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Influence of telomerase activity on bone and soft tissue tumors. 国際誌

Norifumi Umehara, Toshifumi Ozaki, Shinsuke Sugihara, Toshiyuki Kunisada, Yuki Morimoto, Akira Kawai, Keiichiro Nishida, Aki Yoshida, Takuro Murakami, Hajime Inoue

Journal of cancer research and clinical oncology 130 ( 7 ) 411 - 6 2004年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

PURPOSE: Telomeres consisting of a repeating nucleotide sequence (TTAGGG)n are shortened in normal somatic cells. Telomerase is an enzyme that elongates the telomere sequence and is detected in most human cancers and usually not in normal somatic cells. Little is known about telomerase activity in bone and soft tissue tumors. The aim of this study was to investigate the relationship between telomerase activity and clinical factors in bone and soft tissue tumors. METHODS: Telomerase activity was measured using the modified telomeric repeat amplification protocol (TRAP) assay in 115 bone and soft tissue tumors obtained through open biopsy or resection. RESULTS: Telomerase activity was detected in 10% of benign tumors and 44% of malignant tumors (p < 0.001). A higher incidence of telomerase activity was detected in high-grade tumors than in low-grade tumors (p = 0.002). The cumulative metastasis-free and overall survival in telomerase-positive patients was significantly worse than in telomerase-negative patients (p = 0.045 and p = 0.048). CONCLUSION: Our study suggests that telomerase activity is associated with tumor aggressiveness and may be a useful parameter to predict the prognosis of patients with malignant bone and soft tissue tumors.

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Extended total sacrectomy and reconstruction for sacral tumor. 国際誌

Norihide Ohata, Toshifumi Ozaki, Toshiyuki Kunisada, Yuki Morimoto, Masato Tanaka, Hajime Inoue

Spine 29 ( 6 ) E123-6 2004年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

STUDY DESIGN: This case report includes the results of long-term follow-up after extended total sacrectomy in a 13-year-old boy with a sarcoma originating in the sacrum with an extraskeletal extension and infiltration into the left ilium. OBJECTIVE: To report and discuss a case of sacral tumor treated by extended sacrectomy. SUMMARY OF BACKGROUND DATA: Sacral tumors are often at an advanced stage with a large volume at diagnosis. Although total or extended sacrectomy is the only radical means to treat the massive sacral tumor, unavoidable complications in total sacrectomy are serious in the treatment selection. METHODS: Initial histologic findings indicated a synovial sarcoma. Additional genetic analysis redesignated the tumor as an unclassified sarcoma. Preoperative neoadjuvant chemotherapy and radiotherapy were completed. The response to the preoperative treatment appeared as a reduction in tumor size (approximately 50%) on radiographs. After extended sacrectomy, the L5 vertebral body was fixed between the ilia, and the pelvic ring was compressed by the Zielke system. The ISOLA instrumentation system connected the lumbar spine and both ilia. All sacral nerve roots and the L5 root on the left side were cut. RESULTS: At the 5-year follow-up examination, the patient was disease-free, could walk with crutches, and could climb stairs using the handrail and one crutch. CONCLUSIONS: The patient's excellent response to preoperative antitumor treatment was considered crucial to the long-term outcome. But the decision between a radical resection with reconstruction and a less extensive procedure with combined therapy remains controversial.

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Hoechst 33342 is a useful cell tracer for a long-term investigation of articular cartilage repair.

Keisuke Kawasaki, Shinsuke Sugihara, Keiichiro Nishida, Toshifumi Ozaki, Aki Yoshida, Aiji Ohtsuka, Hajime Inoue

Archives of histology and cytology 67 ( 1 ) 13 - 9 2004年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

