Научная статья на тему 'Section 2 new developments in basic and translational Researc'

Section 2 new developments in basic and translational Researc Текст научной статьи по специальности «Биотехнологии в медицине»

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Текст научной работы на тему «Section 2 new developments in basic and translational Researc»

SECTION 2

NEW DEVELOPMENTS IN BASIC AND TRANSLATIONAL RESEARCH

ORAL PRESENTATIONS

ID 90

Enhanced tumor cell kill by combined treatment with Nutlin and adenoviruses encoding p53

H.C.A. Gmat, J.E. Carette, F.H.E. Schagen, W.R. Gerritsen,

G.J.L. Kaspers, P.I.J.M. Wuisman, V.W. van Beusechem V.U. University Medical Center, Department of Orthopedic Surgery, Department of Medical Oncology, Division of Gene Therapy, Pediatric Oncology/Hematology, Amsterdam, Netherlands

Introduction: The tumor suppressor p53 is the central coordinator of cellular responses to stress signals. It is often mutated in bone and soft tissue tumors. p53 function can also be compromised by inhibition of the p53 protein. The major negative regulator of p53 is the mouse double minute 2 protein (MDM2). The highest incidence of MDM2 amplification is found in soft-tissue sarcomas and osteosarcomas. Reactivation of the p53 pathway to control tumor growth is therefore an interesting approach to treat osteosarcoma. Possibilities to reactivate p53 range from expressing an exogenous p53 gene in p53 mutant cancers to antagonizing a p53 inhibitor in p53 wild type cancer cells. Recently, a novel class of highly potent and specific MDM2-antagonists, the Nutlins, was identified. We envisioned that Nutlins could protect both endogenous and exogenousp53 from MDM2-media-ted inactivation.

Material and Methods: We selected six cancer cell lines (4 osteosarcoma cell lines) and analyzed p53 and MDM2 expression via western blotting. Effect of combination treatment was studied with WST-1 assay or crystal violet staining. Apoptosis induction was measured via cell cycle analysis. Viral replication and burst was studied with the adenoX rapid titer kit. Results: Combination treatment of exogenous p53 expression and Nutlin resulted in a significant increased cell kill of p53-negative and p53 wild type cancer cells expressing high or low levels of MDM2. Cytotoxicity was associated with profound cell cycle checkpoint activation and apoptosis induction. We also tested Nutlin in combination with a replicating oncolytic adenovirus expressing p53. Nutlin treatment accelerated lysis of oncolytic adenovirus-infected cancer cells

and viral progeny burst, which augmented the eradication of p53 wild type osteosarcoma cancer cells up to 1,000-fold. Conclusions: These findings suggest that Nutlins are promising compounds to be combined with p53 gene therapy and oncolytic virotherapy for cancer such as osteosarcoma.

ID 74

CD99 and caveolin1: new anticancer proteins for osteosarcoma

K. Scotlandi, M.C. Manara, L. Cantiani, M. Zuntini,

M. Sciandra, P. Picci IOR, Bologna, Italy

Introduction: CD99 is expressed in a variety of solid tumors and is a diagnostic marker of Ewing's sarcoma. Osteosarcomas, however, have never been studied. We report that in

11 different cell lines and 17 clinical samples CD99 expression is either undetectable or very low. Since in osteoblasts CD99 expression is under control of the osteoblast-specific transcription factor Cbfa1 (RUNX2), we tested the hypothesis that CD99 downregulation may have a role in osteosarcoma development and progression.

Material and Methods: Gene transfection of U-2 OS and Saos-2 cell lines was performed to induce forced expresssion of CD99 and caveolin. The clones obtaine dwere analyzed for in vitro parameters related to malignancy as well as for in vivo behaviour. Gene profile of the transfected cells was used to identify critical genes strctly connected to CD99. Functional studies were performed to prove the in vitro involvement of caveolin and c-src. Results: CD99 forced expression in two osteosarcoma cell lines significantly reduced resistance to anoikis, inhibited growth in soft agar and cell migration and led to abrogation of tumorigenic and metastatic ability. CD99 gene transfection induces caveolin-1 up-regulation and the two molecules were found to co-localize on the cell surface. Treatment with antisense oligonucleotides to caveolin-1 abrogates the effects of CD99 on migration. On the other hand transfection of cells with caveolin 1 clearly show how the molecule acts as an oncosuppressor for osteosarcoma. C-src appeared to be negatively regulated either by CD99 or caveolin1. Conclusions: CD99 and caveolin should be downregulated in osteosarcoma to express full malignancy. It is very likely that

CD99 acts by enhancing caveolin-1 and its association of Src to caveolae, therfore leading to subsequent inactivation of Src.

ID 110

Post operative infection and increased patient survival.

Is there a link?

