Gabriella Gamberi

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.

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. Int. J. Cancer (Pred. Oncol.) 84:489–493, 1999.

Cytokeratin expression and distribution in adamantinoma of the long bones and osteofibrous dysplasia of tibia and fibula. An immunohistochemical study correlated to histogenesis

Twenty‐four cases of adamantinoma and 24 cases of osteofibrous dysplasia of the long bones were studied to evaluate the expression and distribution of cytokeratin (CK) subtypes in relation to histogenesis and differentation. The immunohistochemical study was performed on tissue fixed in buffered formalin and embedded in paraffin wax utilizing antibodies to vimentin, factor VIII, epithelial membrane antigen and cytokeratins of different molecular weights. In all cases the vimentin antibody marked positively in stroma, endothelium and osteoblasts, while factor VIII expression was confined to endothelial cells. In 71% of adamantinomas, vimentin showed strong immunoreactivity in the tumour cells of nests and tubules. CKAE1/AE3 and CK19 were strongly expressed in all morphological patterns of adamantinoma emphasizing their epithelial origin, while the antibodies to CK8 and CK18 showed a high percentage of negative responses. In osteofibrous dysplasia the epithelial‐like component was much smaller than in adamantinoma and was present in scattered islands composed of a few cells positive for CKAE1/AE3 and CK19 and negative for other keratins. These results suggest that these two lesions are of a similar histogenesis.

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

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.

Role of MMP-9 and its tissue inhibitor TIMP-1 in human osteosarcoma: findings in 42 patients followed for 1-16 years.

Background The activity of matrix metalloproteinases (MMPs) in degrading extracellular matrix is controlled by activation of proenzymes and inhibition of MMP tissue inhibitors (TIMPs). Patients and methods To assess the proteolytic cascade imbalance in malignancy progression, tissue expression and serum levels of MMP-2, MMP-9 and of their inhibitors TIMP-2 and TIMP-1 respectively were evaluated in 42 selected patients with high-grade osteosarcoma (OS). MMP-2, MMP-9, TIMP-2 and TIMP-1 were studied in biopsies by immunohistochemistry and in serum by ELISA test. Patients were subdivided into 3 groups according to their follow up: continuously disease-free, diagnosis of metastasis during follow-up, and metastasis at diagnosis. Results Immunohistochemistry demonstrated an imbalance between MMPs and TIMPs, with a more evident role for MMP-9 than for MMP-2 in tumor progression. TIMP-1 inhibitor in plasma was higher in patients with osteosarcoma than in a control group. This high value of TIMP-1 was particularly evident in the group of patients who later developed metastases and/or local recurrences, and in those with metastases at diagnosis. Interpretation Our findings confirm the protective action of TIMP-1, as MMP inhibitor, but also show its activity as a growth factor underlining its multifunctional role in OS.

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.

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

Aims and Background Ewings 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 Ewings 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 μm 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 ≥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 Ewings sarcoma.

Molecular Diagnosis in Ewing Family Tumors: The Rizzoli Experience—222 Consecutive Cases in Four Years

The Ewings family of tumors (EFTs) are characterized by chimeric transcripts generated by specific chromosomal rearrangements. The most common fusions are between the EWSR1 gene on chromosome 22 and the ETS family of transcription factors; rarely, FUS (on chromosome 16) substitutes for EWSR1. The detection of specific translocations using molecular analysis is now a routine part of the pathological examination of EFT. Here, we report our experience with molecular diagnosis of EFT during the 4 years (2006-2009) at the Rizzoli Institute. We analyzed 222 consecutive tumors with a presumptive diagnosis of EFT using molecular techniques and IHC. We found five distinct types of EWSR1-FLI1 fusion transcripts resulting from translocation t(11;22), three types of EWSR1-ERG transcripts resulting from t(21;22), and one type of t(2;22) resulting in EWSR1-FEV fusion. Molecular investigation validated 92% of cases ultimately diagnosed as EFT; IHC validated 76% of the cases. Thus, despite the difficulties and limitations associated with both molecular and IHC analysis on fresh and formalin-fixed, paraffin-embedded tissue, a combination of these techniques is the best approach to enhancing the accuracy of EFT diagnosis. We also present our method for choosing which molecular techniques to apply. Finally, we collected the most prevalent breakpoints reported in the literature, indicating which exons are involved, the sequence breakpoints, and the NCBI reference sequences.

Altered G1 phase regulation in osteosarcoma

Alterations in the normal cell cycle lead to abnormal cell proliferation and to tumor development. To explore the role of the cyclin D/Cdk4 complex and the retinoblastoma protein (pRb) in the growth and spread of osteoblastic osteosarcoma (OS), 40 tumor samples were selected. In 17 of these cases, lung metastases occurred during follow‐up. Expression of pRb, cyclin D1 and its catalytic subunit, Cdk4, was studied by immunohistochemistry and immunoblotting. As controls, non‐neoplastic tissues surrounding the tumor were used. The expression level and pattern were compared to clinical outcome. Cdk4 was over‐expressed in 80% of OS, independently of clinical outcome, and showed an intense and uniform distribution in tumor cells compared to normal cells. However, co‐immunoprecipitation of Cdk4 with cyclin D1 revealed low levels of cyclin D/Cdk4 complex; 20 of 40 OS examined had a negative or minimal immunostaining for active pRb. The probability of relapse was significantly higher in pRb‐negative than in the ‐positive patients (p < 0.05). The ratio of unphosphorylated/hyperphosphorylated pRb was lower in relapsed patients than in patients with no evident disease, though the difference was not statistically significant. High levels of pRb/cyclin D1 were found in all samples exhibiting functional pRb expression. Our results show that G1 phase deregulation is involved in formation and development of OS. The expression levels of both pRb and cyclin D1 had a clear correlation with clinical outcome, suggesting that these parameters could be used as prognostic markers. Int. J. Cancer 74:518–522, 1997.

CIC–DUX4 fusion-positive round-cell sarcomas of soft tissue and bone: a single-institution morphological and molecular analysis of seven cases

Round‐cell sarcomas lacking specific translocations represent a diagnostic challenge. The aim of this study was to describe seven cases of CIC–DUX4 fusion‐positive sarcomas, including the first reported example arising primarily in bone.