Koos Boeve

106 Chapter 6 Table 1. Continued Initial (158 tumors) Validation (155 tumors) Tumor front cohesive 55 (35%) NA non-cohesive 102 (65%) missing 1 (1%) Extracapsular spread no 156 (99%) NA yes 2 (1%) * histological status of patients without neck dissection was based on follow-up of at least 2 years. Abbreviations : FOM, floor of mouth; NA, data not available; PO(Ch)RT, postoperative (chemo)radiotherapy. Tissue microarray From 158 tumors, sufficient formaldehyde-fixed paraffin-embedded tissue was available for incorporation in a TMA. From each tumor block, 3 tissue cylinders with a diameter of 0.6 mm were punched out, avoiding areas of necrosis, and arrayed in a recipient paraffin block. The TMAs contain normal tonsillar epithelium as control tissue to ensure similarity of staining quality and intensity between the different blocks. Fluorescence in-situ hybridization Fluorescence in situ hybridization (FISH) was performed on fresh sectioned, 4 micrometer thick paraffin TMA sections. Slides were deparaffinized and pretreated with sodium citrate and protease buffers. Afterward, the slides were dehydrated and hybridized with 15 µL Vysis CCND1 / CEP11 FISHprobe (AbbottMolecular Diagnostics,TheNetherlands) in aThermoBrite (Abbott Laboratories, Chicago, IL) at 37°C overnight. The next day, they were washed in saline-sodium citrate buffers, counterstained with diamidino-phenylindole, dehydrated and mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). One- hundred tumor cell nuclei per tumor were analyzed for the CCND1 gene and CEP11 probe signals at X100 magnification on a Leica DM5500 B microscope system using Application Suite Advanced Fluorescence software (Leica Microsystems, Rijswijk, The Netherlands). The CCND1/CEP11 ratio was calculated to correct for centromere signals. A ratio >1.25 until 2.00 was defined as low-level and a ratio ≥2.00 as high-level amplification. Immunohistochemistry IHC staining for cortactin and FADD was performed manually. For cyclin D1, the Ventana Benchmark Ultra (Ventana Medical Systems, Tucson, AZ) automatically staining procedure