L.M. Jeys, R.J. Grimer, S. Carter, R.M. Tillman, A.A. Abudu Royal Orthopaedic Hospital, Birmingham, UK

Introduction: Despite the advances in adjuvant chemotherapy and surgical techniques, malignant sarcomas involving bone have a significant mortality. Recent basic science and animal studies have shown survival advantages following infections for some tumour types. This study investigates the effect of post operative infection in patients treated for a malignant primary tumour involving bone using endo-prosthetic replacement (EPR).

Material and Methods: A consecutive series of 1264 patients underwent endoprosthetic reconstruction between 1966 and 2000. Patients were excluded from the study for insufficient follow up data (24 patients, 1.9%), reconstruction following excision of a benign tumour (73 patients, 6%) or metastasis (134 patients, 10.6%), death due to causes unrelated to their original tumour (11 patients, 0.9%, mean 9.2 years from the diagnosis). Previous studies showed that 70% of deep infections occur within 1 year from reconstruction. Therefore landmark analysis was performed; all patients infected after 12 months of reconstruction were excluded (32 patients, 2.5%) and those who died within 12 months from diagnosis were excluded (122 patients, 9.7%), leaving 868 patients in the study group. Any survival advantage early infection conveyed could be analysed by Kaplan-meier survival analysis from this landmark point. Results: Overall population survival was 63.3% at 5 years, 58.8% at 10 years and 53.9% at 20 years from landmarking. There were 89 patients (10.3%) who endured an infection within 1 year of implantation. These patients had significantly better survival (p = 0.04) than those without infection. This held true only for the diagnoses of osteosarcoma (p = 0.017) and myeloma (p = 0.01). The 10 year survival for patients with osteosarcoma with infection was 73.5% compared to 53.6% in the non infected group after landmarking.

Conclusions: There was evidence for increased survival after deep post-operative infection in osteosarcoma patients, in keeping with other research. The authors feel this warrants further investigation.

ID 127

Prometastatic role of NG2 proteoglycan-collagen type VI interaction in sarcomas

M.S. Benassi1, G. Gamberi1, L. Pazzaglia1, F. Ponticelli1,

A. Chiechi1, P. Picci1, S. Cattaruzza2, B. Wasserman2,

R. Perris2, P. Braghetta3, P. Bonaldo3

1 Istituto Ortopedico Rizzoli, Bologna, Italy

2 National Cancer Institute, Aviano, Italy

3 Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Padova, Italy

Introduction: Recent evidence suggest that NG2 proteoglycan, previously demonstrated to be a marker associated to

primary malignancy and metastasis in melanoma, promotes tumour dissemination through growth factor sequestering and interaction with collagen type VI that evokes specific intracellular signals.

Material and Methods: In human primary sarcomas and me-tastases we analysed mRNA level and protein immunoreacti-vity of NG2 and collagen type VI to relate gene expression to cell metastatic potential. Paired normal tissue was available. Quantitative PCR reaction was performed on cDNA after reverse transcription of mRNA and protein immunoreactivity was assessed with immunological assays.

Results: In all tumors NG2 gene was up-regulated with 2-AA ct values ranging from 90 to 32591 and mRNA median value was significantly higher compared paired normal tissue (p = 0.0005).Moreover, the marked difference between primary and metastases (p = 0.001) was consistent with the median increase of 11-fold in the latter. Distribution of collagen type VI within tumour population followed a similar trend and a moderate to strong protein expression was associated to high mRNA levels. Experimental sarcoma models in mice confirm the prometastatic role of NG2-collagen type VI interaction by demonstrating that tumour model cells expressing higher levels of NG2 reveal a higher metastatic potential. Ectopic expression of NG2 also confers to cells a higher metastatic ability, but when inoculated into Col VI-deficient mice, metastasis formation is strongly reduced, confirming a direct interplay between molecules in the metastatic process. Conclusions: Positive involvement of NG2 pathway in malignancy progression may have a potential biological value in discriminating high risk patients, with important clinical implications for cancer prognosis and therapy.

ID 79

Telomere biology in giant cell tumor of bone

R.G. Forsyth1, G. de Boeck1, A.H.M. Taminiau2,

P.C.W. Hogendoorn2, D. Uytendaele3, H. Roels3,

M.M. Praet3, S. Bekaert3

1 N. Goormaghtigh, Institute of Pathology, Ghent, Belgium 2LUMC, Leiden, Netherlands

3 UZG, Ghent, Belgium

Introduction: Giant Cell Tumor of Bone (GCTB) is a benign tumor known for its unpredictable clinical behavior of local recurrences and in rare occasions distant metastases. GCTB is composed of uniformly distributed osteoclastic giant cells in a background of mononuclear rounded and spindled-sha-ped cells. Cytogenetically, telomere associations (TAS) are the most common chromosomal aberrations. TAS in general are nearly exclusively found in high-grade malignancies. GCTB has been defined as a polyclonal tumor, but more recently a recurrent aberration was reported which suggests a possible role for a disturbed telomere maintenance. The aim of this study was to further investigate telomere maintenance in GCTB.

