Surprisingly, 2D IEF of Rb from cycling populations of all four tumor cell lines, HCT116 colon carcinoma, H1299 lung adenocarcinoma, U2OS osteosarcoma, and HL60 promyelocytic leukemia, showed the presence of only mono-phosphorylated Rb and hyper-phosphorylated Rb, with no evidence of multi-phosphorylated, hypo-phosphorylated Rb isoforms, even though cyclin D:Cdk4/6 was deregulated in all four tumor cell types (Figure 1F). This evidence concerns the gene RB1 and neoplasm.