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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.

High-grade osteosarcoma is an extremely aggressive neoplasm, where over 80% of patients present with life-threatening micrometastases at diagnosis. Systemic control of the disease is therefore critical for the treatment of these patients and neoadjuvant chemotherapy using various drugs, including doxorubicin (DXR), which has been demonstrated to be the most effective regimen. Multidrug resistance (MDR) to some anticancer agents, including DXR, mediated by the MDR1 gene product P-glycoprotein (Pgp), has been shown to be a major cause of chemotherapy failure in osteosarcoma. We analyzed the effect of a cyclosporine A derivate Valspodar (PSC 833) on MDR human osteosarcoma cells. We also evaluated Pgp expression in sporadic appendicular canine osteosarcoma. Moreover, dogs were treated with combined administration of DXR and PSC 833. Several blood samples were collected for the determination of DXR and PSC 833 levels. PSC 833 induced a complete reversal of the resistant phenotype at concentrations compatible with the clinical use. Pgp was present in 12/18 (66.6%) of the cases. At the time of DXR administration, adequate blood concentrations of PSC 833, to provide a complete MDR reversal, were obtained without clinical or laboratory findings of toxicity. Combination therapy with DXR and PSC 833 allowed a 30% decrease in DXR dose infusion with equivalent therapeutic exposure. The high incidence of Pgp expression in osteosarcoma confers to the study a rationale for an effective regimen based on down-modulation of MDR.

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.

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.

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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., (Copyright 2000 S. Karger AG, Basel)

With the aim of identifying innovative therapeutic strategies for osteosarcoma patients who are refractory to conventional chemotherapy, we analyzed the in vitro effects of the blockage of autocrine circuits. Since the insulin-like growth factor-I receptor (IGF-IR)-mediated loop is relevant to the growth of osteosarcoma, we analyzed the activity of the IGF-IR-blocking antibody alphaIR3 in both sensitive and multidrug-resistant osteosarcoma cell lines. Only limited effects, however, were observed, suggesting the simultaneous existence of other autocrine circuits. Indeed, in a representative panel of 12 human osteosarcoma cell lines, in addition to the IGF-IR-mediated circuit, we demonstrated also a loop mediated by epidermal growth factor receptor as well as the presence of nerve growth factor, low-affinity nerve growth factor receptor as well as tyrosine receptor kinase A in the great majority of osteosarcomas. Therapies based on the inhibition of single circuits may have only limited effects in osteosarcoma, whereas the use of suramin, a drug which, besides other activities, non-selectively interferes with the binding of growth factors to their receptors, appears as a promising alternative, in both sensitive and drug-resistant osteosarcoma cells.

The relationship between P-glycoprotein expression and malignancy is controversial. We have recently found that, in osteosarcoma, multidrug resistance (MDR) is associated with a less aggressive behavior, both in vitro and in clinical settings. In this study, we evaluated whether P-glycoprotein overexpression has a cause-effect relationship with the reduced metastatic potential of MDR cells, or rather reflects a more complex phenotype. MDR1 gene-transfected osteosarcoma cell clones, showing different levels of P-glycoprotein expression, were analysed for their in vitro characteristics and their tumorigenic and metastatic ability in athymic mice. Apart from the different levels of P-glycoprotein, no significant change in the expression of surface antigens or in the differentiative features were observed in the MDR1 gene transfectants compared to the parental cell lines or control clones, obtained by transfection with neo gene alone. In contrast to controls, however, MDR1 transfectants showed a significantly lower ability to grow in semi-solid medium and were completely unable to grow and give lung metastases in athymic mice. These findings indicate that P-glycoprotein overexpression is causally associated with a low malignant potential of osteosarcoma cells, and open new insights on the role and functions of P-glycoprotein activity.

Innovative, more effective 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 have previously shown (K. Scotlandi et al, Cancer Res., 56: 4570-4574, 1996) the existence and the pathogenetic relevance of an autocrine loop that is mediated by the insulin-like growth factor-I receptor (IGF-IR) and is crucial for the survival and proliferation of ES cells in vitro. In this study, we report that the IGF-IR-blocking monoclonal antibody alphaIR3 may also significantly inhibit ES cell growth in vivo. In particular, in almost one-half of the animals tested, after s.c. inoculation with TC-71 ES cells, the blockage of IGF-IR by alphaIR3 induced a complete regression of tumors that developed, which suggests that IGF-IR is valuable as a specific target for novel therapeutic strategies. In addition, suramin, a drug that can interfere with growth factor binding with their receptors, inhibited the tumorigenic and the metastatic ability of TC-71 cells and, therefore, is a promising agent to be combined with conventional cytotoxic drugs for the design of more effective therapeutic regimens.

