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Background: Giant cell tumour of bone (GCTB) is an osteolytic tumour which contains numerous osteoclast-like giant cells and a proliferation of mononuclear stromal cells (MSC). Giant cell-rich osteolytic lesions can also develop in the jaw bones in Noonan syndrome, a cherubism-like developmental abnormality that is transmitted in an autosomal dominant fashion, often because of mutation in the PTPN11 or BRAF genes., Methods: We screened GCTBs for mutations in PTPN11 and BRAF to determine whether GCTBs develop through alterations of genes involved in Noonan syndrome. MSC were isolated from 10 GCTBs., Results: Chromosome banding analysis of these cells revealed telomeric associations (tas) in 7 of the 10 cases. Thus, the cultured cells expressed a cytogenetic abnormality typically found in short-term cultures from GCTBs. Sequencing of DNA extracted from the seven GCTB-derived MSC cultures displaying tas did not identify any mutation in PTPN11 or in exons 9-15 of BRAF., Conclusion: Our findings indicate that the molecular pathways involved in GCTB development are different from those causing Noonan syndrome. The method for isolating and culturing GCTB stromal cells described in this study generated a population of MSC that contained tas, indicating that it is useful for obtaining stromal cells from GCTB and other giant cell-rich lesions, such as giant cell reparative granuloma, for genetic and other studies., (© 2012 John Wiley & Sons A/S.)

Aims: Epidermal growth factor receptor (EGFR) is implicated in bone remodelling. The aim was to determine whether EGFR protein expression contributes to the aggressiveness and recurrence potential of giant cell tumour of bone (GCTB), an osteolytic primary bone tumour that can exhibit markedly variable clinical behaviour., Methods and Results: Immunohistochemical analysis on tissue microarrays (TMA) of 231 primary, 97 recurrent, 17 metastatic and 26 malignant GCTBs was performed using TMA analysis software and whole digital slides allowing validated scoring. EGFR expression was restricted to neoplastic stromal cells and was significantly more frequent in recurrent (71 of 92; 77%) than in non-recurrent GCTBs (86 of 162; 53%) (P = 0.002); and in clinicoradiologically aggressive (31 of 43; 72%) than latent (27 of 54; 50%) cases (P = 0.034). Detecting phosphotyrosine epitopes pY1068 and -pY1173 indicated active EGFR signalling, and finding EGFR ligands EGF and transforming growth factor-α restricted to cells of the monocytic lineage suggested paracrine EGFR activation in stromal cells. In functional studies EGF supported proliferation of GCTB stromal cells, and the addition of EGF and macrophage-colony stimulating factor promoted osteoclastogenesis., Conclusion: In GCTB, EGFR signalling in neoplastic stromal cells may contribute to disease progression through promoting stromal cell proliferation and osteoclastogenesis., (© 2011 Blackwell Publishing Limited.)

Giant cell tumour of bone, a benign but potentially aggressive neoplasm, shows an increasing rate of chromosomal aneusomy that correlates with clinical course. Mechanisms that generate chromosomal instability in giant cell tumour of bone are poorly understood. One possible cause of chromosomal instability is an error in mitotic segregation due to numeric and/or functional abnormalities of centrosomes. Centrosome alteration is a common phenomenon in many cancers and has a major role in the development of chromosomal instability in cancer cells. To gain an insight into the possible mechanism for the generation of chromosomal instability in giant cell tumour of bone, we analysed 100 cases, including 57 primary nonrecurrent, 35 recurrent and 8 malignant giant cell tumour of bone cases. gamma-Tubulin immunohistochemistry was performed on tissue microarrays of 59 formalin-fixed paraffin-embedded cases, whereas pericentrin and gamma-tubulin fluorescent immunocytochemistry was carried out on 41 frozen smears. Fluorescent in situ hybridization was performed on 23 cases of pericentrin immunostained smears, allowing the simultaneous analysis of centrosomes and chromosome aberrations. Centrosome amplification was significantly higher in recurrent and malignant giant cell tumour of bones compared with nonrecurrent tumours (P<0.001). A comparison of the percentage of aneusomic cells with a normal centrosome content (4.7%) with that of aneusomic cells with centrosome amplification (6.4%) revealed no significant association between chromosome number alterations and centrosome aberrations (P=0.31). These findings indicate that centrosome alteration and frequency of aneusomy correlate with clinical behaviour; the lack of an association between centrosome amplification and chromosome number alteration suggests that alternative causative mechanisms produce genetic instability in giant cell tumour of bone.

Genetic instability in relation to clinical behavior was studied in 52 cases of giant cell tumor of bone (GCTB). Ploidy was determined in the mononuclear cell population by using native cell smears and image cytometry. A relocalization technique allowed fluorescent in situ hybridization (FISH) analysis of CD68-negative neoplastic cells for numerical changes of chromosomes X, 3, 4, 6, 11, and telomeric association on 11p. Genome-wide alterations were tested using array comparative genomic hybridization (array-CGH) on magnetically separated CD68-negative tumor cells. CTNNB1, TP53, and BCL2 protein expression was also analyzed in formol-paraffin sections to see if their pathways are involved in the development of chromosomal instability. CD68-positive histiocytes showed no significant numerical chromosome and telomeric alterations. Based on ploidy values and clinical outcome, we could distinguish five groups as follows: diploid nonrecurrent (n = 20), tetraploid nonrecurrent (n = 6), diploid recurrent (n = 5), tetraploid and/or aneuploid recurrent (n = 14), and malignant cases (n = 7). Random individual-cell aneusomy was significantly (P < 0.001) more frequent in the recurrent groups (36.01 +/- 11.94%) than in the benign nonrecurrent cases (10.65 +/- 3.66%). The diploid recurrent group showed significantly (P < 0.001) increased balanced aneusomy compared with the diploid nonrecurrent group and the tetraploid nonrecurrent group represented eusomic polysomy. Array-CGH and FISH showed clonal aberrations almost exclusively in the malignant group. None of the protein markers tested showed significant correlation with elevated aneuploidy/polysomy (P = 0.56). Our results show that ploidy determination combined with FISH analysis may help predicting recurrence potential of GCTB and suggest that chromosomal abnormalities superimposed on telomeric associations could be responsible for an aggressive clinical course.