miRNA­218 targets multiple oncogenes and is a therapeutic target for osteosarcoma.

Survivin is overexpressed in various cancers and is correlated with treatment resistance and prognosis. MicroRNAs (miRNAs) directly regulate several target genes and are potential therapeutic agents for various cancers. The present study evaluated multiple gene targets of miR­218, including survivin, in osteosarcoma and compared the anti­tumor effects of miR­218 with those of YM155, an anti­survivin agent. It assessed the expression levels of miR­218 and survivin in osteosarcoma and osteoblast cell lines, as well as the proliferative, migratory and invasive capacities of cells following treatment with miR­218 or YM155. The form of cell death was assessed using fluorescence­activated cell sorting analysis to examine the expression of invasion ability­related genes. Osteosarcoma cell lines were subcutaneously injected into immunodeficient mice; the mice were then treated with miR­218 or YM155 to assess the anti­tumor effects of these agents. The results showed that miR­218 was downregulated, whereas survivin was overexpressed in the osteosarcoma cell line compared with normal osteoblast cells. The expression of survivin was suppressed upon overexpression of miR­218 (miR­218 group) or administration of YM155 (YM155 group), leading to apoptosis and inhibition of osteosarcoma cell proliferation. Invasion and migration abilities were inhibited in the miR­218 group, but not in the YM155 group. In the animal model, both the miR­218 and YM155 groups showed a reduced tumor volume and decreased survivin expression. In osteosarcoma, miR­218 showed a wider range of therapeutic efficacy compared with YM155, suggesting that miR­218 should be evaluated as a treatment target.

A bent needle tip during irrigation for enchondroma of the distal phalanx: a new curettage tool.

BACKGROUND: We employed a novel curettage tool, a bent needle tip, during irrigation for enchondroma of the distal phalanx. This study aimed to evaluate our new curettage tool for treating enchondroma of the distal phalanx. METHODS: Seven distal phalanx enchondromas were pathologically diagnosed at our institute. We evaluated age, gender, tumor location, affected side, clinical symptoms, Takigawa classification, size, recurrence, complications, residual pain, Tordai score, and follow-up period. We bent an 18G needle tip connected to an extension tube and syringe. The bent needle was inserted through the small hole, and the cavity for bone grafting was adequately filled with injectable calcium phosphate cement through the small hole. RESULTS: There were five centric-type and two giant-type tumors, with a mean size of 52.7%. All patients had clinical symptoms at the initial presentation. All patients showed complete bone healing within 3 months on post-radiological examinations and were Grade 1 according to the Tordai score. CONCLUSIONS: This tool is extremely simple, and both the incision and the cortical window can be small. We recommend a bent needle tip, easily devised in any hospital, as a curettage tool for treating enchondroma in small bones, especially of the distal phalanx.

A thoracic venous aneurysm mimicking a chronic expanding haematoma.

Thoracic venous aneurysms are rare, and bleeding is possible. A 9-year-old female patient presented with a thoracic wall mass. No blood flow was observed in the mass, and a chronic expanding haematoma was suspected based on the differential diagnosis. However, the venous structure was identified in the wall of the mass on pathological examination, and the diagnosis of the venous aneurysm was thereby established. Because the venous aneurysm contains fresh blood and bleeding can be profuse when such lesions are not properly handled during a surgical procedure, making the visibility of the surgeon poor, the venous aneurysm must be included in the differential diagnosis.

MiR-1 Suppresses Proliferation of Osteosarcoma Cells by Up-regulating p21 via PAX3.

BACKGROUND/AIM: miRNA-1(miR-1) is down-regulated in various cancer cells including osteosarcoma cells. This study was conducted to analyze the function of miR-1 in osteosarcoma cells. MATERIALS AND METHODS: miR-1 expression in osteosarcoma cells was evaluated by qRT-PCR. Cell proliferation was evaluated after transfecting miR-1 by WST8 assay and FACS analysis, both in vitro and in vivo. RESULTS: Overexpression of miR-1 suppressed cell proliferation and induced cell-cycle arrest in the G0-G1 phase by increasing p21 levels via a p53-independent pathway. Overexpression of miR-1 down-regulated PAX3, a potential p21-regulating gene. Moreover, knockdown of PAX3 suppressed cell proliferation by increasing p21 levels, and induced arrest at the G0/G1 phase. Administration of miR-1 showed an in vivo antitumor effect. CONCLUSION: Overexpression of miR-1 suppressed cell proliferation and induced arrest in the G0/G1 phase by increasing p21 levels via a p53-independent pathway through PAX3 suppression. These results indicate that miR-1 could be a therapeutic target for osteosarcoma.

