Koos Boeve

107 Cyclin D1 in early oral cancer was used. In short, 4 µm thick paraffin sections were deparaffinized with xylene and rehydrated. Endogenous peroxidase activity was blocked using a 0.3% hydrogen peroxide phosphate-citrate buffer for 15 minutes. Next, the slides were washed in water and subsequently subjected to antigen retrieval by boiling in EDTA buffer, pH 9.0 (cyclin D1 and FADD) or citrate buffer, pH 6.0 (cortactin) for 20 minutes. After cooling down and washing with phosphate buffered saline (PBS) for 5 minutes, tissue slides were incubated with the primary antibody cyclin D1 (clone SP4, USA; dilution 1:100; Cellmarque, Rocklin, CA), primary antibody FADD (556402, dilution 1:100; BD Pharmingen TM , San Jose, CA, USA) or primary antibody Cortactin (610049, dilution 1:200; BD Transduction Laboratories TM , San Jose, CA, USA) for 60 minutes. After washing with PBS (3 times), the slides were incubated with poly- horseradish peroxidase goat goat anti-mouse/rabbit/rat (Bright Vision, Imunologic, Duiven, The Netherlands, ready to use) for 30 minutes followed by washing with PBS (3 times). Slides were then developed with diaminobenzidine for 10 minutes and hematoxylin was used for counterstaining. Oral cancer with known amplification of the 11q13 has been used as positive control (with antibody) and as a negative test (without antibody) control in each test. IHC staining of tumor cells was scored by a dedicated head and neck pathologist (S.M.W.). A core was considered inadequate/lost when the core contained <5% tumor tissue or when more than >95% of the core contained no tissue. For cyclin D1, the percentage of nuclear staining and for both FADD and cortactin intensity of cytoplasmatic staining (0, none; 1, weak; 2, moderate; 3, strong) was scored semi-quantitative. During validation as biomarker, Cyclin D1 expression was also scored by an independent head and neck cancer researcher (K.B.) to assess interobserver agreement. Statistical Analysis To investigate the consistency of IHC staining of cyclin D1, FADD and cortactin within the tumor, we analyzed the intraclass correlation coefficient (ICC) among the 3 scored cores. The ICC is a descriptive statistic that describes how strongly different quantitative measures resemble each other, in this case multiple cores of the same tumor. An ICC <0 reflects ‘poor’ , 0 to 0.20 reflects ‘slight’, 0.21 to 0.4 reflects ‘fair’, 0.41 to 0.60 reflects ‘moderate’, 0.61 to 0.8 reflects ‘substantial’, and above 0.81 reflects ‘almost perfect’ reliability of the measurement. Any measurement should have an ICC of at least 0.6 to be useful with regard to reliability of the result [20]. Correlation between CCND1 copy number results and nuclear cyclin D1 expression was analyzed using the Kruskal-Wallis test. For correlation with occult nodal metastasis, protein expression results were dichotomized. For cyclin D1 protein expression, ROC-curve analysis was used to determine cut-off levels for prediction of occult nodal metastasis. For both CCND1 gene amplification and protein expression the Pearson chi-