Biomedical subjects

G Gamberi

Publications and source records attributed to G Gamberi.

At least 19 recordsLinked to original sources

PubMed2006

Molecular alterations of monophasic synovial sarcoma: loss of chromosome 3p does not alter RASSF1 and MLH1 transcriptional activity.

Differential diagnosis of monophasic synovial sarcoma requires the detection of specific biological markers. In this study we evaluated the presence of molecular alterations in 15 monophasic synovial sarcomas. Multiple changes affecting chromosome arms were detected by CGH-array in all microdissected cases available, and an association between gain or loss of specific regions harbouring cancer progression-associated genes and aneuploid status was found. The most frequent alteration was loss of 3p including 3p21.3-p23 region that, however, did not involve the promoter regions of the corresponding genes, RASSF1 and MLH1. Using Real-Time PCR, mRNA levels of both resulted moderately high compared to normal tissue; however, the weak to absent protein expression suggests RASSF1 and MLH1 post-transcription deregulation. Moreover, immunohistochemical analysis revealed that both mesenchymal and epithelial antigens were present in diploid tumours. These findings confirm the genetic complexity of monophasic synovial sarcoma and underline the need to integrate different analyses for a better knowledge of this tumour, essential to investigate new diagnostic and prognostic markers.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Proteases and interleukin-6 gene analysis in 92 giant cell tumors of bone.

BACKGROUND: Giant cell tumor of bone (GCT) is a benign tumor with a significant tendency to recur locally and rarely to produce pulmonary metastases. It is characterized by the presence of multinucleated osteoclast-like giant cells together with mononuclear spindle-shaped cells. Few prognostic markers have been reported to predict the clinical outcome of GCT patients, so is very important to find the factor that can be implicated in its potential aggressiveness. PATIENTS AND METHODS: Different groups of GCT patients were selected for this study, including patients without evidence of disease and patients who recurred locally or with lung metastasis. The total of 92 tumor samples also included the specimens of the local recurrences and the lung metastases. By using immunohistochemistry and real-time quantitative polymerase chain reaction techniques, the genetic and proteic analyses were performed on the urokinase-type plasminogen activator (u-PA), its receptor (u-PAR) and its inhibitor (PAI-1), which have been described to be frequently implicated in the process of degradation of the extracellular matrix during the metastatic process. Interleukin-6 (IL-6), a cytokine released by GCT cells, which stimulates resorption of bone, was also analyzed. RESULTS: IL-6, u-PA, u-PAR and PAI 1 genes were found amplified, respectively, in 7%, 5%, 8% and 12% of total cases (92). In particular, the percentages of amplified genes were higher in the GCT cells that gave rise to metastases (12 cases) and in the samples of lung metastases (nine cases) compared with the disease-free group of patients (60 cases). CONCLUSIONS: These results suggest a possible association of these factors with a higher biological aggressiveness of GCT. Morever, it appears that increased expression of the IL-6, u-PA, u-PAR and PAI1 proteins might not depend on mutation of the corresponding genes.

Biomarkers, Tumor↗

PubMed2004

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.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMed2003

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.

Adolescent↗

PubMed2001

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.

Adolescent↗

PubMed2001

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.

Adolescent↗

PubMed2000

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.

Adult↗

PubMed2000

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.

Bone Neoplasms↗

PubMed2000

Brain tumors as second malignant neoplasms in patients with osteosarcoma treated with adjuvant and neoadjuvant chemotherapy: report of 2 cases.

A malignant, primary brain tumor developed as Second Malignant Neoplasm (SMN) in 2/490 long-term-survivor osteosarcoma patients treated at our Institute over a 20-yr period. They developed the brain tumor (one astrocytoma and one glioblastoma) 3 and 5 yr after treatment, (chemotherapy and surgery), for localized osteosarcoma of the extremity.

Adult↗

PubMed1999

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.

Blotting, Western↗

PubMed1999

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.

Adolescent↗

PubMed1999

Secondary tumors in bone sarcomas after treatment with chemotherapy.