The repair process of a full-thickness osteochondral defect was observed in a rat model using Hoechst 33342 as a cell tracer. The osteochondral defect was created at the medial femoral condyle of the right knee joints of twelve 11 week old male rats. Three weeks after the surgery, Hoechst 33342 was injected into the same knee joints. Calcein, a marker of the mineralization front, was then injected subcutaneousely twice at seven days and one day before harvesting of the tissue. At six, ten, and fourteen weeks and one year after the surgery, femoral condyles were obtained from the operated knee joints, fixed by alcohol, and embedded in polymethylmethacrylate. The sections were examined by fluorescent and then light microscopy. In the lateral femoral condyle cartilage, Hoechst 33342 labeling of chondrocyte nuclei was observed in all layers of the intact cartilage, and the dye never infiltrated beneath the subchondral bone plate. At 6 weeks after the surgery, Hoechst 33342-positive cells were observed not only in the regenerated fibrous cartilage, but also in the newly formed mineralized tissue in the medial femoral condyle. Interestingly, Hoechst 33342 labeling remained undiminished even one year after the intra-articular injection. The findings of the present study suggest that intra-articular injection of Hoechst 33342 is a useful tracer for long-term investigations of chondrocyte differentiation in vivo.

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Reconstruction with ipsilateral fibula transfer with pasteurized bone after excision of bone sarcoma of the tibia. 国際誌

Toshifumi Ozaki, Kazuo Fujiwara, Toshiyuki Kunisada, Tatsuo Ito, Akira Kawai, Hajime Inoue

Sarcoma 8 ( 2-3 ) 97 - 102 2004年

記述言語：英語掲載種別：研究論文（学術雑誌）

We report a technique of implantation of the ipsilateral vascularized fibula with pasteurized recycled bone after excision of tibia sarcoma in two cases. Plate and screws were used for osteosynthesis of the tibia or talus, vascularized fibula, and pasteurized bone. Microsurgery is not necessary for this reconstruction technique. Two patients who underwent this technique have obtained good functional results without tumor relapse 5 and 6 years after operation. The technique produced excellent results with regard to tibial reconstruction in these cases. We found it to be simple, speedy, safe, and a low cost technique by use of recycled bone.

DOI： 10.1080/13577140400017794

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Amplification of MYCL in atypical ewing tumor. analysis of metaphase and microarray comparative genomic hybridization

Toshifumi Ozaki, Yasuko Nakagawa, Aki Yoshida, Kunihiko Numoto, Shinnsuke Sugihara, Toshiyuki Kunisada, Shuji Hamazaki, Hajime Inoue

Cancer Genomics and Proteomics 1 ( 4 ) 275 - 282 2004年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Institute of Anticancer Research

A 20-year-old man developed a soft tissue mass in his right upper arm and, 3 months later, was referred to our hospital. The tumor cells showed brisk mitotic activity and a large amount of cytoplasmic glycogen was demonstrated with periodic acid Schiff stain. A diagnosis of atypical Ewing sarcoma was made. Chemotherapy according to the VACA protocol, comprising vincristine, actinomycin D, cyclophosphamide and doxorubicin was started. The chemotherapy was effective and a limb salvage procedure was performed by implantation of an autoclaved bone after wide tumor excision. During the postoperative chemotherapy, a local recurrence and multiple metastases developed, and the patient died due to disease progression. Fourteen years later, this tumor sample, preserved in a deepfreeze, was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) to detect the fusion gene. This tumor had an EWS exon 7 to FLI1 exon 6 fusion transcript. Moreover, metaphase and microarray comparative genomic hybridization (CGH) was done to detect chromosomal instabilities. Many gains and losses were noted on metaphase CGH, and MYCL amplification was identified on microarray CGH.

