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Levels of circulating tumor DNA (ctDNA) in liquid biopsies may serve as a sensitive biomarker for real-time, minimally-invasive tumor diagnostics and monitoring. However, detecting ctDNA is challenging, as much fewer than 5% of the cell-free DNA in the blood typically originates from the tumor. To detect lowly abundant ctDNA molecules based on somatic variants, extremely sensitive sequencing methods are required. Here, we describe a new technique, CyclomicsSeq, which is based on Oxford Nanopore sequencing of concatenated copies of a single DNA molecule. Consensus calling of the DNA copies increased the base-calling accuracy ~60×, enabling accurate detection of TP53 mutations at frequencies down to 0.02%. We demonstrate that a TP53-specific CyclomicsSeq assay can be successfully used to monitor tumor burden during treatment for head-and-neck cancer patients. CyclomicsSeq can be applied to any genomic locus and offers an accurate diagnostic liquid biopsy approach that can be implemented in clinical workflows.

Contains fulltext : 251419.pdf (Publisher’s version ) (Open Access) OBJECTIVE: Tumor hypoxia results in worse local control and patient survival. We performed a digital, single-cell-based analysis to compare two biomarkers for hypoxia (hypoxia-inducible factor 1-alpha [HIF-1alpha] and pimonidazole [PIMO]) and their effect on outcome in laryngeal cancer patients treated with accelerated radiotherapy with or without carbogen breathing and nicotinamide (AR versus ARCON). MATERIALS AND METHODS: Immunohistochemical staining was performed for HIF-1alpha and PIMO in consecutive sections of 44 laryngeal cancer patients randomized between AR and ARCON. HIF-1alpha expression and PIMO-binding were correlated using digital image analysis in QuPath. High-density areas for each biomarker were automatically annotated and staining overlap was analyzed. Kaplan-Meier survival analyses for local control, regional control and disease-free survival were performed to predict a response benefit of ARCON over AR alone for each biomarker. RESULTS: 106 Tissue fragments of 44 patients were analyzed. A weak, significant positive correlation was observed between HIF-1alpha and PIMO positivity on fragment level, but not on patient level. A moderate strength correlation (r = 0.705, p < 0.001) was observed between the number of high-density staining areas for both biomarkers. Staining overlap was poor. HIF-1alpha expression, PIMO-binding or a combination could not predict a response benefit of ARCON over AR. CONCLUSION: Digital image analysis to compare positive cell fractions and staining overlap between two hypoxia biomarkers using open-source software is feasible. Our results highlight that there are distinct differences between HIF-1alpha and PIMO as hypoxia biomarkers and therefore suggest co-existence of different forms of hypoxia within a single tumor.

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.

Background: Inevitably, the emergence of COVID-19 has impacted non-COVID care. Because timely diagnosis and treatment are essential, especially for patients with head and neck cancer (HNC) with fast-growing tumours in a functionally and aesthetically important area, we wished to quantify the impact of the COVID-19 pandemic on HNC care in the Netherlands. Material and Methods: This population-based study covered all, in total 8468, newly diagnosed primary HNC cases in the Netherlands in 2018, 2019 and 2020. We compared incidence, patient and tumour characteristics, primary treatment characteristics, and time-to-treatment in the first COVID-19 year 2020 with corresponding periods in 2018 and 2019 (i.e. pre-COVID). Results: The incidence of HNC was nearly 25% less during the first wave (n = 433) than in 2019 (n = 595) and 2018 (n = 598). In April and May 2020, the incidence of oral cavity and laryngeal carcinomas was significantly lower than in pre-COVID years. There were no shifts in tumour stage or alterations in initial treatment modalities. Regardless of the first treatment modality and specific period, the median number of days between first visit to a HNC centre and start of treatment was significantly shorter during the COVID-19 year (26–28 days) than pre-COVID (31–32 days, p < 0.001). Conclusion: The incidence of HNC during the Netherlands’ first COVID-19 wave was significantly lower than expected. The expected increase in incidence during the remainder of 2020 was not observed. Despite the overloaded healthcare system, the standard treatment for HNC patients could be delivered within a shorter time interval.