New oncologic treatments have improved survival in osteosarcoma and Ewing's sarcoma. However, these treatments may cause secondary malignancies after radiotherapy. This study evaluated the incidence of secondary malignancies after neoadjuvant chemotherapy. Between April 1972 and December 1990, 518 osteosarcoma and 299 Ewing's sarcoma patients entered neoadjuvant chemotherapy protocols. Follow-up records of all patients were analyzed and malignant tumors were reported. Nine patients developed another malignancy, including 5 leukemias, 1 astrocytoma, 1 liposarcoma, 1 parotid, and 1 breast carcinoma. Four leukemias were found in patients treated for osteosarcoma with chemotherapy, but not radiotherapy. Only one leukemia developed after Ewing's sarcoma treated with chemotherapy and radiotherapy. The incidence of leukemias is high, while the other tumors can be explained as unrelated cases. Incidence densities for leukemia were calculated for both groups of patients. Treated osteosarcoma patients seem to have a predisposition to develop leukemias, but whether this is chemotherapy induced needs to be investigated.

Adolescent↗

PubMed1999

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.

Apoptosis↗

PubMed1998

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.

Adolescent↗

PubMed1998

Correlation between apoptosis and TP53 status in osteosarcoma.

Samples of 18 osteosarcomas were examined for the occurrence of apoptosis and TP53 status. Apoptosis was investigated by an in situ nick translation technique on paraffin-embedded samples. It was found that apoptosis rarely occurs in osteosarcoma at the early stage of disease, whereas it is frequently activated when tumors are treated with antiblastic drugs. The analysis of the TP53 gene showed no mutation at diagnosis; whereas, during disease progression, four cases showed TP53 mutations. The authors discuss the relation between apoptosis, TP53 status, and therapy.

Adolescent↗

PubMed1998

Adhesion molecules in high-grade soft tissue sarcomas: correlation to clinical outcome.

The extracellular matrix (ECM) forms a framework for cell adhesion, but it also regulates growth and differentiation. Normal and malignant cells interact with the ECM via specific receptors, the integrins. To explore the mechanisms of growth and spread in soft tissue sarcomas the expression of the major ECM molecules and their corresponding integrin receptors were studied by immunohistochemistry in high-grade soft tissue sarcomas: malignant fibrous histiocytoma (20 cases), malignant peripheral nerve sheath tumour (17 cases) and synovial sarcoma (21 cases). The expression pattern was compared with cell proliferation and clinical outcome. Integrins were found to be expressed according to histological pattern. In synovial sarcomas, the epithelial component showed a high alpha 2 but negative or minimal detection of alpha 5 expression, while a weak alpha 2 expression and a moderate alpha 5 expression were found in the spindle cell component. No alpha 2 expression was detected in malignant fibrous histiocytoma, and minimal alpha 5 expression was detected in malignant schwannoma. The alpha 6 expression levels were positively correlated with the occurrence of metastases in all types of sarcomas studied. The expression of ECM molecules was downregulated and irregular in most tumours. In conclusion, the divergent integrin expression pattern could be useful in the diagnosis and classification of soft tissue sarcomas. Furthermore, since high laminin receptor expression correlates with occurrence of metastases, it could become a useful prognostic marker.

Disease-Free Survival↗

PubMed1998

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.

Biopsy↗

PubMed1998

Comparative genomic hybridization of low-grade central osteosarcoma.

Very little is known concerning the cytogenetic and molecular genetic changes of low-grade central osteosarcoma, a rare form of osteosarcoma. In the present study, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number aberrations in 10 samples from 6 patients: 7 typical low-grade central osteosarcomas, one low-grade (Grade II) central osteosarcoma, and two high-grade (III and IV) local recurrences of a low-grade central osteosarcoma Nine samples had aberrations. Six typical low-grade central osteosarcoma samples had a single DNA sequence copy number change per tumor. Three samples from more advanced tumors (a Grade II low-grade central osteosarcoma and local recurrences of Grade III and IV) had a mean of five changes per tumor. Recurrent changes affected these minimal common regions: +12q13-q14 (three tumors), +12p (two tumors), and +6p21.1-p21.3 (two tumors). Nine samples were analyzed for CDK4 and MDM2 expression and SAS amplification. One sample with a gain of chromosome 12 had a very strong expression of MDM2, strong expression of CDK4, and amplification of SAS. One sample with a gain of 12q13-q14 had strong expression of CDK4 and MDM2. Strong expression of CDK4 was found in two additional tumors; one had a gain of 12q13-q21, and the other had no changes in chromosome 12 by CGH. No alterations were detected in the CDK4, MDM2, and SAS panel in three other samples with no changes in chromosome 12 by CGH. In conclusion, the low number of DNA sequence copy number alterations reflects the relatively low malignancy of low-grade central osteosarcoma. This simplicity differs from the complex aberrations seen in conventional high-grade osteosarcomas.

Adolescent↗