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).