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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.

The transcription of MDR1 gene may be increased by mutation or loss of function of p53 gene. In this study, we investigated whether in osteosarcoma, the p53 status is correlated with overexpression of the MDR1 gene product P-glycoprotein. The relationship between P-glycoprotein expression and p53 status was analyzed by immunohistochemistry in 64 primary and 11 metastatic high-grade osteosarcomas. In the same series, we also assessed the nuclear accumulation of MDM2 protein, whose binding to p53 protein provides an alternative mechanism of p53 inactivation. No association was found between mutant-p53 and MDM2 nuclear accumulation either with P-glycoprotein expression or with clinical course. Only increased expression of P-glycoprotein in tumor cells was significantly associated with a poor outcome, further supporting the adverse prognostic value of this marker in osteosarcoma.

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.

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.

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.

Background: The availability of Ki-67 monoclonal antibody has opened new possibilities for an extensive analysis of cell kinetics in human neoplasms. Ki-67 antibody reveals a nuclear antigen that is expressed in proliferating but not in quiescent cells. Although the reliability of Ki-67 immunostaining has been evaluated in different tumor types, little information has been reported on bone neoplasms., Methods: Cell proliferation, as determined by Ki-67 expression, was measured by immunofluorescence on representative cytospins obtained from 205 patients with bone tumors. In each sample, the percentage of Ki-67-positive cells was quantified on at least 500 cells and expressed as Ki-67 labeling index (LI)., Results: Ki-67 LI was lower in benign and low grade lesions as compared with high grade malignant lesions. A correlation between Ki-67 LI and histologic grade was observed in osteosarcoma and chondrosarcoma. In osteosarcoma, among the 43 primary lesions included in this study, 30 patients, all treated with the same regimen of chemotherapy and limb-salvage surgery, were selected to establish the prognostic significance of cell proliferation. The Ki-67 labeling was higher in patients with a good histologic response to chemotherapy. However, at a 24-month follow-up, a worse prognosis was associated with a higher proliferative activity, whereas no correspondence was found between the histologic response to preoperative chemotherapy and the disease free survival, suggesting that in high grade osteosarcoma the biologic aggressiveness expressed by high levels of Ki-67 LI may be clinically more relevant than the responsiveness to antineoplastic agents., Conclusions: In bone tumors, the level of Ki-67 expression correlates with the level of malignancy and is diagnostically and prognostically useful.

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.