Alba Balladelli

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.

Resection of recurrent pulmonary metastases in patients with osteosarcoma

Surgical resection of lung metastases is widely accepted in osteosarcoma patients. Few data exist on treatment of recurrent pulmonary metastases. The authors of the current study retrospectively analyzed patients with osteosarcoma who received surgery for recurrent lung metastases.

High grade osteosarcoma of the extremities with lung metastases at presentation: Treatment with neoadjuvant chemotherapy and simultaneous resection of primary and metastatic lesions

Between 1986 and 2001, 162 patients with extremity osteosarcoma and lung metastases at presentation, were treated by neoadjuvant chemotherapy, simultaneous resection of primary and, when feasible, secondary lesions followed by chemotherapy.

High grade osteosarcoma of the extremities metastatic to the lung: long-term results in 323 patients treated combining surgery and chemotherapy, 1985-2005.

BACKGROUND Approximately one-third of patients with localized osteosarcoma at presentation relapse as well as about three-fourths of the patients with metastases at diagnosis, about 90% of relapses are lung metastases. The role of lung metastasectomy remains to be determined. PATIENTS and methods: Three hundred and twenty three patients, 88 with resectable lung metastases at diagnosis and 235 with localized disease at presentation who relapsed with lung metastases were treated. RESULTS A total of 498 lung surgeries and 607 thoracotomies were performed. The 5 year overall survival was 37%. Final outcome was significantly related to presence or absence of metastasis, time of first relapse and presence of local recurrences. According to stage of the disease, the rate of a 5 year event-free survival (EFS) was 36% for patients with localized disease who later relapsed and 9% for patients with resectable lung metastases at presentation (p<0.0001). However, there were no differences in EFS between patients who underwent two or three thoracotomies and patients who had four or five thoracotomies (7.5 vs 18.7%, p=0.29). CONCLUSIONS In patients with recurrent resectable pulmonary metastases from high grade osteosarcoma treated with adjuvant or neoadjuvant chemotherapy, thoracotomy should always be considered regardless the number of previous lung relapses and the number of secondary pulmonary lesions.

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.

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.

Imaging patterns in elastofibroma dorsi

INTRODUCTION Elastofibroma dorsi is a rare pseudotumor of the soft tissues. Its clinico-radiologic characteristics lead to a correct diagnosis. MATERIAL AND METHODS We followed 43 patients with elastofibroma dorsi with a confirmed histological diagnosis or on the basis of typical imaging pattern (ultrasound, CT, MR) confirmed by evolution. RESULTS Elastofibroma is prevalent in females, its onset occurs around 60 years of age and is most frequently localized in the deep subscapular region (93%), bilateral in 54% of cases. In 7% it was found in an atypical isolated suprascapular region, in 7% it was synchronous to that in the subscapular region. Four ultrasound patterns were detected: Type I (54%) inhomogeneous fasciculated, Type II (22%) inhomogeneous aspecific, Type III (15%) hyperechogeneous, Type IV (9%) hypoechogeneous. Three patterns were detected at CT and MR: Type A (84%) inhomogeneous fasciculated corresponding to Types I and III and partially to Type II ultrasound pattern, Type B (8%) inhomogeneous aspecific corresponding to Type II ultrasound pattern; Type C (8%) homogeneous isodense or isointense to the muscle corresponding to Type IV ultrasound pattern. CONCLUSION A solid, slow-growing lesion, in the deep periscapular region in females aged between 50 and 60 years, with a typical fasciculated pattern is pathognomonic of elastofibroma dorsi and bilateral location convalidates diagnosis. Ultrasound is sufficient to orientate diagnosis. CT and/or MR are reserved only for non-fasciculated ultrasound patterns, when site is atypical or in candidates for surgery. Biopsy is reserved only in cases where integrated imaging shows a non-fasciculated pattern to differentiate it from other malignant lesions.

Ewing’s sarcoma family tumours: DIFFERENCES IN CLINICOPATHOLOGICAL CHARACTERISTICS AT PRESENTATION BETWEEN LOCALISED AND METASTATIC TUMOURS

Despite local treatment with systemic chemotherapy in Ewings sarcoma family tumours (ESFT), patients with detectable metastases at presentation have a markedly worse prognosis than those with apparently localised disease. We investigated the clinical, pathological and laboratory differences in 888 patients with ESFT, 702 with localised disease and 186 with overt metastases at presentation, seen at our institution between 1983 and 2006. Multivariate analyses showed that location in the pelvis, a high level of serum lactic dehydrogenase, the presence of fever and a short interval between the onset of symptoms and diagnosis were indicative of metastatic disease. The rate of overt metastases at presentation was 10% without these four risk factors, 22.7% with one, 31.4% with two, and 50% for those with three or four factors. We concluded that in ESFT the site, the serum level of lactic dehydrogenase, fever, and the interval between the onset of symptoms and diagnosis are indicators of tumours having a particularly aggressive metastatic behaviour.