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Single nucleotide polymorphism in fibroblast growth factor receptor 4 at codon 388 is associated with prognosis in high-grade soft tissue sarcoma. 国際誌

Yuki Morimoto, Toshifumi Ozaki, Mamoru Ouchida, Norifumi Umehara, Norihide Ohata, Aki Yoshida, Kenji Shimizu, Hajime Inoue

Cancer 98 ( 10 ) 2245 - 50 2003年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: A recent study revealed that single nucleotide polymorphism (SNP) at codon 388 (Gly or Arg) of fibroblast growth factor receptor 4 (FGFR4) was associated with prognosis in patients with carcinoma of the breast and colorectal carcinoma. The purpose of the current study was to investigate the correlation between codon 388 SNP and clinical prognosis in patients with sarcoma of the bone and soft tissues. METHODS: Tumor samples were obtained from 143 patients with high-grade bone and soft tissue sarcomas at Okayama University Hospital between 1986-2002, and from 102 healthy volunteers. SNP of codon 388 was detected by sequencing and fragment length of polymerase chain reaction products digested by restriction enzyme. The chi-square test was used to compare genotype distribution and the Kaplan-Meier method was used for survival analysis. RESULTS: With regard to FGFR4 genotypes in the 143 patients studied, 54 (37.8%) were Gly/Gly, 72 (50.3%) were Gly/Arg, and 17 (11.9%) were Arg/Arg, findings that were not significantly different from those of controls (P = 0.97). With regard to cumulative overall and metastasis-free survival, patients with the Gly/Gly genotype were found to have a better prognosis (P = 0.085 and P = 0.27, respectively). FGFR4 SNP was found to be correlated significantly with overall and metastasis-free survival in patients with soft tissue sarcomas (P = 0.029 and P = 0.045, respectively), but not in those patients with bone sarcomas (P = 0.88 and P = 0.75, respectively). CONCLUSIONS: In the current study, the authors found a significant correlation between FGFR4 SNP and prognosis in patients with soft tissue sarcoma, although the samples were comprised of various histologic types. This SNP might be used to improve the prediction of clinical prognosis and lead to new treatment strategies in patients with soft tissue sarcomas.

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Correction of leg-length discrepancy after hip transposition. 国際誌

Robert Rödl, Georg Gosheger, Benedikt Leidinger, Norbert Lindner, Winfried Winkelmann, Toshifumi Ozaki

Clinical orthopaedics and related research ( 416 ) 271 - 7 2003年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Four patients were treated with limb lengthening to correct a leg-length discrepancy that developed after a hip transposition after pelvic resection for sacroma of the pelvis. Three patients had Ewing's sacroma and one patient had osteosarcoma. All patients received chemotherapy; radiotherapy also was administered to the three patients with Ewing's sacroma. Femur lengthening was started at an average of 5.7 years (range, 4.4-6.8 years) after tumor surgery. At the start of elongation, the average age of the patients was 17.3 years (range, 10.3-20.8 years). The average leg-length discrepancy was 10.3 cm (range, 6-12 cm). The average of achieved lengthening was 6.4 cm (range 6-7.5 cm). The average healing index was 32 days (range, 27-40 days) per 1-cm elongation. According to the classification of Paley, two problems and two minor complications were treated by additional interventions. At the final followup, the average functional score was 22 (73%) according to the system of the Musculoskeletal Tumor Society. Leg-length discrepancy after hip transposition can be corrected with distraction osteogenesis. All patients who wore a stiff ankle-foot orthosis before lengthening wore an ordinary shoe lift after lengthening. The problems of the lengthening procedure are similar to the general complication rate of bone lengthening. Because the 5-year survival after resection of a pelvic sarcoma is only 20% to 30%, leg lengthening after hip transposition should be offered only to long-term survivors with at least 5 years event-free survival.

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Splicing isoform of SYT-SSX fusion protein accelerates transcriptional activity and cell proliferation. 国際誌

Yuki Morimoto, Mamoru Ouchida, Toshifumi Ozaki, Akira Kawai, Tatsuo Ito, Aki Yoshida, Hajime Inoue, Kenji Shimizu

Cancer letters 199 ( 1 ) 35 - 43 2003年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

The human SYT-SSX gene has two splicing isoforms (type N and I), the latter of which contains an additional insertion of 93 bases. In the present study, we found increased transcriptional activity of the SYT-SSX type I protein in luciferase assay. When the SYT-SSX cDNAs were transfected to NIH3T3 cells, the type I transformant grew faster than the type N transformant. Furthermore, we evaluated the isoform ratio of the SYT or SYT-SSX transcripts in various tissues. Our results suggest that the SYT-SSX type I protein plays a critical role in the tumorigenesis of synovial sarcomas through increased transcriptional activity.

