Hypoxia Signaling in Cancer: From Basics to Clinical Practice.

Cancer hypoxia, recognized as one of the most important hallmarks of cancer, affects gene expression, metabolism and ultimately tumor biology-related processes. Major causes of cancer hypoxia are deficient or inappropriate vascularization and systemic hypoxia of the patient (frequently induced by anemia), leading to a unique form of genetic reprogramming by hypoxia induced transcription factors (HIF). However, constitutive activation of oncogene-driven signaling pathways may also activate hypoxia signaling independently of oxygen supply. The consequences of HIF activation in tumors are the angiogenic phenotype, a novel metabolic profile and the immunosuppressive microenvironment. Cancer hypoxia and the induced adaptation mechanisms are two of the major causes of therapy resistance. Accordingly, it seems inevitable to combine various therapeutic modalities of cancer patients by existing anti-hypoxic agents such as anti-angiogenics, anti-anemia therapies or specific signaling pathway inhibitors. It is evident that there is an unmet need in cancer patients to develop targeted therapies of hypoxia to improve efficacies of various anti-cancer therapeutic modalities. The case has been opened recently due to the approval of the first-in-class HIF2α inhibitor.

The Function of NM23-H1/NME1 and Its Homologs in Major Processes Linked to Metastasis.

Metastasis suppressor genes (MSGs) inhibit different biological processes during metastatic progression without globally influencing development of the primary tumor. The first MSG, NM23 (non-metastatic clone 23, isoform H1) or now called NME1 (stands for non-metastatic) was identified some decades ago. Since then, ten human NM23 paralogs forming two groups have been discovered. Group I NM23 genes encode enzymes with evolutionarily highly conserved nucleoside diphosphate kinase (NDPK) activity. In this review we summarize how results from NDPKs in model organisms converged on human NM23 studies. Next, we examine the role of NM23-H1 and its homologs within the metastatic cascade, e.g. cell migration and invasion, proliferation and apoptosis. NM23-H1 homologs are well known inhibitors of cell migration. Drosophila studies revealed that AWD, the fly counterpart of NM23-H1 is a negative regulator of cell motility by modulating endocytosis of chemotactic receptors on the surface of migrating cells in cooperation with Shibire/Dynamin; this mechanism has been recently confirmed by human studies. NM23-H1 inhibits proliferation of tumor cells by phosphorylating the MAPK scaffold, kinase suppressor of Ras (KSR), resulting in suppression of MAPK signalling. This mechanism was also observed with the C. elegans homolog, NDK-1, albeit with an inverse effect on MAPK activation. Both NM23-H1 and NDK-1 promote apoptotic cell death. In addition, NDK-1, NM23-H1 and their mouse counterpart NM23-M1 were shown to promote phagocytosis in an evolutionarily conserved manner. In summary, inhibition of cell migration and proliferation, alongside actions in apoptosis and phagocytosis are all mechanisms through which NM23-H1 acts against metastatic progression.

The Effects of Different mTOR Inhibitors in EGFR Inhibitor Resistant Colon Carcinoma Cells.

Several monoclonal antibodies and inhibitors targeting signalling pathways are being used in personalised medicine. Anti-EGFR antibodies seem to be effective, however, therapy resistance often occurs in colon carcinoma cases. mTOR inhibitors (mTORIs) could have a potential role in the breakthrough of therapy resistance. The mTOR activity related protein expression patterns and the in vitro effects of EGFR inhibitors (EGFRIs), mTORIs and their combinations were studied in different colon carcinoma cell lines (with different genetic backgrounds). Alamar Blue test and flow cytometry were used to analyse the in vitro proliferation and apoptotic effects of cetuximab, gefitinib, cisplatin, rapamycin, PP242 and NVP-BEZ235. The expressions of mTOR activity related proteins (p-70S6K, p-S6, Rictor, p-mTOR, Raptor) were studied by Western blot, immunocytochemistry and Duolink staining. The EGFRI resistance of the studied colon carcinoma cell lines related to their known mutations were confirmed, neither gefitinib nor cetuximab inhibited the proliferation or induced apoptosis in vitro. Individual differences in Rictor and Raptor expressions were detected by Western blot and immunocytochemistry beside elevated mTOR activity of these different colon carcinoma cell lines. These expression patterns correlated to the mTORIs sensitivity differences, moreover, mTORIs could enhance the effects of EGFRIs and other in vitro treatments. Our results suggest that mTORI combinations could be helpful in both EGFRI and platinum-based therapy of colon carcinomas. Moreover, we suggest determining both mTOR complex activity and mutations in Akt/mTOR signalling pathways for selecting the appropriate mTORIs and patients in potential future combination treatments.

