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.

Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma.

BACKGROUND: Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours. METHODS: Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell lines. In vitro efficacy of two Aurora kinases-targeting drugs (VX-680 and ZM447439) was evaluated on a panel of four drug-sensitive and six drug-resistant human osteosarcoma cell lines. RESULTS: Human osteosarcoma cell lines proved to be highly sensitive to both drugs. A decreased drug sensitivity was observed in doxorubicin-resistant cell lines, most probably related to ABCB1/MDR1 overexpression. Both drugs variably induced hyperploidy and apoptosis in the majority of cell lines. VX-680 also reduced in vitro cell motility and soft-agar cloning efficiency. Drug association experiments showed that VX-680 positively interacts with all conventional drugs used in osteosarcoma chemotherapy, overcoming the cross-resistance observed in the single-drug treatments. CONCLUSION: Aurora kinase-A and -B represent new candidate therapeutic targets for osteosarcoma. In vitro analysis of the Aurora kinases inhibitors VX-680 and ZM447439 indicated in VX-680 a new promising drug of potential clinical usefulness in association with conventional osteosarcoma chemotherapeutic agents.

High grade malignant peripheral nerve sheath tumors: outcome of 62 patients with localized disease and review of the literature.

Malignant peripheral nerve sheath tumours (MPNST) are rare sarcomas with one of the poorest prognoses of all the soft tissue sarcomas. Information about adjuvant treatment is scarce and not homogeneous for this diagnosis. We analyzed retrospectively the outcome of patients with localized high grade MPNST admitted to our institute from 1969 to 2008. A review of the literature is also reported. Of 62 evaluable patients, 23 were females and 39 males, median age 39 years (17-71), 22/62 had neurofibromatosis type I. Median follow-up was 54 months (range 12-194). A total of 22/62 are alive; 26 patients had surgery alone, 18 received radiation therapy, 12 received radiation therapy and chemotherapy, and 6 received only adjuvant chemotherapy. The 5-year disease-free survival was 30% and 5-year overall survival was 38%. A positive trend for adjuvant radiation, but not for chemotherapy was observed according to univariate analysis only for disease-free survival and overall survival. Multivariate analysis indicated that primary site, size and surgical margins remained significant for disease-free survival and only site and size were significant for overall survival. New drugs employed successfully in advanced mpNSt should be employed also in the adjuvant setting.

Genetic and molecular alterations in rhabdomyosarcoma: mRNA overexpression of MCL1 and MAP2K4 genes.

Rhabdomyosarcoma, the most common soft tissue sarcoma in childhood, belongs to the small round cell tumor family and is classified according to its histopathological features as embryonal, alveolar and pleomorphic. In this study we propose to explore genetic alterations involved in rhabdomyosarcoma tumorigenesis and assess the level of mRNA gene expression of controlling survival signalling pathways. For genetic and molecular analysis, array-based comparative genomic hybridization, combined with Real Time PCR using the comparative method, was performed on 14 primary well-characterized human primary rhabdomyosarcomas. Multiple changes affecting chromosome arms were detected in all cases, including gain or loss of specific regions harbouring cancer progression-associated genes. Evaluation of mRNA levels showed in the majority of cases overexpression of MCL1 and MAP2K4 genes, both involved in cell viability regulation. Our findings on rhabdomyosarcoma samples showed multiple copy number alterations in chromosome regions implicated in malignancy progression and indicated a strong expression of MAP2K4 and MCL1 genes, both involved in different biological functions of complicated signalling pathways.

Amplification of CDK4, MDM2, SAS and GLI genes in leiomyosarcoma, alveolar and embryonal rhabdomyosarcoma.