Additional file 1: Supplementary Table 1. Generalizability sample of pathology data extracted from pathology reports compared to initial cohort. A). Basic clinical characteristics of patients for each year of the sample used in this paper (n = 1502). These are the 3656 patients of which 44% of each year pathology data were extracted and CIS were removed. B). Basic clinical characteristics of the total dataset of patients for each year (n = 3380). These are the 3656 patients with reliable linkage between NKR-PALGA minus 276 CIS patients leaving 3380 patients in total. Supplementary Table 2. Histological characteristics, locoregional recurrences and death of patients treated with tumor-positive lymph nodes (n = 205). Supplementary Table 3. Histological characteristics, locoregional recurrences and death of T3/T4 N0/N+ patients treated with TLE or TLE with RT (n = 98). Supplementary Figure 1. Separate multivariable analysis of extracapsular spread, keratinization and PNI+/VI+/Growth pattern. Each separate histological characteristic was corrected for age, gender, sublocalisation and stage/treatment for locoregional recurrence and death of patients with positive lymph nodes (n = 205). Supplementary Figure 2. Univariable analysis of histological characteristics of the primary tumor for locoregional recurrence and death of T3/T4 N0/N+ patients treated with TLE or TLE with RT (n = 98). * p

Aims Programmed death-ligand 1 (PD-L1) immunostaining is used to predict which non-small-cell lung cancer (NSCLC) patients will respond best to treatment with programmed cell death protein 1/PD-L1 inhibitors. PD-L1 immunostaining is sometimes performed on alcohol-fixed cytological specimens instead of on formalin-fixed paraffin-embedded (FFPE) biopsies or resections. We studied whether ethanol prefixation of clots from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) results in diminished PD-L1 immunostaining as compared with formalin fixation. Methods and results FFPE cell blocks from EBUS-TBNA specimens of 54 NSCLC patients were identified. For each case, paired samples were available, consisting of clots directly immersed in formalin and clots prefixed in Fixcyt (50% ethanol). Serial sections were immunostained for PD-L1 by use of the standardised SP263 assay and the 22C3 antibody as a laboratory-developed test (LDT). PD-L1 positivity was determined with two cut-offs (1% and 50%). Concordance of PD-L1 positivity between the formalin-fixed (gold standard) and ethanol-prefixed material was assessed. When the 22C3 LDT was used, 30% and 36% of the ethanol-prefixed specimens showed false-negative results at the 1% and 50% cut-offs, respectively (kappa 0.64 and 0.68). When SP263 was used, 22% of the ethanol-prefixed specimens showed false-negative results at the 1% cut-off (kappa 0.67). At the 50% cut-off, concordance was higher (kappa 0.91), with 12% of the ethanol-prefixed specimens showing false-negative results. Conclusion Ethanol fixation of EBUS-TBNA specimens prior to formalin fixation can result in a considerable number of false-negative PD-L1 immunostaining results when a 1% cut-off is used and immunostaining is performed with SP263 or the 22C3 LDT. The same applies to use of the 50% cut-off when immunostaining is performed with the 22C3 LDT.

High-grade transformation (HGT) or dedifferentiation has been described in a variety of salivary gland carcinomas, including acinic cell carcinoma, secretory carcinoma, adenoid cystic carcinoma, epithelial-myoepithelial carcinoma, polymorphous adenocarcinoma, low-grade mucoepidermoid carcinoma, and hyalinizing clear cell carcinoma. High-grade (HG) transformed tumors are composed of a conventional low-grade component characterized by specific microscopic and immunohistochemical features for the given entity, intermingled with or juxtaposed to areas of HG morphology. This is usually either poorly differentiated adenocarcinoma, carcinoma not otherwise specified, or undifferentiated carcinoma, in which the original line of differentiation is lost. The HG component is composed of solid nests of anaplastic cells with large vesicular pleomorphic nuclei, prominent nucleoli, and abundant cytoplasm. Frequent mitoses and extensive necrosis may be present. The Ki-67 labeling index is consistently higher in the HG component. The molecular genetic mechanisms responsible for HGT of salivary gland carcinomas are largely unknown, though p53 inactivation and human epidermal growth factor receptor 2 overexpression and/or gene amplification have been demonstrated in the HG component in a few examples, the frequency varies for each histologic type. Salivary gland carcinomas with HGT are more aggressive than conventional carcinomas, with a higher local recurrence rate and a poorer prognosis. They have a high propensity for cervical lymph node metastasis suggesting a need for a wider resection and neck dissection. HGT of salivary gland carcinoma can occur either at initial presentation or less commonly at the time of recurrence, sometimes following postoperative radiotherapy. The potential for HGT in almost any type of salivary gland carcinoma warrants a thorough sampling of all salivary gland malignancies to prevent oversight of a HG component. info:eu-repo/semantics/publishedVersion

