Immunohistochemistry for Rb
Formalin fixed paraffin embedded tumor tissue were collected from all patients prior to enrollment (except for those with DIPG, medulloblastoma, or ATRT). Immunohistochemistry for Rb was performed as previously described17 using a mouse monoclonal anti-Rb antibody (G3-245; BD Biosciences, San Jose, CA) and an automated IHC staining process (Benchmark XT; Ventana Medical Systems, Inc, Tucson, AZ). Briefly, antigen retrieval was performed in Tris, pH 8.0 at 95C for 1 hour, followed by incubation in 3% H2O2 for 16 min, and primary antibody at 1:100 at room temperature for 60 minutes. Tumor Rb1 protein status was denoted as “positive” if ≥20% of tumor cells had positive nuclear staining. Rb1-positive endothelial cells served as an internal positive control. All slides were centrally reviewed by a dedicated neuropathologist (JJP).