Two Revision Surgeries on Cemented Custom-made Tumor Prostheses.

We performed revision surgery in 2 patients for stem fracture of a cemented tumor prosthesis that occurred more than 25 years after the initial surgery. For revision, the global modular replacement system (GMRS) was used. However, as bone cement in the bone could not be adequately removed, stems with respective diameters of 11 and 12.5 mm were used. In revision surgery for cemented tumor prostheses, adequate removal of residual bone cement is optimal. However, when there is a risk of fracture, it may be appropriate to insert a thicker stem after reaming the femoral canal as much as possible, and then fix the stem using the cement-in-cement method.

Lung squamous cell carcinoma with brachial soft tissue metastasis responsive to gefitinib: Report of a rare case.

Metastasis of lung cancer to soft tissue is rare and patient outcomes are generally poor. There are no reports describing soft tissue metastasis in lung squamous cell carcinoma (SCC), in which gefitinib treatment was effective not only for the primary tumor but also the metastatic lesion. A 61-year-old Asian woman presented to our facility with pain and a mass in the brachium. An additional tumor was identified in the lung. As we suspected soft tissue metastasis of lung cancer, an incisional biopsy was performed, yielding a diagnosis of SCC. The brachial tumor continued to grow and became exposed at the biopsy site when the incisional wound dehisced. Because the biopsied specimen was positive for an epidermal growth factor receptor (EGFR) gene mutation, we commenced gefitinib administration. This treatment resulted in the rapid shrinkage of both the brachial metastasis and the primary tumor, followed by healing of the wound. Therefore, tyrosine kinase inhibitors should be used for cases that present EGFR activating mutations independently from the presence of skin and soft tissue metastases.

Secondary malignant giant cell tumor of bone due to malignant transformation 40 years after surgery without radiation therapy, presenting as fever of unknown origin: a case report.

BACKGROUND: Malignant transformation of giant cell tumors of bones, that is, secondary malignant giant cell tumor of bone, is rare. The most common symptoms are local pain and swelling. There are no prior reports of giant cell tumor of bone with fever of unknown origin at the onset. Here we present a case of a secondary malignant giant cell tumor of bone due to malignant transformation 40 years after surgery without radiation therapy, presenting as fever of unknown origin. CASE PRESENTATION: A 75-year-old Asian man presented with a 3-week history of continuous pyrexia and left knee pain and swelling. He had been diagnosed at age 35 years with a giant cell tumor of bone of his left distal femur and underwent bone curettage and avascular fibula grafting at that time. Postoperative radiation therapy was not performed. He remained recurrence-free for 40 years after surgery. At age 75, histopathological findings suggested a secondary malignant giant cell tumor of bone. The tumor specimen expressed tumor necrosis factor-α. Neoplastic fever was suspected, and a naproxen test was conducted. His pyrexia showed immediate resolution. Surgery was performed under a diagnosis of a secondary malignant giant cell tumor of bone with neoplastic fever. His pyrexia and inflammatory activities diminished postoperatively. CONCLUSIONS: This is the first reported case, to the best of our knowledge, of the detection of a secondary malignant giant cell tumor of bone based on fever of unknown origin after long-term (40 years) follow-up. After curettage and bone grafting, giant cell tumor of bone may transform to malignancies within a few years or even decades after surgery. Therefore, meticulous follow-up is essential. The fever might be attributable to the tumor releasing inflammatory cytokines. Not only pain and swelling but also continuous pyrexia may suggest the diagnosis of a secondary malignant giant cell tumor of bone.

MicroRNA-155 expression is independently predictive of outcome in chordoma.