Material and Methods: 19 samples from 19 patients were studied. A combination of immunofluorescence and FISH was performed applying antibodies directed against PML and hTERT and telomere-PNA-probes. TRAP assay and telomere length assay were performed for functional detection of telo-merase activity and alternative telomere lengthening (ALT).

Results: All samples showed positivity for hTERT-PML immunofluoresence. The giant cells, next to the spindle shaped cells, also expressed both markers. The TRAP assay demonstrated a heterogeneous telomerase activity while telomere length assay showed telomere lengths within normal limits indicating the absence of ALT. Confocal microscopy confirmed colocalization of hTERT with PML in association with telomeres.

Conclusions: GCTB demonstrates remarkable telomere maintenance of activated telomerase and inactivated ALT in the presence of normal telomere lengths. ‘Active' hTERT and ‘inactive' PML co localize at the end of chromosomes in association with telomeres. These findings strongly suggest that the hTERT-PML aggregates are part of a structural telomere protective capping mechanism rather than of a telomere lengthening mechanism. The telomere maintenance in GCTB could be considered as an important key factor in its pathogenesis.

ID 80

Osteoclastogenesis in giant cell tumor of bone

R.G. Forsyth1, G. de Boeck1, V.K.V. Verhalle1,

A.H.M. Taminiau2, P.C.W. Hogendoorn2, D. Uytendaele3,

H. Roels3, M.M. Praet3

1 N. Goormaghtigh Institute of Pathology, Ghent, Belgium

2 LUMC, Leiden, Netherlands

3 UZG, Ghent, Belgium

Introduction: Giant Cell Tumor of Bone (GCTB) is a benign tumor notorious for its unpredictable local recurrences and rare distant metastases. Microscopically uniformly distributed osteoclastic giant cells (OCG) are embedded in a background of mononuclear rounded and spindled shaped cells. OCGs are speculated to originate from the fusion of monocyte-macrophage lineage cells. Some reports revealed the expression of several cell markers like CD14 or CD68 in the mononuclear rounded cell population, but none of these are also expressed on OCGs. Recently the expression in both cell types of CXCR4, a unique receptor for SDF-1, was described, indicating chemotaxis of monocytes-macrophages in the process of osteoclastogenesis. CD14 + , CD33+ or CD14 + /CD33 + blood monocytes are destinated as pre-osteoclasts. The macrophage marker CD33 is expressed earlier than CD14, while CD14 is expressed longer compared to CD33 during macrophage development. The aim of this study was to investigate the CD14 / CD33 expression profile in GCTB.

Material and Methods: 19 samples from 19 patients were studied. A double immunofluorescent staining was performed with monoclonal antibodies directed against CD14 and CD33. Results: All samples showed similar expression profiles. The mononuclear rounded cell population was positive for CD14, CD33 or both cell markers. Only large cells with a single round large nucleus were only positive for CD33. These cells were found in the vincity of OCGs. The OCG subpopulation was positive for CD33 only.

Conclusions: Our immunohistochemical results suggest: 1) that osteoclastogenesis in GCTB is the exclusive result of fusion of blood-born CD33 expressing pre-osteoclasts and that osteocla-stogenesis is not the result of fusion of intra-tumoral CD 14 + macrophages; 2) expression of the early marker CD33 by OCGs

suggest that the CD14 + infiltrating subpopulation could be a non-specific phenomenon secondary to the stromal cell induced chemotaxis of blood-born pre-osteoclasts.

ID 188

High grade central chondrosarcoma versus chondromyxoid fibroma

S. Romeo, L.B. Rozeman, J. Oosting, L.H. Hameetman,

A.M. Cleton-Jansen, J.V.M.G. Bovee Leiden University Medical Center,

Department of Pathology, Leiden, Netherlands

Introduction: The presence of polygonal - atypical cells in Chondormyxoid fibroma (CMF) creates difficulty in the differential diagnosis with High Grade Central Chondrosarcoma (HGCCS), especially in biopsy specimens. Nor specific diagnostic tools have been developed to help such a differential diagnosis, neither the molecular mechanisms behind this overlapping morphology have been thoroughly studied. Material and Methods: RNA isolated from 7 CMF and 12 HGCCS was hybridized on a 9k - cDNA microarray enriched for cartilage specific genes and printed in duplicate. For data analysis Linear Model for Microarray Analysis was used. Immunohistochemistry allowed for verification of differentially expressed genes on a larger series of CMF (n = 20) and HGCCS (n = 39).