We evaluated amplification and overrepresentation of CDK4, MDM2, GLI and SAS genes of the 12q13-15 region, in a group of soft tissue sarcomas including leiomyosarcomas (LMS), alveolar rhabdomyosarcomas (ARMS) and embryonal (anaplastic and classic variants) rhabdomyosarcomas (ERMS), to ascertain genomic alterations and possible differences within histologic subtypes of rhabdomyosarcoma (RMS). Quantitative real-time PCR was performed on DNA samples from 29 LMS, 9 ARMS, 7 anaplastic ERMS and 6 classic ERMS. Alteration of one or more of the 12q13-15 genes was revealed in 13/29 LMS (45%) and 12/22 RMS (54%) including 5/9 ARMS (56%), 5/7 anaplastic ERMS (71%) and 2/6 classic ERMS (33%). The potential importance of overproduction of protein products in neoplastic development, led us also to study a possible high expression of cdk4, mdm2 and gli proteins in immunohistochemical staining experiments on paraffin-embedded tissue samples of the same cases. Among LMS and RMS most cases with CDK4, MDM2 and GLI gene alterations also showed a simultaneous high expression of the relative protein. In summary, these results indicate that amplification or overerepresentation of genes at 12q13-15 region involve both LMS and RMS. Moreover these genes alterations reveal predominantly in the alveolar and in the anaplastic variant of the embryonal subtype. These two seem to have a more similar behavior than anaplastic and classic embryonal that are classified in the same subtype.

Tissue and serum loss of metalloproteinase inhibitors in high grade soft tissue sarcomas.

The activity of matrix metalloproteinases (MMPs) in degrading extracellular matrix is controlled by activation of pro-enzymes and inhibition of MMP tissue inhibitors (TIMPs). To assess proteolytic cascade imbalance in malignancy progression, the enzymatic activity of MMP2 and MMP9 and the expression and serum level of their inhibitors, TIMP2 and TIMP1 respectively, was evaluated in selected patients with high-risk soft tissue sarcoma (STS). Gelatinase activity and inhibitor expression was evaluated on 69 biopsies by zymography and immunohistochemistry. TIMP1 and TIMP2 serum concentration was tested in 53 STS patients and in 56 controls using a sandwich enzyme immunoassay. Clinical and biological variables were related to clinical outcome of the patients. A significant gelatinolytic activity was seen in a high percentage of STS. TIMP expression was weak or negative in the majority of samples. The difference between disease-free (p=0.001) and overall survival (p=0.007) curves based on TIMP2 immunoreactivity was statistically significant. TIMP plasma concentration of 53 STS revealed significantly lower levels compared to those of 56 controls (p=0.0001). In conclusion, low levels of negative regulators of proteolysis may be related to tumor biological aggressiveness and used to select patients with poor prognosis to improve cure.

Involvement of INK4A gene products in the pathogenesis and development of human osteosarcoma.

BACKGROUND: The INK4A tumor suppressor gene plays a crucial role in the regulation of the G1 cell cycle phase. It encodes two transcripts, p16 and p14 alternate reading frame (ARF), involved in retinoblastoma protein (pRb)- and p53- cell growth control pathways, respectively. METHODS: To define the role of gene status and molecule expression involved in the INK4A regulatory system, immunohistochemistry, immunoblotting, and polymerase chain reaction (PCR) analysis were performed on 35 primary high grade osteosarcomas (OS). RESULTS: Although p16 and p14ARF proteins were found negative or weakly detectable in 60% and 57% of the cases respectively, INK4A gene analysis of exons 1alpha, 1beta and 2 did not reveal any deletion or mutation. However, methylation status of the 5'CpG promoter region, assessed by methylation-specific PCR, was found in 12 out of 21 OSs with negative or weak p16 expression. A statistical analysis based on pRb/p16 and p53/p14ARF staining status showed that pRb and p16 co-expression was inversely correlated to tumor relapse and was a marker for a more favorable prognosis. A statistically significant inverse correlation was found between wt-p53 and p14ARF expression. In the group of wt-p53 tumors, the loss of p14ARF was associated with a decreased expression of p21 protein, suggesting a down-regulation of the transcriptional activity of p53. CONCLUSIONS: The current results suggest that, in OS, the altered expression of INK4A products plays a primary role in the deregulation of both pRb and p53 cell growth control pathways, contributing to tumor pathogenesis and development.

Metalloproteinase expression and prognosis in soft tissue sarcomas.