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孤立性骨嚢腫の治療成績

中川寧子, 尾崎敏文, 国定俊之, 伊藤達男, 井上一

中部日本整形外科災害外科学会雑誌 46 ( 秋季学会 ) 46 - 46 2003年9月

SYT蛋白とSSX蛋白のyeast two hybrid assayを用いた機能解析

伊藤達男, 大内田守, 尾崎敏文, 国定俊之, 森本裕樹, 大畑範英, 中川寧子, 吉田晶, 伊藤佐智夫, 清水憲二

日本癌学会総会記事 62回 536 - 536 2003年8月

ある種の分子標的薬はSYT-SSX発現細胞株に特異的な抗腫瘍効果を発揮する

沼本邦彦, 伊藤達男, 尾崎敏文, 大内田守, 国定俊之, 伊藤佐智夫, 森本裕樹, 大畑範英, 中川寧子, 清水憲二, 井上一

日本整形外科学会雑誌 77 ( 8 ) S1086 - S1086 2003年8月

cDNA Microarrayを用いたSYT-SSX Fusion Type間の遺伝子発現解析

森本裕樹, 川井章, 尾崎敏文, 伊藤達男, 中田英二, 井上一

日本整形外科学会雑誌 77 ( 8 ) S1086 - S1086 2003年8月

滑膜肉腫に認められる転座遺伝子SYT-SSXのtwo hybrid assayを用いた機能解析

伊藤達男, 尾崎敏文, 大内田守, 国定俊之, 森本裕樹, 大畑範英, 中川寧子, 武田健, 沼本邦彦, 吉田晶, 伊藤佐智夫, 清水憲二, 井上一

日本整形外科学会雑誌 77 ( 6 ) S823 - S823 2003年6月

AI及びBIIIa rotationplasty後の術後リハビリテーション

尾崎敏文, 濱田全紀, 千田益生, 井上一, HillmannAxel

日本整形外科学会雑誌 77 ( 6 ) S834 - S834 2003年6月

Blood loss in patients for total knee arthroplasty. 国際誌

Hiroshi Fujimoto, Toshifumi Ozaki, Kohji Asaumi, Hisayoshi Kato, Keichiro Nishida, Yasuhiro Takahara, Nobuhiro Abe, Hajime Inoue

Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA 11 ( 3 ) 149 - 54 2003年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Ninety-four patients with osteoarthritis (OA) and 180 with rheumatoid arthritis (RA) undergoing unilateral total knee arthroplasty (TKA) were analyzed to clarify the necessity for preoperative autogenous blood deposition or homologous blood transfusion. Two hundred and twenty-four and 50 patients underwent TKA with cement and without cement, respectively. The difference in average blood loss in patients between with (372 ml) and without cementation (449 ml) was significant. In the OA group the average blood loss significantly decreased after cementation but not in the RA group. Although the rate of avoiding transfusion in the OA group did not significantly decrease with the use of cement (92.4% vs. 93.3%), that in the RA group did (80% to 57.1%). Eight of 159 patients with hemoglobin level (Hb) of 11.0 g/dl or higher received homologous blood transfusion. Of these eight patients five had associated disorders. Only one patient with Hb of 12.0 g/dl or higher underwent homologous blood transfusion. Patients with Hb of 12.0 g/dl or higher are not indicated for preoperative autologous blood deposition. In patients with Hb between 11.0 and 12.0 g/dl preoperative blood deposition may be planned after consideration of general condition and complication. Patients with Hb lower than 11.0 g/dl should undergo preoperative blood deposition.

