Expression of matrix metalloproteinases 1, 2, 9 and their tissue inhibitor-1 in cartilage-forming osteal tumors.

The expression of MMP-1, -2, -9 and TIMP-1 was studied in 10 benign cartilage-forming osteal tumors (5 osteochondromas and 5 chondromas) and 39 chondrosarcomas (14 central, 4 periosteal, 7 dedifferentiated, and 14 secondary tumors). No expression of MMP and TIMP-1 was detected in benign cartilage-forming osteal tumors. In chondrosarcomas, the expression of MMP-1 was detected in 84.6%, of MMP-2 in 71.8, of MMP-9 in 97.4, and of TIMP-1 in 82.4% cases, the levels of expression of these markers varied from 10 to 60%. The expression of MMP-1 was not associated with patient gender, maximum size and degree of differentiation of the tumor, but was linked with age. The expression of MMP-1 was more often detected in central and dedifferentiated chondrosarcomas; the expression of MMP-1(+) was significantly associated with 3-year relapse-free and 5-year overall survival of the patients. The expression of MMP-1 in the tumor was associated with unfavorable course of the disease. The values of MMP-2 expression in chondrosarcomas did not reflect the main clinical morphological characteristics of the disease and its prognosis. The level of MMP-9 protein expression in chondrosarcomas ≥40% is prognostically unfavorable, while <40% is a favorable factor for 3-year relapse-free survival. The risk of disease relapse within 1 year after the beginning of therapy was maximum in T3 tumors with expression of MMP-9 protein ≥40%. No relationships between the parameters of TIMP-1 expression in chondrosarcomas and the main clinical morphological characteristics of the disease and its prognosis were detected.

Decreased EXT expression and intracellular accumulation of heparan sulphate proteoglycan in osteochondromas and peripheral chondrosarcomas.

Mutational inactivation of EXT1 or EXT2 is the cause of hereditary multiple osteochondromas. These genes function in heparan sulphate proteoglycan (HSPG) biosynthesis in the Golgi apparatus. Loss of heterozygosity of the EXT1 locus at 8q24 is frequently found in solitary osteochondromas, whereas somatic mutations are rarely found. We investigated the expression of EXT1 and EXT2 (quantitative RT-PCR) and of different HSPGs (immunohistochemistry) in solitary and hereditary osteochondromas and in cases with malignant progression to secondary peripheral chondrosarcoma, in relation to possible mutations and promoter methylation. The mutation status of patients with multiple osteochondromas correlated with decreased EXT1 or EXT2 expression found in their resected tumours. We could not show somatic point mutations or promoter hypermethylation in 17 solitary tumours; however, EXT1 expression was decreased in 15 cases, whereas EXT2 was not. Intracellular accumulation of syndecan-2 and heparan sulphate-bearing isoforms of CD44 (CD44v3) was found in most tumours, which concentrated in the Golgi apparatus as shown by confocal microscopy. This contrasted with the extracellular expression found in normal growth plates. In conclusion, mutational inactivation of either EXT1 or EXT2 leads to loss of mRNA expression of the corresponding gene. We hypothesize that loss of EXT expression disrupts the function of the EXT1/2 complex in HSPG biosynthesis, resulting in the intracellular accumulation of HSPG core proteins that we found in these tumours.

Plasminogen activators and their inhibitor in bone tumors and tumor-like damages.

Expression of urokinase- and tissue-type plasminogen activators and their inhibitor PAI-1 in the cytosolic fraction of 20 osteosarcomas, 20 chondrosarcomas, 13 giant-cell bone tumors, 5 Ewing's sarcomas, and 7 osteochondral exostoses was studied by enzyme immunoassay. The content of urokinase-type plasminogen activator increased, while the concentration of tissue-type plasminogen activator decreased in bone tumors of various histological compositions compared to osteochondral exostoses. A positive correlation was found between PAI-1 content and the volume of osteo- and chondrosarcomas. Expression of urokinase-type plasminogen activator increased in patients with primary osteosarcomas characterized by early generalization of the pathological process.

Chondrosarcoma is not characterized by detectable telomerase activity.