Bone and soft tissue tumors of the head and neck are relatively uncommon tumors that often represent a diagnostic challenge because of the wide range of entities that must be considered in the differential diagnosis. Over the past few years, classification of bone and soft tissue tumors has evolved primarily because of substantial contributions from molecular genetics, with the identification of new markers that are increasingly used to complement histopathologic findings in the routine diagnostic workup. This review focuses on the recently described mesenchymal tumors that preferentially involve the head and neck region, with a focus on the most relevant novel immunohistochemical and molecular findings, including gene fusions and mutations, that can help in the diagnosis and in the assessment of clinical behavior. info:eu-repo/semantics/publishedVersion

During the last 60 years numerous significant attempts have been made to achieve a widely acceptable terminology and histological grading for laryngeal squamous intraepithelial lesions. While dysplasia was included in the pathology of the uterine cervix already in 1953, the term dysplasia was accepted in laryngeal pathology first after the Toronto Centennial Conference on Laryngeal Cancer in 1974. In 1963 Kleinsasser proposed a three-tier classification, and in 1971 Kambic and Lenart proposed a four-tier classification. Since then, four editions of the World Health Organisation (WHO) classification have been proposed (1978, 1991, 2005 and 2017). Several terms such as squamous intraepithelial neoplasia (SIN) and laryngeal intraepithelial neoplasia (LIN) are now being abandoned and replaced by squamous intraepithelial lesions (SIL). The essential change between the 2005 and 2017 WHO classifications is the attempt to induce a simplification from a four- to a two-tier system. The current WHO classification (2017) thus recommends the use of a two-tier system with reasonably clear histopathological criteria for the two groups: low-grade and high-grade dysplasia. Problems with interobserver variability apart, subjectivities and uncertainties remain, but to a lesser degree. Ongoing and additional molecular studies may help to clarify underlying events that will increase our understanding and possibly can facilitate our attempts to obtain an even better classification. The classification needs to be easier for the general pathologist to perform and easier for the clinician to interpret. These two objectives are equally important to provide each patient the best personalised treatment available for squamous intraepithelial lesions.

Additional file 1: Supplementary Figure 1. Differences in PSMA expression between primary, recurrent and metastatic AdCC in 18 patients, in order of PSMA expression of the primary tumour.

Differences in cancer gene copy number alterations between Epstein-Barr virus-positive and Epstein-Barr virus-negative nasopharyngeal carcinoma Marc Lucas Ooft MD, PhD1 | Jolique van Ipenburg MD2 | Rob J. M. van de Loo1 | Rick de Jong1 | Cathy B. Moelans PhD1 | Remco de Bree MD, PhD3 | Martine J. de Herdt Ir4 | Senada Koljenović MD, PhD2 | R. Baatenburg de Jong MD, PhD5 | J. Hardillo MD, PhD5 | Stefan Martin Willems MD, PhD1 1Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands 2Department of Pathology, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands 3Department of Head and Neck Surgical Oncology, University Medical Center Utrecht, Utrecht, The Netherlands 4Department of Otorhinolaryngology - Head and Neck Surgery, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, The Netherlands 5Department of Otorhinolaryngology - Head and Neck Surgery, Erasmus Medical Center Cancer Institute, University Medical Center Rotterdam, Rotterdam, The Netherlands Correspondence Stefan Willems, Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Email: s.m.willems-4@umcutrecht.nl Head & Neck. 2018;40:1986-1998 DOI: 10.1002/hed.25195 In the above-mentioned article, author name “R. Baatenburg de Jong” should be updated to “Robert J. Baatenburg de Jong.” We apologize for the error.

Well-differentiated neuroendocrine carcinoma (also known as "carcinoid") of the larynx is an exceedingly rare tumor that has an epithelial origin. These tumors are malignant and have a low, but definite, risk of metastasis. Although it can be challenging, this tumor should be differentiated from moderately differentiated neuroendocrine carcinoma (also known as "atypical carcinoid"). The clinical and pathologic features of this tumor, as well as treatment and prognosis, are reviewed in detail. info:eu-repo/semantics/publishedVersion

Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)–derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs.Significance:This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813