BACKGROUND: Degradation of extracellular matrix by tumor-associated proteases can promote cell invasion and metastasis. This study assessed the prognostic role of MMP2, MMP9 metalloproteinases, and of the metalloproteinase inhibitor TIMP2, related to disease-free survival (DFS), in soft tissue sarcoma (STS) patients. MATERIALS AND METHODS: Level and distribution of MMP2, MMP9, and TIMP2 expression were evaluated on 73 biopsies by immunohistochemistry and immunoblotting. Biopsies included 29 liposarcomas, 29 synovial sarcomas, and 15 malignant peripheral nerve sheath tumors (MPNST). Association between DFS and overall survival with different variables was assessed. RESULTS: In terms of DFS, increased MMP2 reactivity and lack of TIMP2 expression were significant for poor prognosis in all samples (P = 0.0005 and P = 0.006 respectively). MMP2 correlated to histologic grade (P = 0.005). Lack of TIMP2 expression was a poor prognostic factor for DFS in synovial sarcoma (P = 0.009), while MMP2 and MMP9 correlated with metastasis (P = 0.008 and P = 0.005, respectively) and grade (P = 0.001 and P = 0.04 respectively) in liposarcoma. CONCLUSIONS: These prognostic markers that influence growth and spread of tumor cells might be useful to define tumor aggressiveness and risk of the metastasic event.

Internal low energy atrial cardioversion: efficacy and safety in older patients with chronic persistent atrial fibrillation.

BACKGROUND: Low-energy internal atrial cardioversion is a relatively new technique based on delivery of intracardiac shocks through transvenous catheters placed into the atria or the vessels. OBJECTIVE: The aim of this study was to assess in older and younger patients with chronic persistent atrial fibrillation (AF) the efficacy and safety of transvenous low-energy internal atrial cardioversion performed without routine administration of sedatives or anesthetics. DESIGN: A prospective longitudinal study. SETTING: A cardiological university hospital. PARTICIPANTS: 82 patients, divided into older (> or = 60 years) (n = 49) and younger (n = 33) subjects. MEASUREMENTS: Atrial defibrillation threshold for internal cardioversion, measured as leading edge voltage (V) and delivered energy (J) of effective shocks, percentage of patients maintaining sinus rhythm at short-term (within 3 days) and at long-term follow-up. METHODS: Patients with chronic persistent AF, treated with oral anticoagulants for at least 3 to 4 weeks, were admitted to hospital. Following a clinical work-up, patients were subjected to low-energy internal atrial cardioversion with shock delivery according to a step-up protocol. RESULTS: Internal cardioversion was effective in restoring sinus rhythm in 90% (44/49) of the older patients and in 94% (31/33) of the younger patients. Shocks were effective at a mean energy between 6 and 8 joules (range 0.9-23) and administration of sedatives or anesthetics was required during the procedure in 22% (11/49) of older and in 48% (16/33) of younger patients (P = .026 at chi-square). No major complications occurred during the procedure. Pharmacological prophylaxis of AF recurrences was instituted immediately following the procedure. During inhospital stay and during the follow-up (mean 12 +/- 9 months for older patients and 15 +/- 10 months for younger patients), AF recurred in 39% (17/44) of older patients and in 16% (5/31) of younger subjects (P = .064 at chi-square). CONCLUSIONS: Internal low energy cardioversion is a very effective procedure for restoring sinus rhythm in patients with AF; it can be performed in older patients, and administration of sedatives or anesthetics can be avoided or minimized in a substantial proportion of subjects. Recurrences of AF in the long term tend to be higher in older subjects and intensive prophylaxis with antiarrhythmic drugs is required.

Human osteosarcoma cells release matrix degrading enzymes in response to chemokine activation.

Matrix degrading enzymes released upon autocrine and/or paracrine induction exert a key role in modulating tumor cell behavior. Osteosarcoma is a highly metastatic cancer, with a redundancy of autocrine loops. Here we report that human osteosarcoma cells express a wide array of chemokine receptors and respond to chemokine activation with the release of N-acetyl-beta-D-glucosaminidase and gelatinase/collagenase activity. Of the two cell lines studied, the osteoblast-like MG-63 showed a higher responsivity compared to the less differentiated HOS. This suggests that chemokine modulation of matrix degrading enzymes requires the maintaining of the osteoblastic phenotype and of signaling pathways which occur in normal tissue.

Analysis of 12q13-15 genes in parosteal osteosarcoma.

