Koos Boeve

130 Chapter 7 cytoplasmic (-/+) expression. Markers were independently scored by two observers (KB with MM or LSM). Discordances were discussed with a dedicated head and neck pathologist (BvdV) until consensus was reached. Except for FADD, optimal cut-offs between high and low expression of the markers and N-status were determined using a ROC curve and the average expression of the cores per tumor. The cut-offs were 46% of cytoplasmic expression for cortactin, 73% of nuclear expression for cyclin D1, 4% of any expression (+, ++) of RAB25 and 17% of nuclear expression and 55% of cytoplasmic expression for S100A9. For FADD, cases with at least one core scored as ++ or +++ were defined as high expression. Representative examples of low and high expression detected by immunohistochemical staining are shown in Figure 1. Table 2. Antibody information Antibody Clone Company Catalog No. Website Dilution Cortactin 30 BD BIOSCIENCES 610050 http://www.bdbiosciences.com/us/reagents/ research/antibodies-buffers/cell-biology- reagents/cell-biology-antibodies/purified- mouse-anti-cortactin-30cortactin/p/610050 1:1000 Cyclin D1 SP4-R Roche 790-4508 http://reagent-catalog.roche.com/ product/1530?type=1990 FADD A66-2 BD Biosciences 556402 http://www.bdbiosciences.com/us/ applications/research/apoptosis/purified- antibodies/purified-mouse-anti-human-fadd- a66-2/p/556402 1:100 RAB25 3F12F3 Santa Cruz Biotechnoly Sc-65978 https://www.scbt.com/scbt/product/rab-25- antibody-3f12f3 1:50 S100A9 S36.48 BMA biomedicals T1026 http://www.bma.ch/en/products/t-1026 1:100 Ethical considerations This study used retrospective data and leftover tumor tissue which was collected during treatment according to the national guidelines for oral cavity cancer. Therefore, this research was not a clinical study with human subjects as meant in the Medical Research Involving Human Subjects Act as was concluded by the local Medical Ethics Review Board of the University Medical Center Groningen (M18.225755) and no further approval was required.