Results: Fourthy-two genes showed to be differentially expressed (p<0.01 after false discovery rate correction). CD166 and Cyclin D1 were higher expressed in CMF versus HGCCS, which was immunohistochemically verified (p<0.05). p16, known to be lowly expressed in high grade chondrosarcomas was also evaluated, because of its inhibitory effect on Cyclin D1. Its expression was significantly higher in CMF (p<0.01). Conclusions: We have identified and validated 3 possible markers for the differential diagnosis of CMF versus HGCCS, which can be used on a routine basis: Cyclin D1, CD166 and p16. Higher expression of Cyclin D1 is present in CMF, which is unusual for a benign tumor. However its co-occurrence with high p16 expression might balance their antagonistic actions. The crucial role for loss of p16 expression in HGCCS is confirmed. Down-regulation of adhesion molecules might play a role in malignant progression.

ID 48

Biological reconsrtuction of bone defects

Y. Kollender, J. Bickels, E. Gur, A. Zaretsky, G. Weiss, A. Amir,

O. Merimsky, G. Flusser, A. Nirkin, J. Issakov, I. Meller Tel-Aviv Medical Center, Tel-Aviv, Israel

Introduction: The definition of a situation which describes discontinuity of a normal bone has many synonyms: loss, gap, incontinuity etc. In this review the term Defect will be used. 3 types of bone defects are defined Envelope defects-loss of the soft tissue and periosteum. Partial defect-loss of all bone components in a part of the bone circumference. Complete defect-loss of all the bone components of a whole bone circumference. According to the length dimmention 5 types of bone defects are defined. Intercalary defects, Extended intercalary defect, Osteoarticular defect, Complete whole

bone defect ant Osteoarticular defect with the whole adjunct joint (after an exttaarticular resection).

Material and Methods: The couses of bone defects are: Congenital absence, Post trauma, Post acute or chronic infections, After tumor resection, After local bone disease and post revision surgeries in joint replacement. Reconstruction of a bone defect is depended upon the following factors. Age, nature of the couse, the need for post operative adjuvant therapy (chemo or radio therapy), the size of the defect, the anatomical location, the general health condition of the patient, the cost benefit of the procedure and the availability of the procedure. Results: Reconstruction options and techniques: No materi-als-using scar tissue or callus. Autogenous bone graft: Free microvascularized bone, non vascularized bone such as tibial strut or non vascularized fibula or iliac crest, cancellus bone graft or cancello-cortical bone graft. Pasturized or heated bone, cryosurgeried bone. Allografts of all types such as intercallary or osteoarticular. Synthetic bone subtitutes. Improvized implants composed of hardware and PMMA. Endoprostheses of all kinds. Combinations of all the above. Conclusions: This review stresses few major problems in this complicated subject. The age of the patient plays a major role, the extent of the defect also plays a key role in the decision making. It is obvious that no artificial material may last for ever which means that every prosthesis will need a revision in the future. Biological solutions should be the future for filling most of the bone defects including osteoarticular defects such as Gleno-Humeral joint, Radio-Carpal joint, Elbow joint, the Knee and the Hip joint.

ID 91

Intravenous administration of Ad5-delta24RGD induces regression of osteosarcoma lung metastases

H.C.A. Graat1, M.A. Witlox1, P.I.J.M. Wuisman\

V.W. van Beusechem2, F.H.E. Schagen2, W.R. Gerritsen2,

E.S. Kleinerman3, G.J.L. Kaspers4

1 V.U. University medical center, Department of Orthopedic Surgery, Amsterdam, Netherlands

2 V.U. University medical center; Department of Medical Oncology, Division of Gene Therapy, Amsterdam, Netherlands

3 The University of Texas M. D. Anderson Cancer Center; Division of Pediatrics, Houston, United States

4 V.U. University medical center; Pediatric Oncology/Hematology, Amsterdam, Netherlands

Introduction: A major problem in the treatment of osteosarcoma is the frequent occurrence of metastases in the lungs. Consequently, an effective treatment of lung metastases would be of great benefit for osteosarcoma patients. The purpose of the present study was to determine whether systemic administration of a conditionally replicative adenovirus, Ad5-delta24RGD could suppress the growth of human osteosarcoma lung metastases. For this purpose, we used the established SaOs-lm7 osteosarcoma lung metastases model. Material and Methods: In vitro studies were performed with SaOs-lm7 osteosarcoma tumor cells and cytoxicity was assessed using WST-l assay. Animal experiments were performed on athym/nu/nu mice. The SaOs-lm7 lung metastasis model was obtained from Dr. Kleinerman.