BACKGROUND: Chordoma pathogenesis remains poorly understood. In this study, we aimed to evaluate the relationships between microRNA-155 (miR-155) expression and the clinicopathological features of chordoma patients, and to evaluate the functional role of miR-155 in chordoma. METHODS: The miRNA expression profiles were analyzed using miRNA microarray assays. Regulatory activity of miR-155 was assessed using bioinformatic tools. miR-155 expression levels were validated by reverse transcription-polymerase chain reaction. The relationships between miR-155 expression and the clinicopathological features of chordoma patients were analyzed. Proliferative, migratory and invasive activities were assessed by MTT, wound healing, and Matrigel invasion assays, respectively. RESULTS: The miRNA microarray assay revealed miR-155 to be highly expressed and biologically active in chordoma. miR-155 expression in chordoma tissues was significantly elevated, and this expression correlated significantly with disease stage (p = 0.036) and the presence of metastasis (p = 0.035). miR-155 expression also correlated significantly with poor outcomes for chordoma patients (hazard ratio, 5.32; p = 0.045). Inhibition of miR-155 expression suppressed proliferation, and the migratory and invasive activities of chordoma cells. CONCLUSIONS: We have shown miR-155 expression to independently affect prognosis in chordoma. These results collectively indicate that miR-155 expression may serve not only as a prognostic marker, but also as a potential therapeutic target in chordoma.

Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system.

Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR-Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U-2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR-Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR-Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma.

Inhibition of human osteosarcoma cell migration and invasion by a gene silencer, pyrrole-imidazole polyamide, targeted at the human MMP9 NF-κB binding site.

Osteosarcoma is one of the most prevalent bone tumors, occurring mostly in adolescence. However, no noticeable progress has been achieved in developing new therapeutic agents for this disease. Matrix metalloproteinase 9 (MMP9), a type IV collagenase, is a known anticancer target and is overexpressed in osteosarcomas. MMPs can degrade components of the extracellular matrix and are known to be involved in tumor invasion and metastasis. In the present study, we designed and synthesized a pyrrole-imidazole polyamide (HN.49), a gene-silencing agent that specifically targets the nuclear factor-kappa B (NF-κB) binding site of the human MMP9 promoter. We then examined the effect of HN.49 on the enzyme activity of MMP9 and the migration activity of osteosarcoma cells in vitro. It was clearly shown that HN.49 polyamide reduced the expression level of MMP9 mRNA and the enzymatic activity of MMP-9 in SaOS-2 cells. Moreover, HN.49 polyamide inhibited migration and invasion by SaOS-2 cells in in vitro wound-closure and matrigel-invasion assays. These results indicate that HN.49 may be a potential therapeutic agent for inhibiting the invasion and metastasis of osteosarcoma.

MicroRNA-1 (miR-1) inhibits chordoma cell migration and invasion by targeting slug.

Recent studies have revealed that expression of miRNA-1 (miR-1) is frequently down-regulated in several cancer types including chordoma. Identifying and validating novel targets of miR-1 is useful for understanding the roles of miR-1 in chordoma. We aimed to further investigate the functions of miR-1 in chordoma. Specifically, we assessed whether restoration of miR-1 affects cell migration and invasion in chordoma, and focused on the miR-1 potential target Slug gene. Migratory and invasive activities were assessed by wound healing and Matrigel invasion assays, respectively. Cell proliferation was determined by MTT assay. Slug expression was evaluated by Western blot, immunofluorescence, and immunohistochemistry. Restoration of miR-1 expression suppressed the migratory and invasive activities of chordoma cells. Transfection of miR-1 inhibited cell proliferation both time- and dose-dependently in chordoma. MiR-1 transfected cells showed inhibited Slug expression. Slug was over-expressed in chordoma cell lines and advanced chordoma tissues. In conclusion, we have shown that miR-1 directly targets the Slug gene in chordoma. Restoration of miR-1 suppressed not only proliferation, but also migratory and invasive activities, and reduced the Slug expression in chordoma cells. These results collectively indicate that miR-1/Slug pathway is a potential therapeutic target because of its crucial roles in chordoma cell growth and migration.

Prognostic significance of miRNA-1 (miR-1) expression in patients with chordoma.

Reliable prognostic biomarkers for chordoma have not yet been established. Recent studies revealed that expression of miRNA-1 (miR-1) is frequently downregulated in several cancer types including chordoma. The goal of this follow-up study is to investigate the expression of miR-1 as a prognostic biomarker and further confirm the functional role of miR-1 in chordoma cell growth and proliferation. We determined the relative expression levels of miR-1 and Met in chordoma tissue samples and correlated those to clinical variables. The results showed that miR-1 was downregulated in 93.7% of chordoma tissues and expression was inversely correlated with Met expression. miR-1 expression levels also correlated with clinical prognosis. To characterize and confirm the functional role of miR-1 in the growth and proliferation of chordoma cells, miR-1 precursors were stably transfected into chordoma cell lines UCH-1 and CH-22. Cell Proliferation Assay and MTT were used to evaluate cell growth and proliferation. Restoring expression of miR-1 precursor decreased cell growth and proliferation in UCH-1 and CH-22 cells. These results indicate that suppressed miR-1 expression in chordoma may in part be a driver for tumor growth, and that miR-1 has potential to serve as prognostic biomarker and therapeutic target for chordoma patients.