The region q13-15 of chromosome 12 frequently is altered in human sarcomas, and several genes, such as SAS, CDK4, and MDM2, have been found to be amplified in bone and soft tissue sarcomas. These genes and their products were studied by quantitative polymerase chain reaction and immunohistochemical analysis in 25 parosteal osteosarcoma samples (22 Grades I or II, three dedifferentiated) to evaluate if the possible alterations detected of the genes on chromosome 12 could have a role in the development of this rare bone tumor. Immunohistochemical analysis was performed on formalin fixed, paraffin embedded tumor sections to evaluate CDK4 and MDM2 protein expression. To measure the degree of SAS and CDK4 gene amplification, quantitative polymerase chain reaction was done on deoxyribonucleic acid derived from the same samples. The results showed that CDK4 protein was expressed in 92% of the cases. Strong and uniform CDK4 and MDM2 immunoreactivity was found respectively in three of three and two of three dedifferentiated parosteal osteosarcomas. SAS and CDK4 genes were found to be amplified fourfold in two Grade II tumors and in one dedifferentiated tumor. These findings, which should be investigated further, might suggest a possible role of the chromosome 12 genes in the pathogenesis of parosteal osteosarcoma.

Evaluation of myocardial injury following repeated internal atrial shocks by monitoring serum cardiac troponin I levels.

INTRODUCTION: Electrical shocks delivered for atrial cardioversion (CV) may cause myocardial damage. The aim of this study was to assess the extent of myocardial injury caused by repeated intracardiac shocks delivered for low-energy internal atrial CV. METHODS AND RESULTS: Thirty-five patients with chronic persistent atrial fibrillation (AF) of different etiologies underwent CV with delivery of synchronized biphasic shocks (3.0/3.0 ms) between two catheters positioned in the right atrium and the coronary sinus. Shocks were delivered according to a step-up protocol (50 V, 180 V, then steps of 40 to 56 V up to 500 V, if necessary). In 23 patients, AF was reinduced after baseline CV, and CV was repeated. Myocardial injury was monitored by measuring cardiac troponin I (cTnI) serum concentrations in blood samples taken at baseline and at 2, 4, 8, 12, and 24 h after the procedure, by means of an immunoenzymologic assay (normal values, < or =0.6 ng/mL). A mean (+/- SD) of 6.9 +/- 3.4 shocks per patient were delivered (range, 2 to 17). Shocks delivered in each patient had a maximal energy of 7.3 +/- 4.0 J (range, 1.7 to 15.7). In 20 patients (57%), no evidence of myocardial injury (cTnI level, < or = 0.6 ng/mL) was found. In 13 patients (37%), mildly elevated cTnI levels (range, 0.7 to 1.4 ng/mL) in samples taken 4 to 12 h after CV suggested minor myocardial injury. In two patients (6%), higher cTnI levels were found in samples taken 4 to 8 h after CV (peak, 1.7 and 2.4 ng/mL), indicating a necrotic damage. Patients with no cTnI elevation, with mild cTnI elevation, or with cTnI levels >or =1.5 ng/mL did not differ significantly with respect to the total number of shocks delivered, the mean amount of energy delivered, and the cumulative amount of energy delivered. No clinical complications were observed. CONCLUSIONS: Following internal CV with the delivery of repeated shocks, minor elevations of cTnI serum levels could be detected in a significant proportion of patients, and this suggests subtle asymptomatic minor myocardial injury. The elevations of cTnI levels do not appear to be related to the number of shocks or to the amount of energy delivered.

Presence and expression of the simian virus-40 genome in human giant cell tumors of bone.

SV40 DNA sequences have been found in human tumors, such as mesotheliomas, ependymomas, and bone tumors, suggesting that SV40 may be involved in their etiology. The FOS oncogene could play an important role in bone development because SV40 is able to induce FOS in cell culture. In this study, the presence of SV40 sequences, large T antigen (Tag), and FOS protein expression were investigated in 120 giant cell tumors (GCTs), moderately benign bone tumors that in some cases can progress to a malignant phenotype. Polymerase chain reaction (PCR), using primers that amplify the RB1 pocket binding domain and the intron of Tag, was used to analyze GCT for the presence of SV40 DNA. Tag and FOS protein expression was evaluated by immunohistochemistry. SV40 sequences were found in 30/107 GCTs, and of these, 22/30 samples expressed Tag protein (73%) and 15/30 overexpressed the FOS oncogene (50%). FOS was undetectable in 77 SV40-negative GCTs. Sequence analysis of the amplified DNAs confirmed that the amplified sequences corresponded to SV40 DNA. The correlation between FOS overexpression and SV40-positive GCTs was highly statistically significant (P < 0.001). These results show that SV40 DNA sequences and SV40 Tag are present in GCTs and might induce FOS activity. These data suggest that SV40 might play a role in the development and progression of some GCTs.

Alteration of pRb/p16/cdk4 regulation in human osteosarcoma.