Expression of mTORC1/2-related proteins in primary and brain metastatic lung adenocarcinoma.

Brain metastases (BMs) are common complications of adenocarcinomas (ADCs) of the lung and are associated with a poor prognosis. Although an increasing amount of data indicates that dysregulated activity of mammalian target of rapamycin (mTOR) can influence the metastatic potential of various tumors, the role of mTOR complexes in the development of BMs from ADCs of the lung is largely unknown. To estimate mTOR activity, we studied the expression of mTOR-related proteins (mTORC1: p-mTOR, p-S6; mTORC2: p-mTOR, Rictor) in primary (n=67) and brain metastatic (n=67) lung ADCs, including 15 paired tissue samples, using immunohistochemistry and tissue microarrays. Correlation with clinicopathological parameters was also analyzed. Increased p-mTOR, p-S6, and Rictor expressions were observed in 34%, 33%, and 37% of primary ADCs and in 79%, 70%, and 66% of BMs, respectively. Expression of these markers was significantly higher in BMs as compared with primary carcinomas (P<.0001, P<.0001, P<.001). Rictor expression was significantly higher in primary ADCs of the paired cases with BMs as compared with primary ADCs without BMs (67% versus 28%; P<.01). No other statistically significant correlations were found between mTOR activity and clinicopathological parameters. The increased mTORC1/C2 activity in a subset of pulmonary ADCs and the higher incidence of increased mTORC1/C2 activity in BMs suggest that the immunohistochemistry panel for characterizing mTOR activity and its potential predictive and prognostic role warrants further investigations.

mTOR activity and its prognostic significance in human colorectal carcinoma depending on C1 and C2 complex-related protein expression.

AIMS: Tumour heterogeneity and altered activation of signalling pathways play important roles in therapy resistance. The PI3K/Akt/mTOR signalling network is a well-known regulator of several functions that contribute to tumour growth. mTOR exists in two functionally different multiprotein complexes. We aimed to determine mTOR activity-related proteins in clinically followed, conventionally treated colon carcinomas and to analyse the correlation between clinical data and mTORC1 and mTORC2 activity. METHODS: Immunohistochemistry was performed with different antibodies on tissue microarray blocks from 103 patients with human colorectal adenocarcinoma. mTORC1- and mTORC2-related activity were scored on different stainings including analysis of the expression of Raptor and Rictor-specific elements of mTORC1 and C2 complexes. The staining scores and clinical/survival data were compared and analysed. RESULTS: Detailed characterisation showed stage and grade independent high mTOR activity in 74% of cases. High mTOR activity was present in mTORC1 and/or mTORC2 complexes; >60% of cases had mTORC2-related high mTOR activity. Based on our analysis, high mTOR activity and Rictor overexpression could be markers of a bad prognosis. Combined phosphoprotein and Rictor/Raptor expression evaluation revealed even stronger statistical correlation with prognosis. CONCLUSIONS: The presented staining panel could be appropriate and highly recommended for the accurate specification of mTORC1 and C2 activity of tumour tissues. This could help in the selection of mTOR inhibitors and can provide information about prognosis, which may guide decisions about the intensity of therapy.

Rapamycin can restore the negative regulatory function of transforming growth factor beta 1 in high grade lymphomas.

TGF-ß1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-ß1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-ß signaling, and through these pathways it is able to enhance TGF-ß induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-ß1 combination on cell growth and on TGF-ß and mTOR signalling events in lymphoma cells. Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-ß1 induced apoptosis and restored TGF-ß1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-ß sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin+TGF-ß1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin+TGF-ß1 induced apoptosis was not completely eliminated by a caspase inhibitor. These results suggest that high mTOR activity contributes to TGF-ß resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-ß1.

Expression of certain leukemia/lymphoma related microRNAs and its correlation with prognosis in childhood acute lymphoblastic leukemia.

