CYCLIN D1 AS BIOMARKER FOR OCCULT NODAL METASTASIS CHAPTER 6
than 5% tumor tissue or when more than 95% of the core contained no tissue. For Cyclin D1, 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 (KB) to assess interobserver agreement. Statistical Analysis
To investigate the consistency of immunohistochemical staining of Cyclin D1, FADD and Cortactin within the tumor, we analyzed the Intraclass Correlation Coefficient (ICC) between
the three scored cores. The ICC is a descriptive statistic which 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 ‘slight’, 0.21 to 0.4 ‘fair’, 0.41 to 0.60
‘moderate’, 0.61 to 0.8 ‘substantial’, and above 0.81 ‘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 χ2 test (or Fisher’s exact when appropriate) was used. Binary logistic regression analysis was used to evaluate the value of multiple variables in predicting occult nodal metastases. For interobserver agreement of Cyclin D1 expression during the validation
phase, the ICC between both observers (SMW and KB) was analyzed. If not mentioned otherwise, two-sided p-value < 0.05 was considered as significant. All statistical analyses were performed using SPSS 21.0 Statistical Software (IBM, New York, USA). Ethical justification
Since remaining tissue following the clinical diagnostic process was used, no ethical approval was required according to Dutch national ethical guidelines (www.federa.org). Anonymous or coded use of leftover tissue for scientific purposes is part of standard treatment agreement with patients in our center [21].
119
6