Reactivation of telomerase, an enzyme which elongates human telomeres, is associated with cell immortilization. In approximately 90% of malignant tumours telomerase activity can be demonstrated, whereas in benign tumours it is mostly absent. Chondrosarcomas are relatively rare malignant cartilaginous neoplasms. A small number of chondrosarcomas located centrally in bone arise secondarily to an enchondroma, while the majority of chondrosarcomas developing from the surface arise within the cartilage cap of an osteochondroma. The histological distinction between a benign lesion and low-grade chondrosarcoma is generally considered difficult. To investigate whether the progression towards chondrosarcoma is characterized by reactivation of telomerase activity, this study determined telomerase activity in ten enchondromas, five osteochondromas, and 37 chondrosarcomas using the TRAP assay. In all tumour samples except one, telomerase activity was absent. By adding tumour lysates to the positive control, an increasing inhibition of telomerase activity was found with an increasing chondroid matrix, suggesting that it may contain inhibitory factors. Inhibition due to endogenous RNAse or Taq-polymerase inhibitors was excluded. The lack of detectable telomerase activity in the high-grade component of a dedifferentiated chondrosarcoma without matrix favours the possibility that telomerase is truly absent. Either its true absence or inhibitory effects disabling telomerase detection exclude the telomerase TRAP assay as a diagnostic tool in the differential diagnosis of benign and low-grade malignant cartilaginous tumours.

Telomerase activity in benign bone tumors and tumor-like lesions.

To assess the role and status of telomerase activity in benign bone tumors and tumor-like lesions, we performed telomerase assays in four giant cell tumors of bone, four fibrous dysplasias, three osteochondromas, three aneurysmal bone cysts, two osteoblastomas, one juvenile bone cyst and one myositis ossificans. A very sensitive non-radioactive TRAP assay was applied. Low level activity was detected in 7 of 18 tumor samples (38.9%), and high level activity was not detected in any of the cases. Telomerase activity was observed in all patients with osteochondromas, in two of the three aneurysmal bone cysts, in one of the four giant cell tumors of bone and in one of the four fibrous dysplasias, but not in osteoblastomas, juvenile bone cyst and myositis ossificans. Although the origin of this enzyme is still unclear, it might play a role in precancerous immortalization of benign bone tumors. Other possible reasons explaining the occurrence of telomerase activity, such as migrating lymphocytes or contamination of immortalized non-tumor cells, should not be ruled out. Telomerase activity, however, does exist in those samples having no malignant phenotype, for which reason telomerase assays are not always useful for the clinical and diagnostic approach in benign bone tumors. Determination of the telomerase status in benign lesions may contribute to a better understanding of the regulation mechanism of telomerase activity during progression of bone tumors.

Immunolocalization of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human chondrosarcomas.

We have immunohistochemically examined the localization of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in human chondrosarcomas (CHS) (23 cases) and benign chondroid lesions (BCL) (16 cases of osteochondromas and 11 cases of enchondromas). In CHS, all the MMPs and TIMPs examined were positive. Among them, MMP-1 was immunolocalized in more than 90% of both CHS and BCL, but positive score of MMP-1 was significantly higher in CHS than that in BCL (p < 0.01). Compared with BCL, CHS expressed MMP-3 at a low level, and more often positive in MMP-9. It is possible that chondrosarcoma might have a tendency to lose the ability to secrete MMP-3, which is a metalloproteinase that can degrade cartilage proteoglycans and is related to normal cartilage turnover. MMP-2, TIMP-1 and TIMP-2 were immunolocalized in more than 70% of the cases of both BCL and CHS, but the positive scores of these were not statistically different between the two groups. Interestingly, in several cases of CHS, both MMP-1 and MMP-9 immunostains were observed preferentially within the cells at the marginal areas of cartilaginous lobules. These findings suggest that increased expression of MMP-1 and MMP-9 and decrease in MMP-3 expression are associated with the malignant phenotype of the cartilaginour tumors.

Selective increase in expression of isoform PP1 gamma 1 of type-1 protein phosphatase in chondrosarcoma cells.

The expression of the two catalytic subunits of protein phosphatase (PP) type 1 PP1 gamma 1 and PP1 delta was examined in 4 cases of osteochondroma and 4 cases of enchondroma as a benign cartilaginous tumor, and 4 cases of chondrosarcoma as a malignant cartilaginous tumor using immunohistochemical analysis. The percentage of tumor cells stained positively with antiserum against PP1 catalytic subunit isoform PP1 gamma 1 were significantly higher in chondrosarcoma than in osteochondroma and enchondroma. Furthermore, chondrosarcoma showed markedly high S-phase fraction in the cell cycle of tumor cells, as compared to osteochondroma and enchondroma. These results suggest that PP1 gamma 1 is involved in the accelerated growth of malignant cells in chondrosarcoma.