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Sacral infiltration in pelvic sarcomas: joint infiltration analysis II. 国際誌

Toshifumi Ozaki, Robert Rödl, Georg Gosheger, Christiane Hoffmann, Christopher Poremba, Winfried Winkelmann, Norbert Lindner

Clinical orthopaedics and related research ( 407 ) 152 - 8 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

The incidence and characteristics of sacral infiltration in pelvic sarcomas were analyzed. Fifty-one patients with a pelvic sarcoma (chondrosarcoma, 15 patients; Ewing's sarcoma, 23 patients; and osteosarcoma, 13 patients) abutting the sacroiliac joint had surgical treatment. Tumor infiltration into the sacrum was suspected based on preoperative images in 18 patients; 15 of 18 patients had histologic tumor invasion. There was a significant difference of median volume of sarcomas with and without infiltration. One of 23 Ewing's sarcomas, seven of 15 chondrosarcomas, and seven of 13 osteosarcomas penetrated the sacroiliac joint into the sacrum. Logistic regression test showed that diagnosis was the most important factor influencing sacral infiltration. Twelve tumors infiltrated through the posterior part of the joint, two tumors infiltrated through the anterior part, and one large tumor infiltrated through an unknown route. To obtain wide surgical margins, patients at risk (elderly, large tumor, or diagnosis of osteosarcoma or chondrosarcoma) for sacral involvement may require extended internal hemipelvectomy with the medial margin extending into the sacrum. High quality imaging studies should be used to assess the need for transarticular resection.

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Osteosarcoma of the pelvis: experience of the Cooperative Osteosarcoma Study Group. 国際誌

Toshifumi Ozaki, Silke Flege, Matthias Kevric, Norbert Lindner, Rainer Maas, Günter Delling, Rudolf Schwarz, Arthur R von Hochstetter, Mechthild Salzer-Kuntschik, Wolfgang E Berdel, Heribert Jürgens, G Ulrich Exner, Peter Reichardt, Regine Mayer-Steinacker, Volker Ewerbeck, Rainer Kotz, Winfried Winkelmann, Stefan S Bielack

Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21 ( 2 ) 334 - 41 2003年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

PURPOSE: To define patients and tumor characteristics as well as therapy results, patients with pelvic osteosarcoma who were registered in the Cooperative Osteosarcoma Study Group (COSS) were analyzed. PATIENTS AND METHODS: Sixty-seven patients with a high-grade pelvic osteosarcoma were eligible for this analysis. Fifteen patients had primary metastases. All patients received chemotherapy according to COSS protocols. Thirty-eight patients underwent limb-sparing surgery, 12 patients underwent hemipelvectomy, and 17 patients did not undergo definitive surgery. Eleven patients received irradiation to the primary tumor site: four postoperatively and seven as the only form of local therapy. RESULTS: Local failure occurred in 47 of all 67 patients (70%) and in 31 of 50 patients (62%) who underwent definitive surgery. Five-year overall survival (OS) and progression-free survival rates were 27% and 19%, respectively. Large tumor size (P =.0137), primary metastases (P =.0001), and no or intralesional surgery (P <.0001) were poor prognostic factors. In 30 patients with no or intralesional surgery, 11 patients with radiotherapy had better OS than 19 patients without radiotherapy (P =.0033). Among the variables, primary metastasis, large tumor, no or intralesional surgery, no radiotherapy, existence of primary metastasis (relative risk [RR] = 3.456; P =.0009), surgical margin (intralesional or no surgical excision; RR = 5.619; P <.0001), and no radiotherapy (RR = 4.196; P =.0059) were independent poor prognostic factors. CONCLUSION: An operative approach with wide or marginal margins improves local control and OS. If the surgical margin is intralesional or excision is impossible, additional radiotherapy has a positive influence on prognosis.

