Koos Boeve

129 Molecular biomarkers in SLNB staged early oral cancer tumor slide sections. Finally, 87 cases were available for analysis (82 tumors on two TMA blocks and five whole tumor slide sections). The TMA construction procedure in our center has been described earlier [21]. Briefly, tumors were marked on the HE slides by a pathologist, after which three 0.6 mm cores were taken from the corresponding donor FFPE blocks and added to the recipient TMA FFPE block using a Manual Tissue Arrayer I (Beecher Instruments, Sun Prairie, WI). Both TMA blocks had a unique lay-out with normal tissue samples of liver, testis, cervix, colon and placenta incorporated into the TMAs as negative and positive control tissues. 3 µm thick sections were cut from the TMA blocks for the immunohistochemical staining of the markers. For IHC staining, the first and last sections were HE stained to confirm presence of tumor in the cores of TMA sections. Immunohistochemistry The staining procedures and antibodies have been described before [20,22,23] and are summarized in Table 2. Cyclin D1 was automatically stained using the Ventana Benchmark Ultra (VentanaMedical Systems,Tucson, AZ, US). For the othermarkers: after deparaffinization in xylene and rehydration in a graded alcohol series, sections were treated for antigen retrieval followed by endogenous peroxidase blockage by incubating in 0.3% peroxide solution. Slides were then incubated for one hour with the primary Mouse anti human antibody, followed by 30 minutes incubation of a Rabbit anti Mouse (RAM po ) secondary antibody and 30 minutes incubation of a Goat anti Rabbit tertiary antibody (GAR po ). All antibodies were Horseradish Peroxidase conjugated and diluted in 1% BSA – PBS serum (primary antibody) or diluted in 1:100 1% BSA – PBS – 1%AB serum (RAM po and GAR po ). Slides were developed with 3,3-di-amniobenzidine (DAB) chromogen solution (DAKO, Glostrup, Denmark) and counterstained with hematoxylin. HE, cyclin D1 and FADD stained glass slides were digitized using the NanoZoomer 2.0 HT with a 40x magnification lens and the NDP. view 2 Viewing software U12388-01 (Hamamatsu Photonics, Hamamatsu City, Shizuoku, Japan). Cortactin, RAB25 and S100A9 slides were digitized using the IntelliSite Ultra Fast Scanner with the IntelliSite Image Management System software (Phillips Electronics, Best, The Netherlands). All molecular biomarkers were scored as described earlier [17,20,22,23][Clausen S100A9 in prep]. Cortactin and cyclin D1 were scored as the percentage of tumor cells with a higher cytoplasmic expression (cortactin) or nuclear expression (cyclin D1) compared to normal epithelium or surrounding tissue cells. FADD was scored semi-quantitatively as tumor cells with negative (0), weakly positive (+), positive (++) or strong positive (+++) cytoplasmic expression. RAB25 was scored as percentage of tumor cells with negative (-), moderate positive (+) or strong positive (++) cytoplasmic expression. S100A9 was scored for both percentage of tumor cells with any nuclear (-/+) expression or percentage of any