Successful arthroscopic treatment of pigmented villonodular synovitis of the knee in a patient with congenital deficiency of plasminogen activator inhibitor-1 and recurrent haemarthrosis.

We report the arthroscopic treatment of pigmented villonodular synovitis (PVNS) in a 13-year-old Japanese boy with congenital partial deficiency of plasminogen activator inhibitor-1 (PAI-1). He was admitted to our hospital with recurrent haemarthrosis of his right knee. Characteristic abnormalities of fibrinolysis included shortened euglobulin lysis time, low PAI-1 activity and low PAI-1 antigen levels. In addition, levels of "active PAI" in the plasma, which is a measure of total PAI bound to exogenous plasminogen activator, were very low. These parameters remained low after venous occlusion. The diagnosis of PVNS was established by synovial membrane biopsy, and arthroscopic synovectomy was performed with adjuvant administration of intravenous tranexamic acid. Subsequent bleeding episodes have been well controlled by oral administration of tranexamic acid on demand.

A method for accurate detection of translocation junctions in Ewing family of tumors.

The Ewing family of tumors (ET) generally contain translocations involving the EWS gene and the FLI or ERG genes. Identification of the translocation confirms the diagnosis of ET. Currently, diagnosis of the translocation is made by several methods. In general, these methods require different primer sets for amplifying different translocations and subsequent efforts to identify the amplified product. The need to employ different sets of primers to amplify different translocation junctions presents some limitations. We have developed a method based on PCR with consensus primers followed by direct automated sequencing of the amplified product. With this method we have correctly determined known as well as unknown ET-associated EWS-FLI and EWS-ERG translocations in appropriate specimens. Use of our consensus primers eliminates the need for separate PCRs to amplify EWS-FLI and EWS-ERG translocation junctions, and because direct sequencing is used for confirming the identity of the amplification product, the accuracy of detection becomes 100%. The method might also accurately diagnose ET-associated translocations other than EWS-FLI and EWS-ERG translocations.

Possible involvement of bcl-2 suppression in wild-type p53 gene-dependent cell growth repression in rat osteosarcoma cells.

We recently obtained 3 cloned cell lines demonstrating the p53 mutation from a lung metastatic nodule of a rat transplantable osteosarcoma. In this study, we applied wild-type p53 gene transfer to the rat osteosarcoma cells by lipofection to investigate the effects on cell growth, expression of genes such as waf1/p21, bcl-2, and bax, and nucleosomal DNA fragmentation due to apoptosis. Reconstitution of the p53 gene inhibits cellular growth, and this growth-suppressive effect is partly due to apoptosis involving bcl-2 gene suppression in this tumor type. This rat osteosarcoma model is similar in biologic behavior to human cases and thus is very suitable for further investigation of tumorigenesis and gene therapy for osteosarcoma.

Delay in administration of CDDP until completion of AGM-1470 treatment enhances antimetastatic and antitumor effects.

The efficacy of cis-diammine dichloroplatinum (CDDP) therapy in combination with continuous administration of angiogenesis inhibitor o-(chloroacetyl-carbamoyl) fumagillol (AGM-1470) was evaluated experimentally using a transplantable rat osteosarcoma line previously established in our laboratory. AGM-1470 (2.5 mg/kg body weight/week) was administered by Alzet osmotic pumps for 2 weeks starting from 7 days after tumor inplantation and CDDP (1.25 mg/kg) was given on days 21 and 24. The number of lung metastatic nodules was counted and the wet weights of the primary tumors were measured 5 weeks after tumor inplantation. Values with administration of CDDP 3 days after discontinuation of AGM-1470 were significantly lower than when the two agents were coadministered (P < 0.05). This animal model should facilitate optimization of the timing of combination therapy.

Telomerase activity correlates with growth of transplantable osteosarcomas in rats treated with cis-diammine dichloroplatinum or the angiogenesis inhibitor AGM-1470.