Results: Oncolytic activity of Ad5-delta24RGD on SaOs-lm7 cells was clearly demonstrated in vitro. Furthermore, in vivo toxicity studies in nude mice revealed that repeated intravenous administration of this oncolytic virus did not cause severe weight loss or liver damage. SaOs-lm7 osteosarcoma lung metastases bearing mice were treated at week 1,2 and 3 (group I; 12 mice) or at week 5, 6 and 7 (group II; 12 mice) post tumor cell injection with 1x10e9 plaque forming units (pfu) Ad5-delta 24RGD or PBS. Animals were analyzed ten weeks after tumor cell injection. In group I, mice treated with Ad5-delta24RGD did not significantly differ from PBS injected controls. In contrast, mice treated at weeks 5-7 with Ad5-delta24RGD showed a significantly reduced lung weight (decrease of tumor mass, p<0.05), a significant increase (10%) of total body weight (decrease of disease symptoms, p<0.05) and a reduced amount of lung tumor nodules (median 60 versus at least 174) compared to PBS treated control animals.

Conclusions: These findings suggest that systemic administration of Ad5-delta24RGD might be a promising new treatment strategy for metastatic osteosarcoma and is dependent on an established tumor vasculature.

ID 191

Cell cycle regulation in central chondrosarcoma:

CDK4 amplification associated with tumor progression

Y.H. Schrage, K. Szuhai, P.C. Hogendoorn,

A.H. Taminiau, J.V. Bovee

Leiden University Medical Center, Leiden, Netherlands

Introduction: Central chondrosarcomas (CCS) are malignant cartilage forming tumors in the medullar cavity. CCS is mostly primary, but can be secondary to enchondroma, especially in Ollier disease, demonstrating multiple enchondromas. Amplification of 12q13 and deletion of 9p21 are two of the few consistent genetic aberrations in CCS. Previously, we correlated amplification of 12q13, analysed by array-CGH, with an increase in expression of CDK4 by cDNA array analysis. Moreover, loss of p16 expression is associated with tumour progression.We therefore hypothesize that progression of central chondrosarcoma occurs by upregulation of CDK4, promoting the cell cycle from G1 to S phase. Expression profiling also demonstrated a slight difference in cMYC expression between Ollier disease-related and solitary chondrosarcoma. Material and Methods: mRNA levels of CDK4 and cMYC were studied using quantitative Real Time PCR in phalangeal enchondromas (n = 7) and CCS (grade 1 n=11, grade II n = 7 and grade III n = 9). Normal cartilage and growth plates were used for comparison. Immunohistochemistry was performed for CDK4 on 30 CCS.

Results: Tumors with amplification of 12q13 by array-CGH(n = 4) showed higher expression levels than tumors without amplification (n = 8) (p = 0,001). Tumors showed up-regulation of CDK4 compared to growth plates (p = 0,001). mRNA levels increased with increasing histological grade (p = 0,003). This association could also be demonstrated at protein level (p = 0.060). In contrast, no correlation of cMYC expression levels with histological grade could be found. Ollier disease related tumors show significantly higher expression of cMYC (p = 0.011).

Conclusions: Although expression of the oncogene cMYC does not seem to be important for chondrosarcoma progression, it is

higher expressed in Ollier disease related tumors. In addition to loss of p16, increased CDK4 expression is associated with increasing histological grade, underlining the crucial role of these cell-cycle regulating molecules in CCS progression.

POSTER SESSION

ID 55

Desmoid tumors: analysis of PDGF, PDGFR and gene mutations

B. Liegl1, S. Regauer1, A. Beham1, R. Windhager2,

A. Leithner2, T. Bauernhofer3, C. Gully4

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1 Institute of Pathology, Medical University Graz,

Graz, Austria

2 Department of Orthopaedic Surgery, Medical University Graz, Graz, Austria

3Department of Oncology, Medical University Graz,

Graz, Austria

4 Center for Medical Research, Medical University of Graz, Graz, Austria

Introduction: Therapy with imatinib mesylate (Gleevec) has been proposed as an option for patients with desmoids tumors. Recent reports, however, indicate that desmoids tumors are c-kit negative. The cause for a possible effect of this other tyrosine kinase inhibitors might be based on expression of PDGFa, PDGFP, PDGFRa or PDGFRp. The aim of the present study was to study this pathway by immunohistochemistry.

Material and Methods: We performed immunohistochemi-cal analysis on 124 archived samples of 81 patients with desmoid tumors using antibodies against PDGF-alpha, PDGF-beta, PDGFR-alpha and PDGFR-beta according to standardized procedures. Furthermore mutational analyses were performed on frozen material from 14 patients.

Results: All desmoid tumors showed a positive reaction with antibodies against PDGF-alpha and PDGFR-alpha (> 80% of the tumor cells strong membranous and cytoplasmic staining). With antibodies against PDGF-beta and PDGFR-beta none of the 124 cases showed a specific reaction. Mutational analysis of PDGFR-alpha (exon 11, 12, 17 and 18) and PDGFR-beta (exon 12) on frozen material from 14 patients with desmoid tumors were performed, but no mutations leading to amino-acid changes in the mature protein could be detected. Conclusions: Desmoids tumors might be regarded as mainly PDGF-alpha and PDGFR-alpha positive and PDGF-beta and PDGFR-beta negative tumors. However, as mutational analysis revealed no mutations leading to amino-acid changes the possible effect of imatinib mesylate should be attributed to another pathway.