Cell-cycle regulation depends on a fine balance between cyclin-cyclin-dependent kinase complexes and a family of kinase inhibitors that bind cyclin-cdk complexes and block their activity. To investigate the role of mechanisms regulating cell-cycle progression in human osteosarcomas (OS), pRb/p16/cdk4 expression was analyzed in 39 high-grade OS; 19 of these developed metastasis during follow-up. Positive reaction for functional pRB was shown by 18/39 (46%) OS, while 21/39 (54%) were negative. A higher probability of metastasis was seen in patients with negative pRb expression (p < 0.05). Furthermore, while functional pRb and D1 expression are inversely associated to metastasis occurrence, the presence of D1/cdk4 complex in our study was related to poor prognosis. We found that 10/18 pRb-positive and 14/21 pRb-negative tumors were p16-positive. No significant correlation was found between pRb and p16 expression. On the other hand, high cdk4 levels in p16-positive tumors as compared with p16-negative tumors resulted in a positive association between p16 and cdk4 expression (Chi squared = 5.98; p = 0.01). No extensive p16INK4A genomic alterations were found in tumors lacking p16-protein expression. To determine which mechanisms are involved in the down-regulation of p16 protein, the methylation status of the p16INK4 gene was evaluated on the 15 p16-negative tumors: 8 samples showed 5' CpG-island methylation; 4/8 had a complete methylation status, while in the remaining 4 the gene was only partially methylated. These data confirm the role of the pRb/p16/cdk4 pathway in OS development.

Increased c-myc oncogene expression in Ewing's sarcoma: correlation with Ki67 proliferation index.

AIMS AND BACKGROUND: Ewing's sarcoma is a highly malignant musculoskeletal tumor composed of small round cells. Although important results have been achieved with surgery associated with chemotherapy, recurrent disease is still a major problem. In order to define new prognostic factors useful for therapeutic decision-making, we conducted a study on 38 Ewing's sarcoma samples in which c-myc oncogene expression and Ki67 proliferation index were correlated with clinical outcome. METHODS AND STUDY DESIGN: Nineteen patients developed metastases during follow-up and 10 of these patients died. C-myc and Ki67 protein expression was evaluated by immunohistochemistry performed on 5 microm formalin-fixed and paraffin-embedded sections, while the c-myc mRNA transcript was localized using in situ hybridization. RESULTS: A statistically positive correlation was found between c-myc protein and Ki67 (P = 0.001) and c-myc mRNA and Ki67 expression (P = 0.047). The 38 patients were divided into two groups using as the cutoff 50% of Ki67-positive cells. The disease-free survival and overall survival estimates were 68% and 90%, respectively, in the group of patients with a percentage of Ki67-positive cells <50%, and 25% and 50%, respectively, in the group with a percentage of Ki67-positive cells > or = 50%. The difference between the survival curves was statistically significant (P <0.05 and P <0.01). Furthermore, relapsed patients had a high and uniform expression of c-myc protein and mRNA compared to disease-free patients. CONCLUSION: These results suggest a possible role of the c-myc oncogene and Ki67 antigen in the malignant progression of Ewing's sarcoma.

Expression of G1 phase regulators in MG-63 osteosarcoma cell line.

Cyclins and cyclin-dependent kinases (cdks) form complexes that govern transitions during cell cycle phases. In this study we characterized a human osteosarcoma cell line, MG-63, for the expression level of cyclin D1, cyclin E, cdk4, cdk2, and cell cycle inhibitors pRb and p21. To investigate the role of these proteins we treated MG-63 cells with tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Cell proliferation analysis demonstrated an increased proliferation of MG-63 cells with IL-6, while TNF-alpha acted as an anti-proliferative agent. Immunoblotting revealed an increased expression of p21 with TNF-alpha and its complex with cdk2. TNF-alpha reduced the expression of the cyclin E-cdk2 complex. TNF-alpha did not affect the amount of cyclin D1, cyclin E, cdk4, cdk2, and of cyclin D1-cdk4 complex. IL-6 decreased p21 expression and its complex with cdk2, while it increased the cyclin E-cdk2 complex. Cyclin D1 and cdk4 expression and their complex did not change after IL-6 treatment, nor did cyclin E and cdk2 protein expression. Hyperphosphorylated/dephosphorylated Rb protein ratio was reduced with TNF-alpha whereas it increased with IL-6. These results may suggest an important role of p21 and of cyclin E-cdk2 complex in the G1 phase regulation through pRb phosphorylation in MG-63 cells.