In spite of the improved efficacy of therapy, it still fails in 15-20 % of childhood acute lymphoblastic leukemia (ALL) patients. Recently, altered expression of certain miRNAs (miRs) have been described in ALL with potential effect on prognosis. Presence of certain miRs (miRNA-16, -21, -24, -29b, -128b, -142-3p, -155, -223) was characterized in human lymphoma and leukemia cells by real-time PCR. Expression of miRs in pediatric ALL patients (n = 24) was measured before chemotherapy, at conventional response checkpoints and at relapse. Correlation between altered miR expression and response to prednisolone at day 8 of therapy and long term prognosis was statistically analysed. Overexpression of "oncomiR/inflammamiR"-21 - which is characteristic in different tumors-was missing in human ALL cells. However, higher expression of miR-128b and lower expression of miR-223 is generally characteristic for human ALL cell lines and ALL cells isolated from pediatric patients. Correlation was shown between miR-128b expression and prognosis, prednisolone response and survival data in childhood ALL. Expression of miR-128b and miR-223-both are leukemia specific-changed in parallel with percentage of bone marrow blasts in remission and during relapse. Therefore, we suggest that overexpression of miR-128b and downregulation of miR-223 shows a significant correlation with treatment response and prognosis in childhood ALL.

Major partial response to crizotinib, a dual MET/ALK inhibitor, in a squamous cell lung (SCC) carcinoma patient with de novo c-MET amplification in the absence of ALK rearrangement.

The initial radiotherapy of a 73 years old Caucasian male patient with advanced squamous cell lung carcinoma was terminated due to severe pericarditis. Subsequently, the tumor sample was analyzed for possible targets with comprehensive molecular diagnostics. EGFR, KRAS and PIK3CA genes were wild type, ALK and ROS1 were negative for rearrangement, but c-MET was amplified by fluorescent in situ hybridization. The kinase inhibitor crizotinib is already in clinical use for the treatment of ALK positive non-small cell lung cancers, but it is also known to be a potent c-MET inhibitor. The patient was treated with the standard dose of twice a day 250 mg crizotinib as a monotherapy. Major partial response to therapy was confirmed by chest CT and PET/CT after 8 weeks on therapy. C-MET expression is associated with poor prognosis and resistance to EGFR inhibitors. This case may indicate that c-MET tyrosine kinase inhibitors can be an effective targeted treatment option for squamous cell carcinoma patients, and future clinical trials should be expanded for this patient group as well.

Characteristic mTOR activity in Hodgkin-lymphomas offers a potential therapeutic target in high risk disease--a combined tissue microarray, in vitro and in vivo study.

BACKGROUND: Targeting signaling pathways is an attractive approach in many malignancies. The PI3K/Akt/mTOR pathway is activated in a number of human neoplasms, accompanied by lower overall and/or disease free survival. mTOR kinase inhibitors have been introduced in the therapy of renal cell carcinoma and mantle cell lymphoma, and several trials are currently underway. However, the pathological characterization of mTOR activity in lymphomas is still incomplete. METHODS: mTOR activity and the elements of mTOR complexes were investigated by immunohistochemistry on tissue microarrays representing different human non-Hodgkin-lymphomas (81 cases) and Hodgkin-lymphomas (87 cases). The expression of phospho-mTOR, phospho-4EBP1, phospho-p70S6K, phospho-S6, Rictor, Raptor and Bcl-2, Bcl-xL, Survivin and NF-kappaB-p50 were evaluated, and mTOR activity was statistically analyzed along with 5-year survival data. The in vitro and in vivo effect of the mTOR inhibitor rapamycin was also examined in human Hodgkin-lymphoma cell lines. RESULTS: The majority (>50%) of mantle cell lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma and Hodgkin-lymphoma cases showed higher mTOR activity compared to normal lymphoid tissues. Hodgkin-lymphoma was characterized by high mTOR activity in 93% of the cases, and Bcl-xL and NF-kappaB expression correlated with this mTOR activity. High mTOR activity was observed in the case of both favorable and unfavorable clinical response. Low mTOR activity was accompanied by complete remission and at least 5-year disease free survival in Hodgkin-lymphoma patients. However, statistical analysis did not identify correlation beetween mTOR activity and different clinical data of HL patients, such as survival. We also found that Rictor (mTORC2) was not overexpressed in Hodgkin-lymphoma biopsies and cell lines. Rapamycin inhibited proliferation and induced apoptosis in Hodgkin-lymphoma cells both in vitro and in vivo, moreover, it increased the apoptotic effect of chemotherapeutic agents. CONCLUSIONS: Targeting mTOR activity may be a potential therapeutic tool in lymphomas. The presence of mTOR activity probably indicates that the inclusion of mTOR inhibition in the therapy of Hodgkin-lymphomas may be feasible and beneficial, especially when standard protocols are ineffective, and it may also allow dose reduction in order to decrease late treatment toxicity. Most likely, the combination of mTOR inhibitors with other agents will offer the highest efficiency for achieving the best clinical response.