The disappointingly low survival rate observed in Ewing's sarcoma (ES)/peripheral neuroectodermal tumor (PNET) despite the adoption of aggressive multimodal treatments prompted us to study the existence of autocrine circuits to be used as innovative therapeutic targets. Of the several circuits analyzed, only the insulin-like growth factor receptor (IGF-IR)-mediated loop was found to be constantly present both in cell lines and clinical samples, suggesting a role for this autocrine circuit in the pathogenesis of ES/PNET. The in vitro inhibition of the IGF-IR-mediated circuit by the specific IGF-IR binding antibody alphaIR3 suppressed the growth of ES/PNET cells by decreasing the proliferative rate and increasing apoptosis. alphaIR3 also significantly inhibited the ability of ES/PNET cells to grow in soft agar and to migrate following a chemotactic stimulus. Inactivation of the IGF-IR signaling pathway may therefore be considered as an effective therapeutic modality for patients with ES/PNET.

Overexpression of the hepatocyte growth factor receptor (Met/HGF receptor), a transmembrane tyrosine kinase encoded by the met proto-oncogene, has been associated with tumor progression in different human carcinomas. More recently, the Met/HGF receptor has also been described in tumor cell lines of mesenchymal origin, suggesting the existence of an autocrine loop that may contribute to the pathogenesis of sarcomas. In this study, we analyzed the expression of Met/HGF receptor by Western blotting and immunohistochemistry in frozen samples of 87 primary tumors of bone and soft tissues. Among benign tumors, overexpression was consistently found only in giant-cell tumor, a locally aggressive lesion that may also, although rarely, spread to the lung. Among malignant lesions, the presence of the Met/HGF receptor was detected in a relevant percentage of primaries and in almost all of the recurrences. The highest levels of Met/HGF receptor were found in osteosarcoma, a highly aggressive tumor that typically permeates the host bone and rapidly expands to the soft tissues. On the contrary, only low levels of Met/HGF receptor were found in chondrosarcoma, a slowly growing tumor that usually expands without massive destruction of the surrounding structures. These data indicate an association of Met/HGF expression with local aggressiveness in human mesenchymal tumors. The finding of Met/HGF receptor overexpression in all of the osteosarcomas suggests a role for the met proto-oncogene in the pathogenesis of this tumor.

In osteosarcoma, resistance to chemotherapy and metastatic spread are the most important mechanisms responsible for the failure of current multimodal therapeutic programs. We have shown previously that overexpression of the MDR1 gene product P-glycoprotein is the most important predictor of an adverse clinical course in patients with osteosarcoma. treated with chemotherapy. In this study, we analyzed the relationship between P-glycoprotein expression and local aggressiveness and systemic dissemination of multidrug-resistant (MDR) human osteosarcoma cells. Compared to parental sensitive cells, MDR cells showed a decreased tumorigenicity,and metastatic ability in athymic mice, together with a reduced migratory and invasive ability and a lower homotypic adhesion ability in vitro, suggesting that P-glycoprotein overexpression is associated with a less malignant phenotype. These experimental observations were confirmed by clinical data. In fact, the time of appearance of lung metastases in a series of osteosarcoma patients treated with chemotherapy was significantly shorter in the group of cases with no expression of P-glycoprotein in the primary lesion compared to the group with P-glycoprotein overexpression. Moreover, the incidence of P-glycoprotein overexpression was found to be higher among patients with localized disease at the clinical onset than in patients with evidence of metastasis at the time of diagnosis. These data indicate that, in osteosarcoma, the MDR phenotype is not associated with a more aggressive behavior both in vitro and in clinical settings, suggesting that the previously shown association of the MDR phenotype with a worse outcome in osteosarcoma is not related to a higher metastatic ability of cells with P-glycoprotein overexpression but is more likely due to their lack of responsiveness to cytotoxic drugs.

The identification of Ewing's sarcoma (ES) and peripheral neuroectodermal tumor (PNET) among other small round cell tumors (SRCTs) is a critical issue in musculoskeletal pathology because of the lack of clearly distinctive morphological features. In this study, the authors have compared advantages and limits of two procedures that were recently suggested as additional tools for the identification of ES/PNET, the analysis of p30/32MIC2 antigen by immunohistochemistry, and the evaluation of the fusion products of two specific chromosomal aberrations, the t(11;22)(q24;q12) and the t(21;22)(q22;q12), by reverse transcriptase-polymerase chain reaction (RT-PCR). The authors have analyzed the expression of p30/32MIC2 in 28 cell lines and in 90 tumor samples. p30/32MIC2 was highly expressed in ES/PNET but was also present in all the other cell types. The broad spectrum of positivity for p30/32MIC2 in SRCTs of bone was substantially confirmed by the analysis of tissue samples. In the same material, the authors have evaluated the presence of t(11;22) or t(21;22) transcripts (EWS/FLI-1 and EWS/ERG, respectively) by RT-PCR. These transcripts were found in all the cell lines and tissue samples of ES/PNET, but not in other tumors. The authors' results question the use of p30/32MIC2 immunostaining alone for the identification of ES/PNET and suggest the adoption of RT-PCR as an advantageous alternative. Molecular diagnosis of ES/PNET by RT-PCR is highly specific and can be applied to small amounts of tissue. Moreover, RNA extracted from paraffin-embedded specimens was shown to be suitable for RT-PCR analysis, thus enabling analysis of archival material.