ID 172

Surgery vs radiation therapy for nonmetastatic Ewing's sarcoma: experience of a single institution

N. Fabbri1, G. Bacci1, S. Ferrari1, A. Longhi1, D. Donati1,

M. Manfrini1, P. Picci1, M. Mercuri1, F. Bertoni1, E. Barbieri2

1 Istituto Ortopedico Rizzoli, Bologna, Italy

2 Policlinico S. Orsola, Bologna, Italy

Introduction: The treatment and prognosis of Ewing's sarcoma has substantially changed during the past 30 years and the role of surgery for local control in the multimodal manage-

ment of disease has remarkably increased over the last 2 decades. However, selection bias due to location (extremities vs axial skeleton) and relatively non-homogeneous treatment received by patients in multi-institutional trials may limit objective evaluation and comparison of the relative role of surgery and radiation therapy in this setting. Purpose of this study was to review a large series of patients homogeneously treated at a single institution.

Material and Methods: Between 1979 and 1999, 512 patients with a nonmetastatic ESFT of bone entered 4 different adjuvant and neoadjuvant studies performed at a single institution. 335 patients were treated with surgery alone (196) or surgery followed by reduced doses (44.8 Gy) of radiotherapy (139). The outcome of this group of patients was compared with 177 patients who were locally treated by full-dose (60.8 Gy) radiotherapy.

Results: Local control (88.8% vs 80.2%, p < 0.009) and 5-year DFS (63.8% vs 47.6%, p < 0.0007) were significantly better in patients treated with surgery. In this group of patients surgically managed, those with adequate surgical margins were associated with better results in terms of local control and 5-year DFS (96.6% vs 71,7%, p < 0.0008, and 69.6% vs 46.3%, p < 0.0002). Nevertheless, better results were essentially only observed in patients with tumor located in the extremities. Conclusions: Surgery is better than radiotherapy in case of respectable ESFT of the extremities when adequate surgical margins can reliably be achieved, while in case of inadequate surgical margins adjuvant reduced-dose-radiotherapy is ineffective. If inadequate margins can be anticipated, patients are treated more effectively with full dose radiotherapy.

ID 193

Array-CGH analysis of chondromyxoid fibroma complements conventional cytogenetics

S. Romeo1, J. Oosting1, P.C.W. Hogendoorn1, K. Szuhai2,

R. Sciot3, M. Debiec-Rychter4, F. Mertens5,

1 Department of Pathology, Leiden University Medical Center, Leiden, Netherlands

2 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Netherlands

3 Department of Pathology,

Catholic University of Leuven, Leuven, Belgium

4 Department of Human Genetics,

Catholic University of Leuven, Leuven, Belgium

5 Department of Clinical Genetics,

Lund University Hospital, Lund, Sweden

Introduction: Chondromyxoid fibroma (CMF) is a rare benign cartilaginous tumor of bone mainly occurring in the second decade and affecting long bones. Chromosome banding analysis of CMF has revealed recurrent involvement of 6p23-25, 6q12-15, and 6q23-27 in balanced as well as unbalanced rearrangements. We have used array comparative genomic hybridization (array-CGH) to detect genomic imbalances associated with CMF.

Material and Methods: In the setting of EuroBoNeT, a European network of excellence on rare bone tumours of children and adults, it was possible to collect a relatively large number of frozen samples of CMF. DNA was isolated from 10 tumor samples containing >70% tumor cells. Samples were hybridi-

zed on an array-CGH containing a set of >3500 PAC/BAC clones from the Wellcome Trust Sanger Institute. Chromosome banding analysis was performed on 5 of the 10 cases. Results: Cytogenetics showed chromosomal alterations in 3 cases: 46,XX,del(6)(?q21?q23),add(7)(q21) (case L1789); 46,XX, t(6;17)(q23;p13) (case L1788) and 46,XY,del(6)(q15der(6),t(6;6) (q15;q27),inv(6)(p25q13) (case L1787). Array CGH showed complex interstitial deletion patterns in two cases, involving chromosomes 3 and 6 (case L1765) and chromosomes 6 and 7 (case L1789), respectively. Deleted regions ranged from 1.6 to 14 Mb. Confirmatory FISH confirmed that these deletions were not constitutional. At chromosome band 6q23 there was a small region of overlap in both cases. Intriguingly, this region was involved in a balanced translocation also in case L1788. Conclusions: Array CGH and chromosome banding analyses showed recurrent balanced as well as unbalanced rearrangements of chromosome band 6q23, indicating a crucial role for one or more genes in this region for CMF pathogenesis.