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Chromosomal alterations in osteosarcoma cell lines revealed by comparative genomic hybridization and multicolor karyotyping. 国際誌

Toshifumi Ozaki, Thomas Neumann, Daniel Wai, Karl-Ludwig Schäfer, Franz van Valen, Norbert Lindner, Christina Scheel, Wermer Böcker, Winfried Winkelmann, Barbara Dockhorn-Dworniczak, Jürgen Horst, Christopher Poremba

Cancer genetics and cytogenetics 140 ( 2 ) 145 - 52 2003年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

We characterized the chromosomal alterations in eight osteosarcoma cell lines (OST, HOS, U-2 OS, ZK-58, MG-63, SJSA-1, Saos-2, and MNNG) by comparative genomic hybridization (CGH); gains and losses of DNA sequences were defined as chromosomal regions with a fluorescence ratio, wherein all of the 95% confidence interval was above 1.25 and below 0.75, respectively. In four of 8 cell lines, multicolor karyotyping (MK) was added. CGH revealed the average number of aberrations per cell line was 20.8 (range: 10-31); the average numbers of gains and losses were 11.1 and 9.6, respectively. The frequent gains were identified on 1p21 approximately q24, 1q25-q31, 7p21, 7q31, 8q23 approximately q24, and 14q21; frequent losses were at 18q21 approximately q22, 18q12, 19p, and 3p12 approximately p14. High-level gains were observed on 8q23 approximately q24, 5p, and 1p21 approximately p22. MK revealed the most common translocations in the four cell lines were t(8;9), t(1;3), t(3;5), t(1;13), t(2;6), t(3; 17), t(1;15), t(10;20), and t(6;20). Chromosomes 1, 3, 8, 9, and 20 were most frequently involved in translocation events. The concordance rate of aberrations in CGH and translocations in MK was 76%. MK was useful to identify the chromosomal alterations and as a supplement to the CGH results in three of four chromosomes.

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Genetic imbalances revealed by comparative genomic hybridization in osteosarcomas. 国際誌

Toshifumi Ozaki, Karl-Ludwig Schaefer, Daniel Wai, Horst Buerger, Silke Flege, Norbert Lindner, Matthias Kevric, Raihanatou Diallo, Agnes Bankfalvi, Christian Brinkschmidt, Heribert Juergens, Winfried Winkelmann, Barbara Dockhorn-Dworniczak, Stefan S Bielack, Christopher Poremba

International journal of cancer 102 ( 4 ) 355 - 65 2002年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Osteosarcomas are the most frequent bone sarcomas. The molecular chromosomal aberrations in osteosarcomas were analyzed by comparative genomic hybridization (CGH). We studied 47 frozen tumors (41 primary samples, 6 relapses) in osteosarcoma patients registered in the Cooperative Osteosarcoma Study (COSS) protocol. Genomic imbalances were detected in 40 of 41 primary tumors and 6 of 6 relapsed tumors. Gains were more frequent than losses (ratio of 1.3:1). The median number of changes was 16 and 12 in primary and relapsed osteosarcomas, respectively. The median number of aberrations in primary high-grade osteosarcomas (17.0) was significantly higher than in low- or intermediate-grade osteosarcoma subtypes (3.0) (p = 0.038). The most frequent gains included 8q, 1p21-p31 and 1q21-q24, and the most frequent losses were 10q, 5q and 13q. High-level gains were observed on 8q23-q24, 17p13 and 1q21-q24. A gain of 19p (p < 0.001) or loss of 9p (p = 0.027) was more frequent in poor responders than in good responders. Univariate analysis revealed that patients with primary metastases (p = 0.002), poor histologic responses (p = 0.005), high-level gains of 19p (p = 0.012) or losses of 13q14 (p = 0.042) had significantly lower event-free survival (EFS), whereas patients with a loss of 5q (p = 0.007) or a loss of 10q21-22 (p = 0.017) had significantly higher EFS than patients without these aberrations. Multivariate analysis demonstrated that primary metastasis, loss of 13q14 and loss of 5q were independent prognostic factors. The findings of our study seem to be useful for evaluating the prognosis of patients and may finally lead to treatment strategies based on genetic background of osteosarcoma.

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骨軟部腫瘍におけるレセプターチロシンキナーゼ(c-kit)の発現

吉田晶, 川井章, 内藤訓子, 森本裕樹, 伊藤達男, 大畑範英, 国定俊之, 尾崎敏文, 別府保男

日本癌学会総会記事 61回 397 - 397 2002年10月

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