OBJECTIVE: The objective of this study is to determine at a national level whether patients with metastatic non-small cell lung cancer (NSCLC) are adequately tested for EGFR mutations and ALK rearrangement, because targeted therapy is tailored to the results of molecular diagnostics. DESIGN: Retrospective cohort study. METHOD: Data from all patients with metastatic non-squamous NSCLC diagnosed in 2013 or 2015 were identified from the Netherlands Cancer Registry, and coupled with data from the Netherlands national pathology registry (PALGA). Using information extracted from PALGA we determined what percentage of the tumours were tested for EGFR or KRAS mutations and ALK rearrangement, identified the variables that were associated with the performance of molecular diagnostics and investigated the differences between 48 laboratories. RESULTS: A total of 6,619 patients were included (2013: n = 3,195; 2015: n = 3,424). In 2013, EGFR or KRAS testing was performed on 73.1% of the tumours (variation between laboratories 30.6-91.7%); in 2015 this was 78.9% (variation 40.0-91.0%). In 2013 49.5% of the tumours without EGFR or KRAS mutations underwent ALK testing (variation between laboratories 6.3-100%) and in 2015 ALK testing was performed on 77.4% (32.5-100%). In 2015, 6 and 7 laboratories tested significantly fewer EGFR and ALK tests, respectively, than the national average. CONCLUSION: In 2013, molecular testing for EGFR and KRAS mutations and, in particular, for ALK rearrangement was suboptimal. EGFR and ALK testing was performed significantly more often in 2015. Despite this increase, there is room for improvement in a number of laboratories and hospitals, considering that some patients were possibly wrongly not eligible for targeted therapy.

Salivary duct carcinoma (SDC) is a subtype of salivary gland cancer with a dismal prognosis and a need for better prognostication and novel treatments. The aim of this national cohort study was to investigate clinical outcome, prognostic factors, androgen receptor (AR) and human epidermal growth factor receptor 2 (HER2) expression. SDC patients diagnosed between 1990 and 2014 were identified by the Nationwide Network and Registry of Histo‐ and Cytopathology in the Netherlands (PALGA). Subsequently, medical records were evaluated and pathological diagnoses reviewed. Data were analyzed for overall survival (OS), disease‐free survival (DFS), distant metastasis‐free survival (DMFS) and prognostic factors. AR was evaluated by immunohistochemistry (IHC), HER2 by IHC and fluorescent in‐situ hybridization. A total of 177 patients were included. The median age was 65 years, 75% were male. At diagnosis, 68% presented with lymph node metastases and 6% with distant metastases. Median OS, DFS and DMFS were 51, 23 and 26 months, respectively. In patients presenting without distant metastases, the absolute number of positive lymph nodes was associated with poor OS and DMFS in a multivariable analysis. AR and HER2 were positive in 161/168 (96%) and 44/153 (29%) tumors, respectively, and were not prognostic factors. SDC has a dismal prognosis with primary lymph node involvement in the majority of patients. The absolute number of lymph node metastases was found to be the only prognostic factor for DMFS and OS. AR expression and—to a lesser extent—HER2 expression hold promise for systemic treatment in the metastatic and eventually adjuvant setting., What's new? Salivary duct carcinoma (SDC) is a rare and often fatal malignancy. Little is known about associations between its pathological features and clinical outcome. In this study, clinicopathological factors were analyzed for 177 patients diagnosed with SDC in The Netherlands between 1990 and 2014. The data show that median overall survival (OS) and distant metastasis‐free survival (DMFS) were 51 and 26 months, respectively. At diagnosis, 68% of patients presented with lymph node metastases. Lymph node positivity was associated with poor OS and poor DMFS. The absolute number of metastatic lymph nodes was the only significant prognostic factor for survival in a multivariate analysis. Androgen receptor and human epidermal growth factor 2 (HER2) were positive in 96% and 29%, respectively and were not a prognostic factor.

Objective: Staging of laryngeal cancer largely depends on cartilage invasion. Presence of cartilage invasion affects treatment choice and prognosis. On MRI and contrast-enhanced CT (CECT) it may be challenging to differentiate cartilage invasion from inflammation. The purpose of this study is to compare the diagnostic properties of dynamic contrast-enhanced CT (DCECT) and CECT for visual detection of cartilage invasion in laryngeal cancer. Study Design: Prospective cohort study. Methods: Patients with T3 or T4 laryngeal squamous cell carcinoma treated with total laryngectomy were evaluated using 0.625 mm slice CT. DCECT derived permeability and blood volume maps and CECT images were visually evaluated for the presence of invasion of the cartilaginous T-stage subsites of laryngeal cancer, by detecting continuity with the tumor-bulk of increased permeability, increased blood volume, and enhancement. Histological evaluation of the surgical total laryngectomy specimen served as the gold standard. Sensitivity, specificity, negative predictive value, and positive predictive value were calculated and compared using the McNemar and Chi-squared test. Results: From 14 included patients, a total of 462 subsites were available for T-stage analysis, of which 84 were cartilage. The median time between CT imaging and total laryngectomy was 1 day (range 1-34 days). There was no significant difference in the detection of cartilage invasion between DCECT and CECT. The sensitivity of CECT was better for all subsites combined (0.85 vs. 0.75; p

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.