To determine the role of telomerase activity in the growth of tumors in rats undergoing chemotherapy, a comparison of the volumes of telomerase-positive transplantable osteosarcomas was made in rats treated with the antineoplastic agent cis-diammine dichloroplatinum (CDDP) or the angiogenesis inhibitor O-(chloroacetylcarbamoyl)fumagillol (AGM-1470). Male F344 rats, 8 weeks old, received transplants of macroscopic lung metastatic nodules into the subcutaneous back space and treatment was started on day 14 thereafter. CDDP was injected i.v. at doses of 0, 0.625, 1.25 and 2.5 mg/kg body weight (b.w.) and AGM-1470 was administered at total doses of 0, 2.5, 5 and 10 mg/kg b.w. over 2 weeks by osmotic pumps, also implanted into the subcutaneous back space, but remote from the transplanted tumors. On day 28, all animals were killed for measurement of transplanted tumor size and determination of telomerase activities by telomeric repeat amplification protocol (TRAP) assay. The results showed telomerase activity to be highly correlated with the treated/non-treated (T/C) tumor size ratio (r = 0.96, P < 0.0001). In a second experiment, CDDP at 2.5 mg/kg b.w. and AGM-1470 at 10 mg/kg b.w., these being the most effective doses, were given as in the first experiment, and animals were serially killed on days 14, 21, 28, 35 and 42. Tumors in rats treated with CDDP and AGM-1470 showed 18.2% and 20.5% of the control telomerase activity on days 35 and 21, respectively, when tumor growth was inhibited. However, on day 42, the activities increased to 46.5% and 92.5%, this correlating with re-growth (r = 0.73, P < 0.0001). These results suggest that decline of telomerase activity may be involved in tumor growth retardation induced by chemotherapeutic agents. This possibility clearly warrants further mechanistic studies.

Heterogeneous pattern of gene expression in cloned cell lines established from a rat transplantable osteosarcoma lung metastatic nodule.

We have established three cloned cell lines (COS1NR, COS2NR and COS4NR) from the lung metastatic nodule of a highly metastatic variant of rat transplantable osteosarcoma, C-SLM. All three clones shared the same morphological characteristics and tumorigenicity, but their growth rates in vitro and metastatic ability in vivo differed from each other. Single-strand conformation polymorphism (SSCP) analysis revealed all three clones to have the same p53 gene mutation and parent C-SLM tumor. On the other hand, Northern blot analysis showed a different pattern of expression for the genes, c-fos, c-jun, c-Ha-ras, transin (rat stromelysin), bone Gla protein (osteocalsin) and nm23/NDP kinase. These results indicate the presence of a heterogeneous cell population in terms of the different pattern of gene expression in a lung metastatic nodule of rat osteosarcoma and the present newly established cell lines will be useful for further investigation of the biological behavior of osteosarcomas.

Efficacy of CDDP and AGM-1470 chemotherapy against lung metastasis in rat osteosarcoma depends on the timing of combined administration.

The efficacy of combination therapy with cis-diammine-dichloroplatinum (II) (CDDP) and o-(chloroacetyl-carbamoyl) fumagillol (AGM-1470) was evaluated experimentally using a transplantable rat osteosarcoma line, previously established in our laboratory, with a high potential for metastasis. Tumor-bearing male Fischer 344 rats were administered CDDP (2.5 mg/kg) together with, or after discontinuation of, AGM-1470 treatment (10 mg/kg/body weight/week). When CDDP was administered three days after discontinuation of AGM-1470 the most pronounced antimetastatic effects were observed, although the antitumor effect was approximately the same.

Increased telomerase activity is not directly related to metastatic potential in rat transplantable osteosarcomas.

Previously, we reported the establishment of two transplantable osteosarcomas in rats, one induced by local application of a carcinogen, 4-hydroxyamino quinoline 1-oxide (4-HAQO), and another which developed spontaneously, and their subdivision into four lines with high and low metastatic potential to the lung. In the present study, activation of telomerase was investigated by the telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. Telomerase activity was found to be elevated in all four lines without any link to the metastatic potential. Thus the spontaneous osteosarcoma (SOS) and derived metastatic lesions (S-SLM) demonstrated a 20.1-23.5-fold increase and the chemical carcinogen (4HAQO)-induced osteosarcoma (COS) and metastatic lesions (C-SLM) were 18.4-19.1-fold elevated as compared to the value for abdominal muscle. The results suggest that activation of telomerase occurs in rat osteosarcomas but that it is not directly involved in determining their metastatic potential.

Vascularized fibular graft for reconstruction of the wrist after excision of giant cell tumor.