ID 87

Expression of P-catenin and p53 are prognostic factors in deep aggressive fibromatosis

C. Gebert1, J. Hardes1, H. Ahrens1, W. Winkelmann1,

C. August2, H. Buerger2, G. Gosheger2

1 University Hospital Muenster, Muenster, Germany

2 Department of Pathology, Muenster, Germany

Introduction: Aggressive fibromatosis is a rare fibroblastic, mesenchymal tumour, characterized by a locally highly destructive growth pattern without the formation of distant metastasis. Up to date the treatment of these tumours is based on a wide surgical resection followed under certain circumstances by an adjuvant radiotherapeutic and/or chemotherapeutic treatment. Unfortunately, at the present state the local recurrence rates are rather high. The overexpression of P-catenin is frequently seen in aggressive fibromatosis. However, the prognostic value of P-catenin overexpression is unclear. Also, little knowledge exists about potential molecular markers for new targetted therapies.

Material and Methods: A tissue array of 37 cases of deep aggressive fibromatosis was constructed and subjected to immunohistochemical analysis for P-catenin, p53, SMA, desmin, Ki-67, c-erbB2, EGFR, c-kit, CD34 and S-100. For 23 patients a complete clinical follow-up was available.

Results: Nuclear P-catenin expression was associated with an increased rate of local tumour recurrence (60.0% 1-year and 0% 5-year-event free survival; p<0.05). Furthermore, p53 expression was associated with an increased risk of tumour recurrence (50% 1-year event free survival rate and 0% 5-years event free survival rate p<0.05). The coexpression of p53 and P-catenin was significantly correlated (p<0.05). A statistically significant association between Mib-1 and p53 or P-catenin expression could not be seen, respectively. An expression of EGFR, c-erbB2 or c-kit could also not be observed. Conclusions: Our results show for the first time that the overexpression of P-catenin and p53 are associated with a decreased event-free survival and an increased rate of tumour recurrence in deep aggressive fibromatosis. The potential value of this molecule for a more exact definition of the resection margins remains an issue of further research.

ID 69

Fas-antigen (sFas) in patients with bone tumors

I.V. Babkina, N.E. Kushlinsky, Yu.N. Soloviev, D.A. Borisov N.N. Blokhin Russian Cancer Research Center,

Moscow, Russian Federation

Introduction: The goal of this study was the analysis of sFas concentrations in the blood serum of healthy peoples and patients with osteosarcoma and benign bone tumors. Material and Methods: We examined 32 untreated patients with osteosarcoma, 11 - with benign bone neoplasms (osteoblastoma - 2, chondroma - 1, chondroblastoma - 2, osteochondroma - 3, lipoma - 1, aneurismal bone cyst - 2) and 25 practically healthy subjects. sFas was measured by enzyme immunoassay in the serum using tests created in Russian N.N. Blokhin Cancer Research Centre of RAMS and in M.M. Shemjakin and Ju.A. Ovchinnikov Institute of Bioorga-nic Chemistry RAS.

Results: All samples from patients with benign bone neoplasms contained sFas. Frequency of sFas disclosure in the serum from healthy donors comprised 36%, from patients with osteosarcoma - 87%. sFas levels in healthy donors (0,86±0,30 ng/ml) were lower than in patients with osteosarcoma (3,72±0,89 ng/ml), and benign bone tumors (4,86±1,50 ng/ml). Maximal concentrations of sFas were found in osteoblastoma (11,2 ng/ml), lipoma (11,5 ng/ml) and chondroma (15,6 ng/ml) There were no significant differences in sFas concentrations between patients with osteosarcoma and benign bone tumors. Initial sFas levels were higher in the serum of osteosarcoma pa-tients with metastasizing (4,83±0,56 ng/ml) than in patients without disease progression in the follow-up period (1,55±0,17 ng/ml). Conclusions: These findings suggest that pretreatment sFas levels in the serum of osteosarcoma patients can provide additional prognostic information for the evolution of the disease.

ID 71

sVCAM-1 in the serum of some bone tumors patients

I.V. Babkina, Yu.N. Soloviev, N.E. Kushlinsky,

E.Yu. Russo, I.N. Kuznetsov, D.A. Borisov N.N. Blokhin Russian Cancer Research Center,

Moscow, Russian Federation

Introduction: The aim of the study — comparative analysis of sVCAM-1 concentrations in the serum of practically healthy persons and patients with bone neoplasms.

Material and Methods: We studied sVCAM-1in the serum obtained from 65 untreated bone tumor patients (34 - osteosarcoma, 19 - chondrosarcoma, 12 - Ewing's sarcoma) and in the serum of 25 practically healthy subjects. sVCAM-1 was measured by enzyme immunoassay in the serum using tests from "Bender MedSystems" (Austria).