Soft tissue sarcomas comprise a heterogeneous group of mesenchymal tumors accounting for less than one-percent of adult neoplasms. In the last few years, the use of adjuvant chemotherapy has been proposed for the treatment of these lesions in order to obtain a better systemic control, but its usefulness is still controversial. In this study, we evaluated whether P-glycoprotein, a membrane protein strictly associated with multidrug resistance, is overexpressed in soft tissue sarcomas. By using human multidrug resistant sarcoma cell lines as controls, we analyzed P-glycoprotein expression in 34 primary and in 23 relapsed soft tissue sarcomas of the extremities. Overexpression of P-glycoprotein was found in 6 out of 34 primaries (18%) and in 8 out of 23 relapses (35%). In particular, in malignant fibrous histiocytoma, the most frequent soft tissue sarcoma of adults, P-glycoprotein overexpression was found in 23% of primary untreated cases, in agreement with the reported relapse rate of this tumor after surgery and chemotherapy. These data suggest that, in soft tissue sarcomas, overexpression of P-glycoprotein may be of prognostic value and that the assessment of P-glycoprotein expression may be useful for the design of chemotherapy protocols.

Background: Increased levels of P-glycoprotein occur in some osteosarcomas. In this study we determined the relation between P-glycoprotein status and outcome in patients with high-grade osteosarcoma., Methods: P-glycoprotein status was determined immunohistochemically in specimens of osteosarcoma of the extremities (stage II) from 92 patients who were treated with surgery and chemotherapy. The P-glycoprotein status was analyzed in relation to the length of event-free survival., Results: The presence of increased levels of P-glycoprotein in the osteosarcoma was significantly associated with a decreased probability of remaining event-free after diagnosis (P = 0.002). In a multivariate analysis, P-glycoprotein status (P = 0.001) and the extent of tumor necrosis after preoperative chemotherapy (P = 0.04) were independent predictors of clinical outcome. The risk of adverse events was increased substantially (rate ratio, 3.37; 95 percent confidence interval, 1.60 to 7.10) among patients with increased levels of P-glycoprotein in tumor cells, as compared with patients who did not have increased levels of P-glycoprotein in tumor cells., Conclusions: In patients with high-grade osteosarcoma treated with surgery and chemotherapy, the presence of increased levels of P-glycoprotein in tumor cells is associated with a significantly increased risk of adverse events and is independent of the extent of necrosis after preoperative chemotherapy.

N-methylformamide (NMF), a powerful differentiating agent, has been extensively used in experimental and preclinical cancer chemotherapy studies, alone or in association with conventional anti-cancer drugs. To evaluate the use of this molecule in the treatment of osteosarcoma (OS), we have analyzed the effects of NMF and doxorubicin (DXR) on DXR-sensitive and -resistant human OS cell lines. Our study shows that NMF exerts remarkable effects on cell proliferation and, in Saos-2 and SARG cells, also induces differentiation, as shown by increasing alkaline phosphatase activity. Moreover, NMF increases the cytotoxic activity of DXR when administered after the drug, in both DXR-sensitive and -resistant cells. However, when this agent is given before DXR, it enhances P-glycoprotein expression in U-2 OS cell lines. This over-expression is associated with reduced DXR accumulation within cells and with significant enhancement of resistance to DXR.

Multidrug resistant variants of two human osteosarcoma cell lines (U-2 OS and Saos-2) were selected by continuous exposure to doxorubicin. The in vitro and in vivo growth characteristics of these sublines as well as the expression of osteoblastic markers and of some surface antigens were analyzed. Resistant variants showed a higher doubling time and a lower cloning efficiency, and a lower metastatic ability after i.v. injection than corresponding parental cell lines. All the sublines showed overexpression of p-glycoprotein (referred to as p170). The level of expression of this protein in the different cell lines was directly related to the degree of resistance as shown by the in vitro sensitivity to doxorubicin and other multidrug-related drugs. In sublines showing the highest levels of resistance (over 300-fold), p170 overexpression was associated with mdr 1 gene amplification. These are the first multidrug resistant human osteosarcoma cell lines ever reported. They may be used as a model for further investigations into the mechanisms of drug resistance in osteosarcoma and as a standard for the assessment of chemosensitivity in clinical samples.

The reliability of a simple method evaluating the pattern of subcellular binding of Adriamycin (Adriamycin binding assay, ABA) as an index of sensitivity was demonstrated in different primary cultures and in sensitive and resistant cell lines of human osteosarcoma. After exposure to Adriamycin (10 micrograms/ml for 30 min at 37 degrees C), living sensitive cells showed selective intranuclear uptake of the drug, whereas in resistant cells no distinct subcellular distribution was observed. The binding pattern of Adriamycin in sensitive and in highly resistant cells was inversely related to the expression of P-glycoprotein. However, low levels of resistance in vitro, not detectable by increased levels of expression of P-glycoprotein, were revealed by ABA. The use of ABA in combination with the estimate of P-glycoprotein expression is recommended in clinical practice as an accurate means for predicting the sensitivity of osteosarcoma to Adriamycin.