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

Purpose: 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) uptake is a marker of metabolic activity and is therefore used to measure the inflammatory state of several tissues. This radionuclide marker is transported through the cell membrane via glucose transport proteins (GLUTs). The aim of this study is to investigate whether insulin resistance (IR) or inflammation plays a role in [18F]FDG uptake in adipose tissue (AT). Procedures: This study consisted of an in vivo clinical part and an ex vivo mechanistic part. In the clinical part, [18F]FDG uptake in abdominal visceral AT (VAT) and subcutaneous AT (SAT) was determined using PET/CT imaging in 44 patients with early type 2 diabetes mellitus (T2DM) (age 63 [54–66] years, HbA1c [6.3 ± 0.4 %], HOMA-IR 5.1[3.1–8.5]). Plasma levels were measured with ELISA. In the mechanistic part, AT biopsies obtained from 8 patients were ex vivo incubated with [18F]FDG followed by autoradiography. Next, a qRT-PCR analysis was performed to determine GLUT and cytokine mRNA expression levels. Immunohistochemistry was performed to determine CD68+ macrophage infiltration and GLUT4 protein expression in AT. Results: In vivo VAT [18F]FDG uptake in patients with T2DM was inversely correlated with HOMA-IR (r = − 0.32, p = 0.034), and positively related to adiponectin plasma levels (r = 0.43, p = 0.003). Ex vivo [18F]FDG uptake in VAT was not related to CD68+ macrophage infiltration, and IL-1ß and IL-6 mRNA expression levels. Ex vivo VAT [18F]FDG uptake was positively related to GLUT4 (r = 0.83, p = 0.042), inversely to GLUT3 (r = − 0.83, p = 0.042) and not related to GLUT1 mRNA expression levels. Conclusions: In vivo [18F]FDG uptake in VAT from patients with T2DM is positively correlated with adiponectin levels and inversely with IR. Ex vivo [18F]FDG uptake in AT is associated with GLUT4 expression but not with pro-inflammatory markers. The effect of IR should be taken into account when interpreting data of [18F]FDG uptake as a marker for AT inflammation. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to establish the prognostic value of the epidermal growth factor receptor (EGFR) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on local control in patients with early stage supraglottic laryngeal squamous cell carcinoma (LSCC) treated with radiotherapy only. Study design: Retrospective cohort study. Methods: Immunohistochemical staining for EGFR and PTEN was performed on pretreatment biopsies of a selected well‐defined homogeneous group of 52 patients with T1–T2 supraglottic LSCC treated with radiotherapy between 1990 and 2008. Kaplan–Meier analysis and univariate and multivariate Cox Regression analyses were performed to correlate clinical data and expression levels of EGFR and PTEN with local control. Results: Kaplan–Meier survival analysis and Cox Regression analysis showed a significant association between PTEN expression and local control (hazard ratio [HR] = 3.26, 95% confidence interval [CI] = 1.14–9.33, P =.027) and between lymph node status and local control (HR = 3.60, 95% CI = 1.26–10.31, P =.017). Both were independent prognostic factors in a multivariate analysis (HR = 3.28, 95% CI = 1.14–9.39, P =.027 and HR = 3.62, 95% CI = 1.26–10.37, P =.017, respectively). There was no significant association between EGFR expression and local control (HR = 1.32, 95% CI = 1.17–10.14, P =.79). Conclusion: This study showed an association between both high PTEN expression and the presence of lymph node metastasis and deteriorated local control in early stage supraglottic LSCC treated with radiotherapy. Level of Evidence: NA [ABSTRACT FROM AUTHOR]

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance. [ABSTRACT FROM AUTHOR]

Purpose: We recently reported on the identification of the Fas-associated death domain (FADD) as a possible driver of the chromosome 11q13 amplicon and the association between increased FADD expression and disease-specific survival in advanced-stage laryngeal carcinoma. The aim of this study was to examine whether expression of FADD and its Ser194-phosphorylated isoform (pFADD) predicts local control in patients with early-stage glottic carcinoma primarily treated with radiotherapy only. Methods and Materials: Immunohistochemical staining for FADD and pFADD was performed on pretreatment biopsy specimens of 92 patients with T1–T2 glottic squamous cell carcinoma primarily treated with radiotherapy between 1996 and 2005. Cox regression analysis was used to correlate expression levels with local control. Results: High levels of pFADD were associated with significantly better local control (hazard ratio, 2.40; 95% confidence interval, 1.04–5.55; p = 0.040). FADD overexpression showed a trend toward better local control (hazard ratio, 3.656; 95% confidence interval, 0.853–15.663; p = 0.081). Multivariate Cox regression analysis showed that high pFADD expression was the best predictor of local control after radiotherapy. Conclusions: This study showed that expression of phosphorylated FADD is a new prognostic biomarker for better local control after radiotherapy in patients with early-stage glottic carcinomas. [Copyright &y& Elsevier]

Purpose: Leiomyosarcomas (LMS) of deep and superficial tissues were examined to identify prognostic markers explaining their different biological behaviour and to define differences between cutaneous and subcutaneous LMS. LMS and leiomyomas (LM) of the skin were compared to find consistent differences that could aid in the (sometimes difficult) diagnosis. Patients: Material was obtained from 27 patients with a deep LMS, 14 with a superficial LMS, and 21 with a LM. Methods: Proliferation markers (mitotic and Ki-67 indices), DNA ploidy, size, grade, and the amount of apoptosis were studied. Statistical analysis was performed and survival curves were constructed by the Kaplan-Meier method and compared by the log-rank test. Results: Superficial LMS were smaller than deep LMS (p < 0.05), and the overall survival of patients with a superficial LMS was better than with a deep LMS (p < 0.05).Within the group of superficial LMS only entirely subcutaneous, and not cutaneous tumors metastasized. No differences were found in the other examined parameters. Proliferation and apoptotic indices were significantly higher in superficial LMS compared to superficial LM. Discussion: The difference in clinical outcome between patients with a superficial and deep LMS, seems to be related to site and size.The metastatic potential of subcutaneous LMS, however, seems to be related to location alone and not to size.The amount of apoptosis and proliferation can be used as additional criteria in the differentiation between superficial LMS and LM. [ABSTRACT FROM AUTHOR]

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

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