IDH mutations in G2-3 conventional central bone chondrosarcoma: a mono institutional experience.

BACKGROUND: Heterozygous isocitrate dehydrogenase (IDH) mutations occur in about half of conventional central bone chondrosarcomas (CCBC). Aim of this study was to assess the frequency and prognostic impact of IDH mutations in high grade CCBC patients. METHODS: 64 patients with G2 and G3 CCBC were included. DNA extraction, PCR amplification of IDH1/2 exon 4s, and sequencing analysis with Sanger were performed. RESULTS: IDH mutations were detected in 24/54 patients (44%): IDH1 in 18, IDH2 in 4, and both IDH1/2 in 2 patients. The frequency of mutations was 37% in G2 vs. 69% in G3 (p = 0.039), and 100% in three Ollier disease associated chondrosarcoma. 5-year overall survival (OS) at 124 months (range 1-166) was 51%, with no significant difference based on the IDH mutational status: 61% in IDHmut vs. 44% in IDH wild type (IDHwt). The 5-year relapse free survival (RFS) was 33% (95% CI:10-57) for IDHmut vs. 57% (95%CI: 30-77) for IDHwt. Progression free survival (PFS) was 25% (95%CI:1-65) IDHmut vs. 16% (95%CI: 0.7-52) IDHwt. 55% (5/9) of IDHmut G2 became higher grade at the recurrence, as compared with 25% (3/12) of G2 IDHwt. CONCLUSIONS: This study shows a higher frequency of IDH mutations in G3 CCBC as compared with G2. No significant differences in OS, RFS, and PFS by mutational status were detected. After relapse, a higher rate of G3 for IDH mutated CCBC was observed.

Primary extraskeletal myxoid chondrosarcoma of bone: Report of three cases and review of the literature.

Extraskeletal myxoid chondrosarcoma is a rare neoplasm of soft tissue. The usual location is in deep parts of the proximal extremities and limb girdles in middle-aged adults. The bone location as primary location is extremely rare and few cases are reported. We present three cases arising in bone with molecular confirmation using both RT-PCR and FISH analysis. Patients include two men and one woman with an age of 62, 69 and 73 years old. The mean size of the lesion was 13cm (range 8-18cm). Tumors arose in the iliac bone in two cases and in the proximal humerus in the other case. At time of diagnosis the three cases show bone cortex and soft tissue involvement. On imaging, lesions have a lobular pattern, are purely lytic, but take up contrast medium after injection. Two patients are alive with disease (local recurrence and lung metastasis) after five years and five years and six months, respectively and one patient died of disease two years after the diagnosis. The primary extraskeletal myxoid chondrosarcoma of bone seems to have a more aggressive behavior than the soft tissue counterpart. The molecular confirmation of diagnosis using RT-PCR is necessary to do the differential diagnosis with other entities, in particular with myoepithelioma that shows similar morphological features and EWSR1 and FUS genes rearrangement.

Sclerosing epithelioid fibrosarcoma of the thigh: report of two cases with synchronous bone metastases.

We report two cases of sclerosing epithelioid fibrosarcoma occurring in the deep soft tissue of the thigh, confirmed by molecular analysis and associated with bone metastases in the lumbar vertebrae and the iliac wing at the time of diagnosis. Synchronous bone metastases of sclerosing epithelioid fibrosarcoma are extremely difficult to diagnose because clinical and radiological features are not specific. In addition, the range of differential diagnoses is very wide, including metastatic carcinoma and osteosarcoma. At present, all but three published cases of sclerosing epithelioid fibrosarcoma with bone metastases showed bone metastases during follow-up. We confirm in our two cases that the distinct pattern of immunohistochemical staining for MUC4, associated with the absence of staining for both SATB2, a marker of osteoblastic differentiation, and pan-cytokeratin, allows differentiating between sclerosing epithelioid fibrosarcoma and metastatic carcinoma or osteosarcoma.

Primary malignant myoepithelioma of the distal femur.