Mammalian target of rapamycin (mTOR) activity dependent phospho-protein expression in childhood acute lymphoblastic leukemia (ALL).

Modern treatment strategies have improved the prognosis of childhood ALL; however, treatment still fails in 25-30% of patients. Further improvement of treatment may depend on the development of targeted therapies. mTOR kinase, a central mediator of several signaling pathways, has recently attracted remarkable attention as a potential target in pediatric ALL. However, limited data exists about the activity of mTOR. In the present study, the amount of mTOR activity dependent phospho-proteins was characterized by ELISA in human leukemia cell lines and in lymphoblasts from childhood ALL patients (n = 49). Expression was measured before and during chemotherapy and at relapses. Leukemia cell lines exhibited increased mTOR activity, indicated by phospho-S6 ribosomal protein (p-S6) and phosphorylated eukaryotic initiation factor 4E binding protein (p-4EBP1). Elevated p-4EBP1 protein levels were detected in ALL samples at diagnosis; efficacy of chemotherapy was followed by the decrease of mTOR activity dependent protein phosphorylation. Optical density (OD) for p-4EBP1 (ELISA) was significantly higher in patients with poor prognosis at diagnosis, and in the samples of relapsed patients. Our results suggest that measuring mTOR activity related phospho-proteins such as p-4EBP1 by ELISA may help to identify patients with poor prognosis before treatment, and to detect early relapses. Determining mTOR activity in leukemic cells may also be a useful tool for selecting patients who may benefit from future mTOR inhibitor treatments.

Activity and complexes of mTOR in diffuse large B-cell lymphomas--a tissue microarray study.

Diffuse large B-cell lymphoma is a heterogeneous group of diseases with different responses to therapy. Targeting mTOR (mammalian target of rapamycin) offers a new approach to improve the treatment. mTOR inhibitors are being developed and are in clinical use in mantle cell lymphoma therapy and clinical trials are ongoing in other high-grade lymphomas as well. However, there is limited data about mTOR activity and the expression of its different complexes in diffuse large B-cell lymphomas. Tissue microarray blocks were constructed from paraffin-embedded biopsy specimens. More than 700 immunohistochemical stainings (mTOR signaling-related proteins and phosphoproteins, markers for lymphoma classification) were evaluated from 68 diffuse large B-cell lymphoma biopsies from conventionally treated and followed patients. Approximately 30% of cases were characterized as germinal center-derived diffuse large B-cell lymphomas, which showed virtually no mTOR activity, as determined by phospho-ribosomal S6 expression, the most sensitive marker of mTOR activity. In about 80% of non-germinal center-derived diffuse large B-cell lymphoma cases, positivity of mTOR-related phosphoproteins was observed, denoting mTOR activity. Moreover, Rictor (a characteristic protein of the mTOR complex2) was overexpressed in 43% of all diffuse large B-cell lymphomas and in 63% of mTOR-active non-germinal center diffuse large B-cell lymphoma samples. Rictor overexpression with mTOR activity indicated significantly worse survival for patients than mTOR inactivity or mTOR activity with low Rictor expression. These results suggest that mTOR activity is characteristic in most non-germinal center-derived diffuse large B-cell lymphomas with potentially variable mTOR-inhibitor sensitivity. Taken together, mTOR inhibitors may be useful in addition to regular therapy in diffuse large B-cell lymphomas, however, patient and inhibitor selection criteria must be carefully considered.

Denosumab--a powerful RANKL inhibitor to stop lytic metastases and other bone loss actions by osteoclasts.

Denosumab is a perfect example on the targeted anticancer therapy. The inhibition of RANKL activity suppressed the osteoclasts' resorptive function and so prevented skeletal related events. This effect is useful not only against bone metastases, but also in the treatment of other diseases caused by bone loss. In different solid tumors with bone metastasis the quality of life also improved, although the overall survival usually showed no change. On the market the main competitors for denosumab are still the bisphosphonates (questions of costs and reimbursement are not discussed) and some potential new agents e.g. Src kinases (as dasatinib, saracatinib, bosutinib), cathepsin K inhibitors, (e.g. odanacatib), and new selective estrogen receptor modulators (e.g. bazedoxifene, lasofoxifene). Nevertheless, today denosumab is one of the most powerful agents in bone-saving area.

[mTOR complexes -- molecular spiders in molecular networks]. / mTOR-komplexek -- molekuláris pókok a molekuláris hálókban.

