Immunohistochemistry

Five μm tissue slides were sectioned on APES coated slides, and deparaffinized utilizing xylene (2x10min) and ethanol (2x5min). The slides were rinsed with PBS before blocking with 0.3% H2O2 for 30 min. In-situ hybridization for EBV encoded RNA (EBER) was performed with the Novocastra^™ Epstein-Barr virus ISH Kit using standard methods [14]. Monoclonal antibodies to the HLA-class I heavy chain (HC-10, kindly provided by prof. dr. J. Neefjes, the Netherlands Cancer Institute, Amsterdam, the Netherlands) and polyclonal rabbit anti human B2 microglobulin (B2M, DAKO, Glostrup, Denmark), at dilutions of 1:500 and 1:400 respectively, were used to mark HLA class I expression. Monoclonal antibody to CR3/43 (DAKO) that binds to the HLA-II chain of HLA-DP, HLA-DQ, and HLA-DR at a dilution of 1:200 was used to mark HLA class II expression. CD30 was used to aid in identifying HRS cells. Tissues were stained using the Leica Bond 3 staining platform. Scoring for EBER and HLA expression was performed if at least 50 HRS cells were present in all three cores combined. A case was considered positive for HLA if there was membranous staining in more than 50% of the HRS cells.