Epigenetic lockdown of CDKN1A (p21) and CDKN2A (p16) characterises the neoplastic spindle cell component of giant cell tumours of bone

Giant cell tumour of bone (GCTB) comprises the eponymous osteoclastic multinucleated giant cells eliciting bone lysis, an H3F3A‐mutated neoplastic mononucleated fibroblast‐like cell population, and H3F3A wild‐type mononucleated stromal cells. In this study, we characterised four new cell lines from GCTB. Furthermore, we compared the genome‐wide DNA methylation profile of 13 such tumours and three further cell lines with giant cell‐rich lesions comprising three H3F3B‐mutated chondroblastomas, three USP6‐rearranged aneurysmal bone cysts, three non‐ossifying fibromas, two hyperparathyroidism‐associated brown tumours as well as mesenchymal stem cells, osteoblasts, and osteoclasts. In an unsupervised analysis, we delineated GCTB and chondroblastomas from the other analysed tumour entities. Using comparative methylation analysis, we demonstrated that the methylation pattern of the cell lines approximately equals that of H3F3A‐mutated stromal cells in tissue. These patterns more resemble that of osteoblasts than that of mesenchymal stem cells, which argues for the osteoblast as the cell of origin of giant cell tumours of bone. Using enrichment analysis, we detected distinct hypermethylated clusters containing histone and collagen genes as well as target genes of the tumour suppressor p53. We found that the promotor regions of CDKN1A, CDKN2A, and IGFBP3 are methylated more strongly in GCTB than in the other giant cell‐containing lesions, mesenchymal stem cells, osteoblasts, and osteoclasts (p