PI3K route is one of the most outstanding signal transduction pathways, which has a key role in the decision-making processes and functions of a cell. In this network mTOR (mammalian target of rapamycin) is a well-known integrator. mTOR forms two complexes, and their increased activity is present in many human tumors. Therefore, mTOR inhibitors became more and more important in the targeted therapy, first of all in the treatment of renal cancer, neuroendocrine pancreatic cancer and certain astrocytomas, and many trials are going on in other tumor types. The therapeutic results are obvious, but problems also occur, which lead to new strategies and to the development of new drugs in order to create more individualised cancer therapy.

Epidermal growth factor receptor signalling contributes to osteoblastic stromal cell proliferation, osteoclastogenesis and disease progression in giant cell tumour of bone.

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.

Mitotic lymphoma cells are characterized by high expression of phosphorylated ribosomal S6 protein.

Growth factors and mitogens influence signaling pathways and often induce the activity of p70S6 kinase (p70S6K), which in turn phosphorylates the ribosomal S6 protein (S6). Although recent data are rather conflicting, the overall view suggests that phosphorylated S6 is a regulator of global protein synthesis, cell proliferation, cell size and glucose homeostasis. In the present work, emphasis was given to cell cycle-dependent activation of S6 focusing mainly on human lymphoid and lymphoma cells. Paraffin-embedded human tissue blocks from lymph node and different tumor biopsies as well as in vitro cell lines were investigated by immunohistochemistry, immunocytochemistry, flow cytometry and Western blotting using antibodies directed against phospho-S6, phospho-mTOR, phospho-p70S6K and phospho-Histone H3. To enrich the cell number in different phases of the cell cycle, nocodazole, staurosporine or rapamycin were used in cell cultures. We observed strong phospho-S6 positivity by immunostainings in the dividing lymphoid cells of reactive lymph nodes and in lymphoma cells cultured in vitro. Phospho-S6 protein levels were shown to be elevated throughout mitosis in lymphoma cells; however, the high expression of phospho-S6 in mitotic cells was not a general hallmark of tumor cell types studied so far: phospho-S6-negative mitotic cells were detected in several carcinoma and sarcoma biopsies. These observations may have practical implications as they raise the possibility to consider p70S6K and/or S6 as a potential therapeutic target-besides mTOR-in certain lymphomas and perhaps in clinical immunosuppression.

Integrating molecular diagnostics into anticancer drug discovery.

In the 1990s, the breast cancer drug trastuzumab (Herceptin; Genentech/Roche)--an antibody specific for human epidermal growth factor receptor 2 (HER2; also known as ERBB2)--was approved based on trials in which HER2 expression levels were used to select patients in clinical trials. This provided support for analogous efforts for drugs that target the epidermal growth factor receptor (EGFR). However, the development of these drugs, such as cetuximab (Erbitux; Bristol-Myers Squibb/Lilly) and gefitinib (Iressa; AstraZeneca), has revealed that EGFR expression is an insufficient and unreliable biomarker to select patients for EGFR-targeted therapies in both lung and colon cancer. Indeed, evidence on patient populations that are likely to respond to such therapies, on the basis of specific mutations in proteins of the targeted pathway, has only recently been clinically validated and incorporated into some of the drug labels. This article highlights lessons learned from the development of the first drugs targeting the EGFR family and discusses strategies to decrease the risk of failure in clinical development by more effectively integrating molecular diagnostics into anticancer drug discovery.

Panitumumab: an arrow on target.

Several options are available today in the treatment of advanced colorectal cancer: traditional chemotherapeutic regimens, targeted therapies, and their combinations. Panitumumab is a new, fully human anti-EGFR monoclonal antibody, what is well-tolerated, effective as a single agent in chemotherapy refractory patients and in different combinations. The clinical response is restricted to tumors with wild-type RAS, therefore the RAS status should be checked before treatment.

High resolution melting curve analysis of DNA sequence alterations of various sizes.

In the diagnostic workflow we need to think in algorithms, containing more assays. One of the most important task in the management of cancer patient is to detect nucleic acid sequence changes in clinical specimens. Before using the most expensive method to analyze direct our targets, a screening assay is needed to reduce the number of samples. In the detection of gene-sequence alterations classical screening methods are available, as SSCP, DGGE or TGGE, (Finke Exp Clin Endocrinol Diabetes 104:92-97, 1996; Lessa and Applebaum Mol Ecol 2:119-129, 1993) however these are very time consuming processes. At this time in the molecular lab the real-time PCR equipments are very popular and with the function of melting curve analysis it can be a very convenient, simple and cost-efficient screening method.

Centrosome abnormalities in giant cell tumour of bone: possible association with chromosomal instability.

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.