A 55-year-old male presented with a 1-month history of localized pain caused by an osteolytic and destructive lesion in the right distal femur. Histologically, the tumour consisted of spindle cells intermingled with epithelioid eosinophilic cells arranged in small cords embedded in a hyalinized-to-chondromyxoid stroma. Electron microscopy and immunohistochemistry showed features of myoepithelial differentiation. RT-PCR failed to demonstrate chimeric transcripts of extraskeletal myxoid chondrosarcoma. The final diagnosis was primary malignant myoepithelioma of bone. The patient is alive with lung metastases 13 months after surgery. Primary malignant myoepithelioma of bone is an exceptionally rare neoplasm that should be considered in the differential diagnosis with the more aggressive myxoid spindle cell sarcomas.

CD99 isoforms dictate opposite functions in tumour malignancy and metastases by activating or repressing c-Src kinase activity.

CD99 gene encodes two distinct proteins, produced by alternative splicing of CD99 gene transcript. Full-length CD99 isoform (CD99wt) is formed by an extracellular domain, followed by a transmembrane domain and a 36 amino-acid intracytoplasmic domain, which is partially deleted in the truncated, short form (CD99sh). A differential expression of these two CD99 molecules can lead to distinct functional outcomes in lymphocytes. To investigate the functional effects of CD99 molecules on malignancy, forced overexpression of the two CD99 isoforms was induced in osteosarcoma and prostate cancer cells. The two isoforms exhibited opposite functions: the major form dramatically inhibits anchorage-independent growth, anoikis resistance, migration and metastasis, whereas the CD99sh remarkably favours the phenomena. A mechanistic analysis of CD99-transfected osteosarcoma cells points to involvement of c-Src family kinase activity in regulating CD99 functions in malignancy. Ser168 residue of CD99 plays a pivotal role in the reversion of the malignant phenotype. Our findings highlight the involvement of CD99 in crucial processes of cancer malignancy, serving as a curtain raiser for this, so far neglected molecule. In addition, a dualistic role for the two CD99 isoforms was shown in agreement with what was observed for other cell adhesion molecules.

The molecular mechanisms responsible for resistance to ET-743 (Trabectidin; Yondelis) in the Ewing's sarcoma cell line, TC-71.

Identification of new active agents against sarcoma is considered an important challenge in medical oncology. ET-743 (Trabectidin; Yondelis) has recently emerged as the first active drug developed against sarcoma in the last two decades, with promising results especially against soft-tissue sarcoma and Ewing's sarcoma (ES). In this study, we analyzed the molecular mechanisms responsible for resistance to ET-743 in ES cells. Three resistant cell variants (TC/ET 3 nM, TC/ET 6 nM and TC/ET 12 nM) were obtained, showing 28-, 47- and 102-fold increase in ET-743 resistance. Cross-resistance to other drugs was analyzed. Comparative genomic hybridization and cDNA microarray technology were employed to characterize and compare the gene expression profile of two TC/ET variants with the parental cell line. TC/ET cells show a conventional multidrug resistance phenotype and P-glycoprotein overexpression was found to significantly contribute to ET-743 resistance. However, functional studies with the cyclosporine analogue, PSC-833, indicate that other mechanisms are involved in resistance to ET-743. The gene expression profile of TC/ET cells indicated, among up-regulated genes, an increase in expression of insulin-like growth factor receptor-I (IGF-IR) and one of its major intracellular mediators, insulin receptor substrate-1. Functional studies using a neutralizing antibody anti-IGF-IR confirmed involvement of this signaling pathway in resistance to ET-743. Simultaneous blockage of both P-glycoprotein and IGF-IR completely restored sensitivity to ET-743 in ES cells. Overall, these findings provide impetus for future studies testing the therapeutic value of new specific inhibitors of P-glycoprotein and IGF-IR, which could represent a concrete therapeutic option for ES patients refractory to conventional agents.

Effectiveness of Type I interferons in the treatment of multidrug resistant osteosarcoma cells.