Background: In this study, our aim was to identify molecular aberrations predictive for response to everolimus, an mTOR inhibitor, regardless of tumor type. Methods: To generate hypotheses about potential markers for sensitivity to mTOR inhibition, drug sensitivity and genomic profiles of 835 cell lines were analyzed. Subsequently, a multicenter study was conducted. Patients with advanced solid tumors lacking standard of care treatment options were included and underwent a pre-treatment tumor biopsy to enable DNA sequencing of 1,977 genes, derive copy number profiles and determine activation status of pS6 and pERK. Treatment benefit was determined according to TTP ratio and RECIST. We tested for associations between treatment benefit and single molecular aberrations, clusters of aberrations and pathway perturbation. Results: Cell line screens indicated several genes, such as PTEN (P = 0.016; Wald test), to be associated with sensitivity to mTOR inhibition. Subsequently 73 patients were included, of which 59 started treatment with everolimus. Response and molecular data were available from 43 patients. PTEN aberrations, i.e. copy number loss or mutation, were associated with treatment benefit (P = 0.046; Fisher's exact test). Conclusion: Loss-of-function aberrations in PTEN potentially represent a tumor type agnostic biomarker for benefit from everolimus and warrants further confirmation in subsequent studies.

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. (C) 2016 Wiley Periodicals, Inc.

When resecting head and neck tumours, the aim is mostly to strive for en bloc resection with sufficiently large tumour-free margins. If this is not possible, as is frequently the case with transoral laser microsurgery and endonasal endoscopic surgery, multiblock resection can be carried out by cutting through the tumour. With this approach, it is also essential that the final resection margins are tumour-negative. In such cases, surgeon and pathologist together should pay extra attention to tissue orientation and outer resection margins.

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.

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.

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.

Background: Validation of magnetic resonance imaging (MRI) and development of guidelines for the delineation of the gross tumor volume (GTV) is of utmost importance to benefit from the visibility of anatomical details on MR images and to achieve an accurate GTV delineation. In the ideal situation, the GTV delineation corresponds to the histopathologically determined ‘true tumor volume’. Consequently, we developed guidelines for GTV delineation of laryngeal and hypopharyngeal tumors on MRI and determined the accuracy of the resulting delineation of the tumor outline on histopathology as gold standard. Material and methods: Twenty-seven patients with T3 or T4 laryngeal/hypopharyngeal cancer underwent a MRI scan before laryngectomy. Hematoxylin and eosin sections were obtained from surgical specimens and tumor was delineated by one pathologist. GTV was delineated on MR images by three independent observers in two sessions. The first session (del1) was performed according to clinical practice. In the second session (del2) guidelines were used. The reconstructed specimen was registered to the MR images for comparison of the delineated GTVs to the tumor on histopathology. Volumes and overlap parameters were analyzed. A target margin needed to assure tumor coverage was determined. Results: The median GTVs (del1: 19.4 cm3, del2: 15.8 cm3) were larger than the tumor volume on pathology (10.5 cm3). Comparable target margins were needed for both delineation sessions to assure tumor coverage. By adding these margins to the GTVs, the target volumes for del1 (median: 81.3 cm3) were significantly larger than for del2 (median: 64.2 cm3) (p ≤ 0.0001) with similar tumor coverage. Conclusions: In clinical radiotherapy practice, the delineated GTV on MRI is twice as large as the tumor volume. Validated delineation guidelines lead to a significant decrease in the overestimation of the tumor volume.

Objectives: Although TP53 mutations in head and neck squamous cell carcinoma (HNSCC) have been extensively studied, their association with the different subsites in the head and neck region has never been described. Methods: Sanger sequence analysis evaluating exons 4-9 in the TP53 gene was performed on 116 HNSCC patients. The exon location, exact codon and corresponding substitution in relation to the anatomical site (subsite) of the HNSCC were evaluated. Results: We found nonsynonymous TP53 mutations in 70% (81/116) of the patients. In oral cavity carcinomas, most mutations occurred in exon 7 (37%). In oropharyngeal and laryngeal tumors, mutations were mainly found in exons 6 and 7. The most common mutation was located in codon 220, and all of these were an Y220C mutation. Five out of nine (56%) Y220C mutations occurred in oropharyngeal tumors. Additionally, 22% of all mutations observed in oropharyngeal squamous cell carcinoma (OPSCC) consisted of Y220C mutations. Conclusion: In this study, the subsite-related distribution of TP53 mutations underlines the biological diversity between tumors arising from different anatomical regions in the head and neck region. Moreover, the Y220C mutation was by far the most prevalent TP53 mutation in HNSCC and a relative hotspot mutation in the oropharynx. (C) 2015 S. Karger AG, Basel