSCC) is associated with human papillomavirus (HPV). HPV-positive OPSCC is considered a distinct molecular entity with a better prognosis than HPV-negative cases of OPSCC. However, the exact pathogenic mechanisms underlying the differences in clinical and molecular behavior between HPV-positive and HPV-negative OPSCC remain poorly understood. Epigenetic events play an important role in the development of cancer. Hypermethylation of DNA in promoter regions and global hypomethylation are 2 epigenetic changes that have been frequently observed in human cancers. It is suggested that heterogeneous epigenetic changes play a role in the clinical and biological differences between HPV-positive and HPV-negative tumors. Unraveling the differences in methylation profiles of HPV-associated OPSCC may provide for promising clinical applications and may pave the road for personalized cancer treatment. This systematic review aims to assess the current state of knowledge regarding differences in promoter hypermethylation and global methylation between HPV-positive and HPV-negative OPSCC.

Oncogenic fusion genes that involve kinases have proven to be effective targets for therapy in a wide range of cancers. Unfortunately, the diagnostic approaches required to identify these events are struggling to keep pace with the diverse array of genetic alterations that occur in cancer. Diagnostic screening in solid tumours is particularly challenging, as many fusion genes occur with a low frequency. To overcome these limitations, we developed a capture enrichment strategy to enable high-throughput transcript sequencing of the human kinome. This approach provides a global overview of kinase fusion events, irrespective of the identity of the fusion partner. To demonstrate the utility of this system, we profiled 100 non-small cell lung cancers and identified numerous genetic alterations impacting fibroblast growth factor receptor 3 (FGFR3) in lung squamous cell carcinoma and a novel ALK fusion partner in lung adenocarcinoma., (© 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)