Analysis of SAS gene and CDK4 and MDM2 proteins in low-grade osteosarcoma.

The region q13-15 of chromosome 12 contains SAS, CDK4, and MDM2 genes that are rearranged or amplified in a variety of human sarcomas. This study evaluated SAS gene amplification, and MDM2 and CDK4 protein expression in 20 tumor samples of central low-grade osteosarcoma (16 primary, 3 recurrences, 1 lung metastasis). SAS amplification was analyzed by quantitative polymerase chain reaction (PCR), while from the same paraffin-embedded samples, MDM2 and CDK4 protein expression was evaluated by immunohistochemistry. MDM2 and CDK4 proteins were found strongly expressed in 35% and 65%, respectively, of the samples. SAS was found amplified in 15% of the samples. These findings indicate that these genes may be involved in tumorigenesis and progression of low-grade osteosarcoma.

C-myc and c-fos in human osteosarcoma: prognostic value of mRNA and protein expression.

The c-myc and c-fos proto-oncogenes have several putative functions, including regulation of cell growth. In many neoplasms c-myc overexpression has been linked to poor prognosis. In order to study the role of c-myc and c-fos expression on the tumorigenesis, and the metastatic spread of osteosarcoma, frozen and paraffin-embedded tissue 38 primary osteosarcoma and 10 lung metastases were analyzed. The mRNA analysis was performed by quantitative reverse transcription-polymerase chain reaction and in situ hybridization. The protein expression was studied by Western blot analysis and immunohistochemistry. C-myc and c-fos were found overexpressed in a high percentage of the relapsed tumors and of the metastases, and overexpression of both oncogenes in the same tumor was strongly correlated to the development of metastases (p < 0.05), as 6 of the 7 primary tumors overexpressing both the oncogenes gave metastases. In conclusion, both c-myc and c-fos are involved in the growth and spread of osteosarcoma and a synchronous overexpression of both oncogenes is highly significant for a metastatic potential of a primary tumor.

Prognostic significance of cyclin expression in human osteosarcoma.

To evaluate the distribution of cyclin protein expression, in relation to cell proliferation rate and clinical behavior, an immunohistochemical study was performed on 92 tumor samples of patients with high grade osteosarcoma (OS). A large cyclin A- and cyclin E-positive fraction was found respectively in 59% and 47% of the osteosarcomas, while immunostaining for cyclin D1 was weak or absent in most tumor samples. A positive, statistically significant correlation was found between A and E cyclins and Ki67 expression (p<0.001). Disease-free survival (DFS) analysis included 69 of the 92 patients. A significantly higher probability of metastasis was seen in patients lacking cyclin D1 compared to those in which cyclin D1 was positive (p<0.01). Conversely, patients with >40% of cyclin A-positive cells relapsed more frequently than those with <40% of cyclin A-positive cells (p<0.05). The multivariate analysis demonstrated that cyclin A had a lower predective risk in terms of disease-free survival as opposed to the loss of cyclin D1 that is considered a powerful prognostic factor.

Prognostic relevance of C-myc gene expression in giant-cell tumor of bone.

Giant-cell tumor is a primary bone tumor, of uncertain origin, with the potential capacity to metastasize. To study the role of c-myc and c-fos oncogene overexpression in the tumorigenesis and metastatic spread of giant-cell tumors, 32 primary tumors were collected; of these, 19 remained disease-free and 13 metastasized to the lung. Samples of lung metastasis from these 13 patients were also available for study. The expression of c-myc and c-fos mRNA was studied by reverse transcription-polymerase chain reaction and by in situ hybridization. The expression of protein was studied by Western blot analysis and by immunohistochemistry. C-myc mRNA was overexpressed in 12 (38%) of the 32 primary tumors. Thirteen primary tumors metastasized to the lung; in nine (69%) of these, c-myc mRNA was overexpressed. The c-myc protein was overexpressed in seven (54%) of the 13 tumors that metastasized to the lung. C-fos was overexpressed in only one lung metastasis. A strong correlation between the overexpression of c-myc, and the occurrence of metastases was found: thus, c-myc seems a powerful prognosticator in giant-cell tumor. C-myc was overexpressed both in giant cells and in mononuclear cells, suggesting that both cell types are involved in the progression of this tumor.