Seven patients with a giant-cell tumor involving the distal end of the radius were treated with en bloc resection and reconstruction with a free vascularized fibular graft. Two patients with stage 2 disease of Enneking's surgical staging and grade 2 of Campanacci's radiographic grading system were reconstructed with an articular fibular head graft. Five patients with stage 3 and grade 3 disease underwent wrist arthrodesis using fibular shaft transfer. There was radiographic evidence of bone union at the host-graft junctions in all cases. No local recurrence was seen in any of the patients at the most recent follow-up examinations. There were six good and one excellent functional results. Wrist arthroplasty using a vascularized fibula head graft is the best procedure for a stage 2 or grade 2 giant-cell tumor of the distal end of the radius. In cases of stage 3 or grade 3 disease, wrist arthrodesis using a vascularized fibular shaft graft is indicated.

p53 mutation and absence of mdm2 amplification and Ki-ras mutation in 4-hydroxyamino quinoline 1-oxide induced transplantable osteosarcomas in rats.

Previously, we reported the establishment of two transplantable osteosarcomas, one induced by local application of a carcinogen, 4-hydroxyamino quinoline 1-oxide(4-HAQO), and another which developed spontaneously in rats, and their subdivision into four lines with high and low metastatic potential to the lung. In the present study, mutations of p53 and Ki-ras genes were investigated by PCR and SSCP followed by direct sequencing, and the amplification of the mdm2 gene was assessed by Southern blot analysis. Mutations of p53 in exon 7 were detected in 4-HAQO-induced transplantable osteosarcomas, but not their spontaneous counterparts, irrespective of the metastatic potentials. Direct sequencing revealed a CGC to CAC transition with an amino acid change of Arg to His, at codon 246. Neither Ki-ras mutations nor mdm2 amplification were detected in any of the transplantable tumors. The results suggest that while p53 mutations occurred during osteosarcoma development by 4-HAQO without mdm2 amplification and Ki-ras mutation does not contribute to osteosarcoma development in rats.

Closed rupture of the flexor digitorum profundus tendon of the little finger caused by calcification of the triangular-fibrocartilage.

A case of rupture of flexor digitorum profundus tendon of the little finger caused by calcification of the triangular fibrocartilage (TFC) is reported. At operation, a round defect of the TFC and rupture of the flexor digitorum profundus tendon (FDP) of the little finger were observed. The defect of TFC was repaired using the palmaris tendon and FDP of the little finger was woven into FDP of the ring finger. Eleven months after operation, the patient had almost full flexion and extension of the distal and proximal interphalangeal joints.

[A device for rapid elevation of plasma methotrexate (MTX) concentration and its maintenance in high-dose MTX therapy].

For 5 osteosarcomas and one synovial sarcoma, 24 courses of high-dose methotrexate (MTX) therapy were performed. At MTX doses of 300 mg/kg with several infusion patterns, the plasma MTX concentrations were measured by fluorescence polarization immunoassay. In the various types of infusions, five hours oblique and five hours bolus infusions were well maintained at 1,000 micromol/l for several hours. In particular, five hours bolus infusion of MTX needed only 20 minutes to reach 822 micromol/l and maintained 1,000 micromol/l during almost the entire infusion period. Optimization of chemotherapy for sarcomas by MTX requires individual adaptation of the infusion pattern.

Efficacy of slow-releasing anticancer drug delivery systems on transplantable osteosarcomas in rats.

New drug delivery systems for cis-diamminedichloroplatinum (CDDP) incorporated into vehicles, such as polymethylmethacrylate (PMMA), fibrin glue (F.G.), alpha-tricalciumphosphate (TCP) and ethylenevinyleacetate copolymer (Polymer) were examined using a rat osteosarcoma model. The materials containing CDDP were directly implanted into the tumors or subcutaneous tissue of rats, and the inhibitory effects on tumor growth and lung metastasis were evaluated. Data on in vitro kinetics of CDDP release revealed good results for both TCP and F.G., and the release pattern from TCP to be most appropriate for a slow-releasing drug delivery system. This was supported by the results of the implantation experiments, whereby the direct implantation of TCP containing CDDP (CDDP-TCP) into tumors, gave significantly better inhibitions of tumor growth and metastasis than either non-treatment (P < 0.01) or subcutaneous implantation (P < 0.05). In a second experiment, using different administration procedures, different inhibitory effects on tumor growth and lung metastatic potency were observed with intra-arterial and intravenous CDDP administration, as well as with CDDP-TCP implanted subcutaneously. Suppression effects of CDDP (10 mg/kg)-TCP directly implanted into tumors were equal to those of intra-arterial (2.5 mg/kg) and intravenous (5.0 mg/kg) administrations. The present results suggest CDDP-TCP implantation to be effective as a slow-release drug delivery system for inhibiting tumor growth and metastasis, and that it should be a useful adjuvant to conventional i.v. or i.a. chemotherapy.