P-glycoprotein overexpression is an important adverse prognostic marker for osteosarcoma (OS) patients, which is associated with higher risk for developing metastases as a consequence of the limited responsiveness to standard treatments of P-glycoprotein overexpressing OS cells. The use of cytokines has been advocated as a possible therapeutic approach to overcome multidrug resistance (MDR), being active on cell lines that are resistant to conventional drugs. In this study, we evaluated in vitro effects of interferons (IFNs) on MDR P-glycoprotein overexpressing OS cells. Type I IFNs, but not IFNgamma, showed tangible inhibitory effects on OS cell growth, which were higher in MDR cell lines compared to parental cells. The higher sensitivity of P-glycoprotein overexpressing cells to Type I IFNs correlates with higher expression of the activator of the transcription (STAT)-2 and (STAT)-3, two intracellular mediators of the IFNalpha and IFNbeta signaling pathways, whereas no differences were observed with respect to the expression or activation of the Type I IFN receptor and STAT-1. Exposure of OS MDR cells to Type I IFN decreased the expression of P-glycoprotein. This effect resulted in a significantly increased chemosensitivity of MDR cells to doxorubicin. Therefore, our data support the use of IFNalpha or IFNbeta in the treatment of osteosarcoma patients who overexpress P-glycoprotein in their primary tumors, and respond insufficiently to current therapeutic regimens.

Analysis of dihydrofolate reductase and reduced folate carrier gene status in relation to methotrexate resistance in osteosarcoma cells.

BACKGROUND: To evaluate the impact of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes on methotrexate (MTX) resistance in osteosarcoma cells in relation to retinoblastoma (RB1) gene status. MATERIALS AND METHODS: A series of human osteosarcoma cell lines-either sensitive or resistant to MTX-and 16 osteosarcoma tumour samples were used in this study. RESULTS: In U-2OS MTX-resistant variants, and in other RB1-positive cell lines, MTX resistance was associated with increased levels of DHFR and with a slight decrease of RFC gene expression. In Saos-2 MTX-resistant variants, and in another RB1-negative cell line, development of MTX resistance was associated with a decrease in expression of RFC, without any significant involvement of DHFR. In osteosarcoma clinical samples, amplification of the DHFR gene at clinical onset appeared to be more frequent in RB1-positive compared with RB1-negative tumours. CONCLUSIONS: Amplification of the DHFR gene may occur more frequently in the presence of RB1-mediated negative regulation of its activity and can be present at clinical onset in osteosarcoma patients. Simultaneous evaluation of RFC, DHFR and RB1 gene status at the time of diagnosis may become the basis for the identification of potentially MTX-unresponsive osteosarcoma patients, who could benefit from treatment protocols with alternative antifolate drugs.

Impact of IGF-I/IGF-IR circuit on the angiogenetic properties of Ewing's sarcoma cells.

The insulin-like growth factor receptor I (IGF-I)-mediated circuit is a major autocrine loop for Ewing's sarcoma (ES) cells, and plays a role the pathogenesis and malignancy of this tumor. IGF-I receptor (IGF-IR) has emerged as a good therapeutic site for ES patients. In this study, we analyzed the impact of strategies targeting the IGF-IR on the regulation of VEGFs, which are of fundamental importance in angiogenesis, and TGFbeta, CTGF and Cyr61, which are factors primarily involved in skeletal growth control and angiogenesis. IGF-I increases expression of VEGF-A, TGFbeta, CTGF and Cyr61 mRNA. However, only the modulation of VEGF-A expression appears to be mediated by IGF-IR. Functional assays on endothelial cells indicate a strict correlation between survival and proliferation of HUVECs and VEGF-A levels, confirming a major role for this factor in angiogenesis. Blockage of IGF-IR functions by neutralizing antibody or antisense strategies significantly reduced the expression and secretion of VEGF-A by ES cells, and supernatants of treated cells were unable to sustain the survival and proliferation of HUVECs. Analysis of the signaling mechanisms involved in constitutive or IGF-induced expression and secretion of VEGF-A indicated that PI3-K and MAPK signaling pathways are both required for VEGF expression and production in ES cells. Selective inhibitors LY294002 or PD98059 were highly effective in reducing the ability of ES cells to produce VEGF-A and stimulate survival and proliferation of HUVECs. Taken together, these findings add a new activity to the IGF-I repertoire in ES and highlight how disruption of IGF-IR functions may constitute an effective tool for the control of neovascularization in this tumor.

Structure-based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism.