Long-term outcome following surgical treatment of sacral chordoma.

BACKGROUND: Sixteen sacral chordoma surgeries performed at a single institution during the 1983-2008 period were retrospectively studied. Our aim is to assess surgical treatment and long-term outcomes. METHODS: Fifteen patients underwent primary wide excision, and one intralesional excision using ethanol for local control and radiation therapy (RT). A combined anteroposterior approach for large tumors above S2, and wide excision was performed with the modified threadwire-saw (MT-saw) after 1997. RESULTS: Fourteen of the 15 patients had wide margins, one a wide margin with contamination. The MT-saw was facilitated sacral excision with wide margins. Eleven patients are alive for 5-28 years. Five patients died before 10 years, two patients experienced sepsis, and one of another disease. Two patients died of local recurrence (LR) and another of multiple metastases after intralesional excision and wide excision with contamination, respectively. LR and complications occurred 4 each of 11 patients with tumors ≥ 10 cm, neither with tumors < 10 cm. The overall 5- and 10-year survival rate with wide surgical margins was 13/16 (81.3%) and 8/13 (61.5%). CONCLUSIONS: A combined anteroposterior approach for large tumors, and the MT-saw facilitates sacral excision with wide margins. Wide excision is recommended for younger patients.

Revision of tumor prosthesis of the knee joint.

BACKGROUND: Among 40 patients with primary malignant tumors of the knee joint who underwent reconstruction of the affected limb with tumor prosthesis, revision was required in 7 due to stem breakage or loosening. SUBJECTS AND METHODS: In the 7 cases undergoing revision, conditions and background factors at the time of breakage, the breakage site, time of revision, models of previous and new prostheses, stem diameters before and after revision, details of the revision (blood loss, operative time), and the presence or absence of adjuvant therapy were determined. RESULTS: The replacement site was the distal femur in 5 and proximal tibia in 2. Revision was performed 6 years and 2 months after the previous prosthesis placement on average. The broken prosthesis model was KMFTR in 4 and HMRS and the physio-hinge type in one each. Revision due to loosening was performed in a case requiring replacement with Growing Kotz prosthesis. The model was switched to HMRS in 3, and the stem diameter was changed to 12 mm in 3 KMFTR breakage cases. The mean stem diameters were 11.2 and 10.2 mm in the non-revision and revision groups. The respective resection rates were 36 and 45%. The mean functional evaluation was 70.1% before and 76.2% after revision. CONCLUSION: To reduce the risk of tumor prosthesis breakage, the amount of bone resection should be limited to 30% or less in the affected bone, the stem diameter should be at least 12 mm, and the stem shape should be fitted to the anatomical shape of the femur.

A comparative study for wide excision of malignant tumors distal to S2.

BACKGROUND AND AIM: We conducted a retrospective study comparing 5 patients (Group A) who underwent posterior excision of tumors distal to S2 using a modified threadwire saw (MT-saw) with 5 similar patients (Group B) who underwent tumor excision using chisels and airtomes. PATIENTS AND METHODS: The data of 10 patients were obtained from intra-operative records. A flexible silver guide probe connected to an MT-saw by a suture thread was devised for use in osteotomy passing through the S1 sacral canal in the lateral sacrum. Operative time, blood loss and excisional margins were compared between the two groups. RESULTS: Group A had a shorter average operative time (2 hours 24 minutes) and smaller average blood loss (2,124.6 ml) than Group B. In Group A, one patient incurred an S1 nerve root injury, and all patients had wide histological margins. In Group B, one patient had an S1 nerve root injury and another tumor contamination due to a fracture. CONCLUSION: The MT-saw greatly facilitated lateral sacral osteotomy and reduced the risk of tumor cell spread, permitting faster, safer excision with a wider margin.

Survivin expression levels as independent predictors of survival for osteosarcoma patients.

