Koos Boeve

128 Chapter 7 Table 1. Continued True N negative True N positive p-value n (%) n (%) high expression 14 (25) 12 (40) Cyclin D1 low expression 26 (46) 11 (37) 0.497 high expression 31 (54) 19 (63) FADD low expression 39 (70) 15 (50) 0.101 high expression 17 (30) 15 (50) RAB25 low expression 23 (40) 8 (27) 0.245 high expression 34 (60) 22 (73) S100A9 nuclear low expression 17 (30) 10 (33) 0.809 high expression 40 (70) 20 (67) S100A9 cytoplasmic low expression 23 (40) 10 (33) 0.643 high expression 34 (60) 20 (67) True N-status is determined by the combination of postoperative pathological lymph node status (pN) combined with regional recurrence (false negatives). Four patients with a negative SLNB (pN0) were diagnosed with a regional recurrence and counted as true N positives. Molecular expression was dichotomized for cortactin, cyclin D1, RAB25 and S100A9 using a ROC-analysis. FADD was semi-quantitatively scored. Abbreviations: IQR, interquartile range; TNM, American Joint Committee of Cancer TNM classification. The SLNB protocol used was described before [7]. Briefly, one day before surgery a radioactive tracer ( 99m Tc-nannocolloid) was peritumorally injected, followed by dynamic and static lymphoscintigraphy and Single Photon Emission Computed Tomography (SPECT)-CT scanning. The next day during surgery, SLNs were located and harvested using a handheld gamma-probe. Postoperatively, the SLNs were histopathological assessed with step-serial- sectioning and slides from each level were stained for hematoxylin and eosin (HE) and additional cytokeratin (CK AE1-AE3, clone AE1/AE3, Ventana Medical Systems, Tucson, AZ). Tissue microarray construction All HE slides of the primary tumor were revised by a dedicated head and neck pathologist to assess tumor cell presence. Twenty-six of the 113 tumors were too small or had no redundant tissue available for TMA construction. Five other tumors were not suitable for inclusion in the TMA as a result of limited tumor size, but could be assessed using whole