The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined using synchrotron X-ray diffraction data from a vitrified crystal (1.85 A resolution, 99.3% completeness, data redundancy 4.6, R-factor 17.3%, PDB code 6UBP). A distance of 3.5 A separates the two Ni ions in the active site. The binding mode of the inhibitor involves the formation of four coordination bonds with the two Ni ions: one phosphate oxygen atom symmetrically bridges the two metal ions (1.9-2.0 A), while two of the remaining phosphate oxygen atoms bind to the Ni atoms at 2.4 A. The fourth phosphate oxygen is directed into the active site channel. Analysis of the H-bonding network around the bound inhibitor indicates that phosphate is bound as the H2PO4- anion, and that an additional proton is present on the Odelta2 atom of Asp(alpha363), an active site residue involved in Ni coordination through Odelta1. The flexible flap flanking the active site cavity is in the open conformation. Analysis of the complex reveals why phosphate is a relatively weak inhibitor and why sulfate does not bind to the nickels in the active site. The implications of the results for the understanding of the urease catalytic mechanism are reviewed. A novel alternative for the proton donor is presented.

Purification, crystallization and preliminary X-ray analysis of an acetylxylan esterase from Bacillus pumilus.

The gene encoding for acetylxylan esterase from Bacillus pumilus has been cloned and expressed in Escherichia coli. The recombinant protein has been purified to homogeneity and crystallized. The crystals obtained are of regular shape of dimensions 0.05 x 0.05 x 0.05 mm with R32 symmetry and diffract to 2.0 A using synchrotron radiation.

Simultaneous paired analysis of numerical chromosomal aberrations and DNA content in osteosarcoma.

Relatively little is known about the biologic relevance of numerical chromosomal changes in relation to DNA content in osteosarcoma. In this study, by using a series of human osteosarcoma cell lines, we standardized a method for the assessment, on the same nuclei specimen, of both specific chromosome copy numbers by fluorescence in situ hybridization (FISH) and the DNA content by static cytofluorometry or image cytometry. On the same cell lines, we also evaluated the DNA content by using flow cytometry and the chromosome number distribution by metaphase analysis. Comparison between these different methods showed that DNA ploidy level as determined by FISH or metaphase analysis is frequently lower than the ploidy pattern as defined by cytometric methods. By using comparative genomic hybridization, we were able to demonstrate that these discrepancies were due to the presence of several unbalanced chromosome aberrations, specifically gains and high-level amplifications, which affect the total DNA content with less effect on the total chromosome number. Thus, evaluation of DNA ploidy in osteosarcoma cells is needed for a correct interpretation of FISH or cytogenetic data concerning numerical chromosomal changes. Evaluation of tumor ploidy in a series of clinical samples demonstrated that in high-grade osteosarcoma, flow cytometry sometimes may give false results because of the presence of high proportions of contaminating, nonneoplastic cells that cannot be excluded from the flow cytometric assessment but that do not interfere with the evaluation of DNA ploidy by static cytofluorometry or image cytometry, in which only tumor cells are selected for the analysis. The possibility of using this method to evaluate, on the same nuclei sample, both specific chromosomal aberrations and DNA ploidy may allow a better determination of numerical chromosomal changes that may be relevant for the biologic behavior of osteosarcoma.

Inhibition of insulin-like growth factor I receptor increases the antitumor activity of doxorubicin and vincristine against Ewing's sarcoma cells.

Innovative treatment modalities are needed for Ewing's sarcoma (ES), a neoplasm with a disappointingly low survival rate despite the use of aggressive multimodal therapeutic approaches. We and others (D. Yee et al., J. Clin. Investig., 86: 1806-1814, 1990; K. Scotlandi et al., Cancer Res., 56: 4570-4574, 1996) have previously shown the existence and the pathogenetic relevance of an autocrine loop, mediated by the insulin-like growth factor-I receptor (IGF-IR), which is crucial for survival and proliferation of ES cells in vitro. Moreover, we reported that the IGF-IR-blocking monoclonal antibody (MAb), alphaIR3, as well as suramin, a drug that can interfere with growth factor by binding to the receptors, inhibited both the tumorigenic and the metastatic ability of ES cells in athymic mice. In this study, we analyzed whether agents that can block the IGF-IR-mediated loop are of value in association with conventional cytotoxic drugs for the design of more effective therapeutic regimens. Both alphaIR3 MAb and suramin treatment significantly increased the antitumor in vitro effects of doxorubicin and vincristine, two drugs with a leader action on ES. These findings were obtained by both simultaneous and sequential treatments. Analysis of the proliferation rate and of apoptosis revealed that alphaIR3 MAb and suramin significantly enhanced the G(1)-phase rate induced by doxorubicin, without substantially affecting doxorubicin-G(2)-M-blockage of cell cycle, and significantly increased the induction of apoptosis, which confirmed that the specific blockage of IGF-IR deprives ES cells of an important tool for the prevention of drug-induced apoptosis. Moreover, combination treatments of doxorubicin plus alphaIR3 MAb significantly increase the doxorubicin-induced impairment of the ability of ES cells to form colonies in soft agar. In conclusion, we showed that, in ES, the blockage of IGF-IR by a neutralizing MAb or by suramin may greatly potentiate the antitumor activity of conventional chemotherapeutic drugs.