Efficacy of the angiogenesis inhibitor O-(chloroacetyl-carbamoyl)fumagillol (AGM-1470) on osteosarcoma growth and lung metastasis in rats.

The efficacy of the anti-angiogenic agent, O-(chloroacetyl-carbamoyl)fumagillol (AGM-1470), against primary tumor growth and spontaneous lung metastasis was evaluated experimentally using a transplantable osteosarcoma line in rats previously established in our laboratory. Male Fischer 344 rats bearing the tumor with a high potential for metastasis received intermittent or continuous subcutaneous administrations of AGM-1470. Both treatment regimens resulted in significant inhibitions of spontaneous lung metastasis and primary tumor growth in a dose-dependent manner, with continuous administration of AGM-1470 exerting the most pronounced inhibitory effects on both parameters.

Ultrastructural cytochemical demonstration of proteoglycans and calcium in the extracellular matrix of chondroblastomas.

To clarify the characteristics of the extracellular matrix of chondroblastomas, six cases were studied under the electron microscope, with special reference to proteoglycans and calcium in the cellular areas. In ruthenium hexammine trichloride (RHT)-stained sections the matrix was observed to be composed of rounded or polygonal fine granules and unbanded thin filaments that appeared to link neighboring granules together. Treatment with potassium-pyroantimonate showed intracellular accumulation of precipitates, mainly localized within the cisternae of the rough endoplasmic reticulum as well as in the extracellular matrix. The presence of calcium in the precipitates was confirmed using x-ray energy dispersive analysis. These findings, similar to characteristic features observed in calcifying systems, support the theory that chondroblastomas are of chondrogenic origin.

Electron microscopic evidence of a viral nature for osteoclast inclusions in Paget's disease of bone.

Circumstantial evidence from electron microscopic and immunological studies support the view that Paget's disease of bone represents a slow virus infection. However, there is only limited information available regarding its electron microscopic, enzyme and immunocytochemical characteristics. Two cases were studied using electron microscopy with particular emphasis on the inclusions in osteoclasts. Detailed ultrastructural and cytochemical studies including immuno-electron microscopy were performed. Some osteoclasts demonstrated specific virus-like structures composed of aggregations of microtubules in the nucleus and cytoplasm. The structures were easily digested by trypsin or protease, and were sensitive to RNase, which provided substantial evidence of a proteinaceous nature and inclusion of ribonucleic acid. Immunocytochemical examination identified binding of anti-respiratory syncytial virus and anti-measles virus antibodies in the tissue obtained from one of the two cases examined. The presence of viral antigens in structures in the cytoplasm of Pagetic osteoclasts supports the theory of paramyxovirus involvement in this disease.

Increased expression of nucleoside diphosphate kinase/nm23 and c-Ha-ras mRNA is associated with spontaneous lung metastasis in rat-transplantable osteosarcomas.

The relationship between expression of nucleoside diphosphate kinase (NDP kinase)/nm23, c-Ha-ras, and c-myc genes and metastatic potential was assessed in rat-transplantable osteosarcoma lines, derived from spontaneous and chemical carcinogen (4-hydroxyamino quinoline 1-oxide)-induced osteosarcomas in Fischer 344/NS1c rats. These osteosarcomas possess metastatic potential and highly metastatic lines spontaneous osteosarcoma-selected lung metastatic lesions and 4-hydroxyamino-quinoline 1-oxide-induced osteosarcoma-selected lung metastatic lesions were respectively established by selectively transplanting lung metastatic lesions. Northern blot analysis revealed that the levels of NDP kinase/nm23 and c-Ha-ras gene expression were increased in line with metastatic ability; thus transcript levels were remarkably greater in both spontaneous osteosarcoma-selected lung metastatic lesions and 4-hydroxyamino-quinoline 1-oxide-induced osteosarcoma-selected lung metastatic lesions highly metastatic lines than in their respective low metastatic spontaneous and chemical carcinogen (4-hydroxyamino quinoline 1-oxide)-induced osteosarcoma counterparts. c-myc mRNA expression was observed in all tumor lines, without any correlation with metastatic ability. Southern blot analysis did not show evidence of gross rearrangement or amplification of NDP kinase/nm23, c-Ha-ras, or c-myc genes suggesting regulation of their gene expression at the transcriptional and/or posttranscriptional level. These results indicate that NDP kinase/nm23 and c-Ha-ras might be cooperatively involved in a positive manner in signal transduction processes, especially involving G-protein reactions, responsible for metastasis of rat-transplantable osteosarcomas.