Survivin expression has been detected in various cancers and correlations have been recognized between the level of expression of this gene in tumors and prognosis. However, the aforementioned authors did not evaluate correlations between prognosis and survivin expression levels using surgically resected samples. In this study, we retrospectively investigated outcomes by examining the correlations between expression of this gene and clinicopathological parameters. Biopsy and resected specimens from which paraffin-embedded tissues could be extracted, were available from 16 patients in our hospital. We used the RT-PCR method and conducted a densitometric analysis to determine the ratio of survivin relative to h-GAPDH as an internal marker. Expression of survivin mRNA was detected in all samples. There was a significant negative correlation between survivin expression levels and duration of follow up, in months, using the Spearman's rank for the initial biopsy samples (rho=-0.775, p<0.01) and those obtained after chemotherapy (rho=-0.687, p<0.01). Moreover, Cox multivariate regression identified the survivin expression levels in both biopsy and post-chemotherapy samples as independent predictors of survival. We conclude that survivin levels in both initial biopsy and post-chemotherapy samples are useful prognostic indicators.

Clinical significance of a wide excision policy for sacrococcygeal chordoma.

PURPOSE: Surgical treatment of sacrococcygeal chordoma is very difficult and the recurrence rate is high. We investigated the outcomes of wide excision in 12 sacrococcygeal chordoma patients treated between 1983 and 2003. METHODS: The 12 patients underwent primary wide excision. The conventional procedure was wide excision employing a chisel and airtome for sacral tumours, and in 1997 a new threadwire saw procedure was introduced. All 12 patients were included in a retrospective analysis of tumour- and treatment-related variables, and outcomes. RESULTS: Six patients underwent surgery with a combined anterior-posterior approach at the cephalad sacral border. In 12 patients, 11 lesions were broadly excised, and in the 1 remaining patient the intralesional margin showed tumour contamination due to a fracture macroscopically. The latter patient received post-operative radiation therapy, but suffered a recurrence 8.0 years later. Three patients died before final follow-up: one from subsequent metastases, two elderly patients from complications. For the 10 chordoma patients, i.e. excluding the two who died due to complications, the 5, 10 and 20 years recurrence rates were 100, 66.7 and 66.7%, respectively. For the 12 patients, overall 5, 10 and 20 years survival rates were 83.3, 55.6 and 55.6%, respectively. CONCLUSIONS: Our results suggest that large chordoma should be widely excised, using a modified threadwire saw, with a combination of anterior-posterior procedures.

Survivin as a prognostic factor for osteosarcoma patients.

PURPOSE: Survivin is one of the apoptosis inhibitor genes and is rarely expressed in adult -tissues. However, survivin expression has been detected in various human cancers and -correlations have been recognized between the level of expression of this gene in tumors and prognosis. In this study, we investigated the correlations between survivin mRNA expression in osteosarcoma tissues and clinicopathological parameters. METHODS: There were 22 osteosarcoma patients in our hospital with paraffin-embedded -tissues which could be extracted from biopsy specimens. We used the RT-PCR method after extracting total RNA and conducted a densitometric analysis to determine the ratio of survivin relative to h-GAPDH as an internal marker. RESULTS: Expression of survivin mRNA was detected in all osteosarcoma samples. Patients with metastasis had high survivin mRNA levels in initial biopsy specimens (p<0.01). Moreover, there was a statistically significant difference in survivin mRNA expression between -patients with and without metastasis (p<0.01). CONCLUSION: We concluded that high levels of survivin mRNA expression suggest poor prognosis for osteosarcoma patients.

Correlation between the histological grade of chondrosarcoma and the expression of MMPs, ADAMTSs and TIMPs.

BACKGROUND: The malignant degree of human chondrosarcoma can be difficult to determine using only histological findings. We, therefore, assessed the expression of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM) with thrombospondin motifs (ADAMTSs) and tissue inhibitor of metalloproteinases (TIMPs) in chondrosarcoma and ascertained the relationships to histological degree of malignancy and prognosis. MATERIALS AND METHODS: In 28 chondrosarcoma cases, immunostaining was performed using antibodies against MMP 2, 3, 7, 9, 13, ADAMTS 4, 5 and TIMP 1, 2, 3. RESULTS: The chondrosarcoma were classified into groups of 7, 15 and 6 cases based on histologically malignant grade I, II and III, respectively. All target proteins were expressed in chondrosarcoma. Positive correlations (p < 0.05) existed between immunostaining scores and histological grades for all proteins except MMP 9, with strong correlations (p < 0.01) for MMPs 2, 3 and 13, both ADAMTSs and all 3 TIMPs. No correlation existed between prognosis and immunostaining scores. CONCLUSION: These target proteins could, thus, indicate the degree of malignancy in human chondrosarcoma.