Crystal structure of oxidized Bacillus pasteurii cytochrome c553 at 0.97-A resolution.

This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolated from a Gram-positive bacterium. Bacillus pasteurii cytochrome c(553), characterized by a low reduction potential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes, is a useful case study for understanding the structure-function relationships for this class of electron-transfer proteins. Diffraction data on a single crystal of cytochrome c(553) were obtained using synchrotron radiation at 100 K. The structure was determined at 0.97-A resolution using ab initio phasing and independently at 1.70 A in an MAD experiment. In both experiments, the structure solution exploited the presence of a single Fe atom as anomalous scatterer in the protein. For the 0.97-A data, the phasing was based on a single data set. This is the most precise structure of a heme protein to date. The crystallized cytochrome c(553) contains only 71 of the 92 residues expected from the intact protein sequence, lacking the first 21 amino acids at the N-terminus. This feature is consistent with previous evidence that this tail, responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purification procedure. The heme prosthetic group in B. pasteurii cytochrome c(553) is surrounded by three alpha-helices in a compact arrangement. The largely exposed c-type heme group features a His-Met axial coordination of the Fe(III) ion. The protein is characterized by a very asymmetric charge distribution, with the exposed heme edge located on a surface patch devoid of net charges. A structural search of a representative set of protein structures reveals that B. pasteurii cytochrome c(553) is most similar to Pseudomonas cytochromes c(551), followed by cytochromes c(6), Desulfovibrio cytochrome c(553), cytochromes c(552) from thermophiles, and cytochromes c from eukaryotes. Notwithstanding a low sequence homology, a structure-based alignment of these cytochromes shows conservation of three helical regions, with different additional secondary structure motifs characterizing each protein. In B. pasteurii cytochrome c(553), these motifs are represented by the shortest interhelix connecting fragments observed for this group of proteins. The possible relationships between heme solvent accessibility and the electrochemical reduction potential are discussed.

CD99 engagement: an effective therapeutic strategy for Ewing tumors.

CD99 is a Mr 32,000 transmembrane molecule that shows a high level of expression on cells of the hemopoietic system as well as on Ewing tumor cells. Within the hematopoietic system, CD99 has been implicated in cell adhesion and cell death, participating in this way in the differentiation of T-cell precursors. In this study, we demonstrate that engagement of CD99 significantly inhibits the in vitro and in vivo growth ability of Ewing tumor cells by delivering an apoptotic stimulus and reducing the malignant potential of these cells. Moreover, we show that anti-CD99 monoclonal antibodies may be advantageously used in association with conventional anticancer agents. These results provide a novel entry site for therapeutic intervention, which may have application in the care of patients with Ewing tumor, and warrant additional studies to clarify the molecular mechanisms activated by CD99 engagement.

Identification of EWS/FLI-1 transcripts in giant-cell tumor of bone.

The EWS/FLI-1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing's sarcoma. We demonstrate the presence of EWS/FLI-1 hybrid transcripts also in giant-cell tumor, a bone neoplasm featuring intermediate characteristics between benign and malignant lesions. Chimeric products were detected by semi-nested PCR after 2 cycles of amplification in 13/15 cases of giant-cell tumor, and their presence was confirmed by Southern and Western blots and fluorescence in situ hybridization. Moreover, 3/8 primary cultures of giant-cell tumor showed the same type of hybrid transcript observed in the original tumor sample. Sequencing of PCR products confirmed the presence of EWS and FLI-1 sequences in these products. Detection of EWS/FLI-1 fusion transcripts in giant-cell tumor of bone provides a model for the study of the transforming mechanisms of the EWS/FLI-1 fusion gene in mesenchymal tumors.

The complex of Bacillus pasteurii urease with acetohydroxamate anion from X-ray data at 1.55 A resolution.

