Aims Giant cell tumour of bone (GCTB) is histologically defined as a lesion containing reactive giant cells and a neoplastic mononuclear cell population; in up to 92% GCTB is characterized by a specific mutation of the histone gene H3F3A. The cellular composition ranges from giant cell‐rich to giant cell‐poor tumours. The diagnosis of GCTB can be challenging and several other lesions need to be excluded, e.g., aneurysmal bone cysts, non‐ossifying fibromas, chondroblastomas, brown tumours, and giant‐cell‐rich osteosarcomas. Our aim was to analyse the clinical history, imaging, molecular pathology, and histology of three H3F3A‐mutated bone tumours without detectable giant cells. None of the patients received denosumab therapy. Methods and results Diagnostic material was obtained by curettage or resection and/or biopsy. Common histomorphologic features of all three reported lesions were fibrocytic, oval cells in a background of osteoid while multinuclear giant cells were not detected as confirmed by CD68‐immunohistochemistry. We used immunohistochemistry and Sanger‐sequencing to demonstrate positivity for H3.3 p.G34W mutation. Differential diagnoses were systematically excluded based on histomorphology, immunohistochemistry and FISH. The imaging (radiograph, CT, MRI) for all three cases is presented and discussed. Conclusions Therefore, we believe that these GCTB without giant cells expand one end of the heterogeneous range of GCTB. Due to the lack of giant cells the correct diagnosis of GCTB is challenging or even impossible based on pure histological grounds. In these cases, detection of the characteristic H3F3A gene mutation (G34W‐specific antibody RM263 or sequencing) is extremely helpful to diagnose those lesions without giant cells as a giant cell tumour of bone.

Giant cell tumor of bone (GCTB) is an osteolytic tumor driven by an H3F3A-mutated mononuclear cell with the accumulation of osteoclastic giant cells. We analyzed tissue from 13 patients with recurrence and 25 patients with denosumab therapy, including two cases of malignant transformation. We found a decrease in the total number of cells (p = 0.03), but not in the individual cell populations when comparing primary and recurrence. The patients treated with denosumab showed induction of osteoid formation increasing during therapy. The total number of cells was reduced (p < 0.0001) and the number of H3F3A-mutated tumor cells decreased (p = 0.0001), while the H3F3A wild-type population remained stable. The KI-67 proliferation rate dropped from 10% to 1% and Runx2- and SATB2-positive cells were reduced. The two cases of malignant transformation revealed a loss of the H3F3A-mutated cells, while the KI-67 rate increased. Changes in RUNX2 and SATB2 expression were higher in one sarcoma, while in the other RUNX2 was decreased and SATB2-positive cells were completely lost. We conclude that denosumab has a strong impact on the morphology of GCTB. KI-67, RUNX2 and SATB2 expression differed depending on the benign or malignant course of the tumor under denosumab therapy.

Background/Objectives: Flow-cytometric readouts in acute myeloid leukemia (AML) may depend on specimen source and processing. We assessed whether sample source (peripheral blood [PB] vs. bone marrow [BM]) and cryopreservation influence immune-evasion-related markers on AML blasts. Methods: Paired PB and BM samples from 19 adults were analyzed fresh and after cryopreservation. Viable CD45^dim singlets were assessed for ULBP1-6, MICA/B, CD34, CD44, CD70, CD80, and CD184 using median fluorescence intensity (MFI) and relative fluorescence index (RFI). Paired comparisons used two-sided Wilcoxon signed-rank tests with Benjamini–Hochberg correction across six markers within each endpoint and comparison. Discordance was also assessed using a predefined conventional 20% reference threshold. Results: After adjustment, RFI did not differ between fresh and cryopreserved samples within PB or BM, or between fresh PB and BM. In cryopreserved samples, PB and BM differed for CD70 (q = 0.042) and ULBP1-6 (q = 0.028). Cryopreservation increased MFI for CD184 and ULBP1-6 in PB and BM and for MICA/B in PB; no PB-BM MFI difference remained significant. Threshold-based classification was discordant for CD34, CD44, CD80, CD184, MICA/B, and ULBP1-6 in 5.3–38.9% of pairs, whereas CD70 showed none. Conclusions: Cohort-level differences were limited to specific markers and conditions, while patient-level threshold discordance persisted. Sample source and cryopreservation should therefore be considered when interpreting threshold-based classifications.