The structure of Bacillus pasteurii urease inhibited with acetohydroxamic acid was solved and refined anisotropically using synchrotron X-ray cryogenic diffraction data (1.55 A resolution, 99.5% completeness, data redundancy = 26, R-factor = 15.1%, PDB code 4UBP). The two Ni ions in the active site are separated by a distance of 3.53 A. The structure clearly shows the binding mode of the inhibitor anion, symmetrically bridging the two Ni ions in the active site through the hydroxamate oxygen and chelating one Ni ion through the carbonyl oxygen. The flexible flap flanking the active site cavity is in the open conformation. The possible implications of the results on structure-based molecular design of new urease inhibitors are discussed.

Prognostic significance of nuclear accumulation of c-myc and mdm2 proteins in synovial sarcoma of the extremities.

We retrospectively analysed a group of primary tumours from 32 patients with synovial sarcomas of the extremities by using immunohistochemistry on paraffin-embedded tissue samples in order to investigate the prognostic significance of the nuclear accumulation of c-myc and mdm2 proteins. Nuclear positive immunostaining for c-myc or mdm2 proteins were revealed in 9/32 cases (28%) and in 6/30 cases (20%), respectively. Since c-myc protein appears to be a key factor for keeping cells in an active proliferative stage, we also analysed the growth compartment of each tumour by using the MIB1 monoclonal antibody, specific for Ki-67 antigen. A high MIB1 index was found in 8/31 cases (26%) but was not associated either with c-myc or mdm2 nuclear positivity. Analysis of clinical outcome was performed in a subgroup of 27 patients with a minimum follow-up of 24 months. Among the clinicopathologic parameters and the biological markers, only the nuclear accumulation of c-myc was significantly associated with a higher relapse rate (p = 0.03). Accordingly, survival analysis confirmed a trend toward a poor event-free survival rate and a worse outcome in c-myc-positive cases (p = 0.12). These data demonstrate that assessment of c-myc nuclear accumulation can be useful to identify high-risk subsets of patients with synovial sarcoma of the extremities.

Reversal of malignant phenotype in human osteosarcoma cells transduced with the alkaline phosphatase gene.

Alkaline phosphatases are a family of glycoproteins that are able to hydrolize various monophosphate esters at a high pH optimum. Liver/bone/kidney (L/B/K) alkaline phosphatase (ALP) is one of the four major isoenzymes that belong to this family. Apart from its role in normal bone mineralization, other functions of L/B/K ALP remain obscure, both in physiological and in neoplastic conditions, including the bone-forming tumor osteosarcoma. In this study, we transfected the U-2 OS osteosarcoma cell line, which does not show any basal expression of this enzyme, with the full-length gene of L/B/K ALP, and analyzed the in vitro and in vivo features of four transfectants showing different expression of L/B/K ALP. A reduced in vitro ability to invade Matrigel and to grow in a semi-solid medium, together with a lower tumorigenic and metastatic ability in athymic mice, was found to be associated with a high level of cell surface L/B/K ALP activity. Moreover, L/B/K ALP transfectants showed a reduced secretion of matrix metalloproteinase-9 enzyme. These findings indicate a loss of aggressiveness of osteosarcoma cells after the expression of L/B/K ALP on their surface and suggest a new role for this enzyme.

Murine model for skeletal metastases of Ewing's sarcoma.

Ewing's sarcoma shows a strong tendency to metastasize to the lungs or the skeleton, or both. A peculiar feature of the secondary involvement of bone with this tumor is that it may also appear in the absence of clinically evident lung metastases, both at clinical presentation and during the course of the disease. Although osseous metastases are critically relevant for prognosis, the pathogenesis of this peculiar feature of Ewing's sarcoma is poorly understood, partly due to the lack of appropriate experimental in vivo models. We show that the intravenous injection of TC-71 Ewing's sarcoma cells into athymic 4-5-week-old Crl/nu/nu (CD1) BR mice reproducibly colonizes specific sites of the skeleton in addition to the lungs and lymph nodes. The distribution and the morphologic appearance of these experimental bone metastases mimic the pattern of skeletal involvement observed in humans. This experimental model of bone metastasis of Ewing's sarcoma may be the basis for future studies aimed at understanding the pathophysiology and treatment of Ewing's sarcoma.