Results: All samples contained sVCAM-1. sVCAM-1 levels in healthy donors (242,5±23,2 pg/ml) were lower than in patients with osteosarcoma (537,3±81,2 pg/ml), Ewing tumor (399,2±56,5 pg/ml) and chondrosarcoma (340,1±58,1 pg/ml). There were no significant differences in sVCAM-1 concentrations in patients with various types of affected bone. Serum sVCAM-1 levels did not depend on the size of tumor. As demonstrated by statistical analysis, sVCAM-1 levels in bone tu-

mor patients at the generalization stage (549,6±153,9 pg/ml) were higher than in patients with localized stage (418,8± 39,8 pg/ml). But bone tumor patients with sVCAM-1 level <400 pg/ml had poorer prognosis for disease-free survival (less

12 month), than patients with sVCAM-1 level >400 pg/ml. Conclusions: These findings suggest that sVCAM-1 levels may be related to bone tumor pathogenesis.

ID 72

Vascular endothelial growth factor (VEGF) in the sera of bone neoplasms' patients

N.E. Kushlinsky, Yu.N. Soloviev, I.N. Kuznetsov,

I.V. Bulycheva, E.Yu. Russo, D.A. Borisov, M.D. Aliev N.N. Blokhin Russian Cancer Research Center,

Moscow, Russian Federation

Introduction: Aim of the study: comparative analysis of VEGF content in blood serum of bone tumor patients and control healthy subjects.

Material and Methods: 110 primary bone tumor patients (70 male and 40 female) aged 5 - 66 years were enclosed in the study. In all patients clinical and radiographic diagnosis was confirmed by histologic examination. The following morphologic variants were distinguished: osteosarcoma (40), juxtacortical osteogenic sarcoma (1), periosteal osteosarcoma (1), chondrosarcoma (20), secondary chondrosarcoma (4), malignant fibrous histiocytoma (7), giant-cell bone tumor (6), Ewing's tumor (22), osteochondroma (3), aneurismal bone cyst (3), bone fibrous dysplasia (3). The patients were presented at first diagnosis and did not receive any specific treatment before biochemical investigation. Sera of practically healthy persons aged 5 - 60 years (32 male and 20 female) were used as control. VEGF in blood sera was measured by standard ELISA using «R&D» (USA) kits.

Results: VEGF was detected in all serum samples studied — both in bone tumor patients and in the control group. Highly statistically significant differencein VEGF levels (p = 0,000001) was revealed between the patients (377,8±35,1 pg/ml) and control group (100,0±7,5 pg/ml). At the same time, VEGF levels in patients' sera varied in the range from 50 to 2700 pg/ml. No differences were found between VEGF levels in male and female sera, both in control and in the total bone tumor patients' group. No significant differences were revealed depending on malignant and benign tumors' localization either in tubular (451,8± 47,5 pg/ml), or in flat (288,6±67,2 pg/ml) bones. VEGF level in the sera of osteosarcoma patients comprised 392,2±51,4 pg/ml, in the sera of primary chondrosarcoma patients - 384,3± 83,2 pg/ml, in secondary chondrosarcoma - 284,9±144,8 pg/ml, in Ewing's tumor - 455,5±144,5 pg/ml, and were significantly higher than in the control group. The lowest VEGF levels were detected in the sera of giant-cell bone tumor patients (242,8±73,4 pg/ml). VEGF levels in patients with benign tumors and tumor-like bone lesions (320,8±37,4 pg/ml) were slightly lower than in patients with malignant tumors, but significantly higher than in the control group. No associations were found

between pretreatment serum VEGF levels and the degree of therapeutic pathomorphosis in osteosarcoma patients (p = 0,8). Thus, at grade 1 pathomorphosis (10 patients) median VEGF levels comprised 150,5 pg/ml, at grade 2 (20 patients) — 280,7 pg/ml, at grade 3 (8 patients) — 227,8 pg/ml, and at grade

4 (4 patients) — 299,2 pg/ml. In osteosarcoma patients a 2-fold increase in relapse-free survival was observed if initial serum VEGF level was lower than 200 pg/ml.

Conclusions: Increased VEGF concentration was detected in the sera of patients with primary malignant and benign bone tumors as compared to healthy persons. In osteosarcoma patients relapse-free survival was two-fold worse if initial serum VEGF level exceeded 200 pg/ml.

ID 81

Biochemical markers of bone turnover in cancer patients with metastatic bone disease

N.V. Lyubimova1, G.V. Kozarskaya1, A.P. Morozov1,

M.V. Pashkov2

1 N.N. Blokhin Russian Cancer Research Center,

Moscow, Russian Federation

2 N.N. Burdenko Medical Academy,

Voronezh, Russian Federation

Introduction: The osteoclast-specific tartrate-resistant acid phosphatase 5b (TRAP 5b) and osteoblast-specific bone alkaline phosphatase (BAP) have been considered as biochemical markers of bone resorption and formation This study was undertaken to evaluate the utility of bone metabolism markers TRAP 5b and BAP in breast and prostate cancer patients with bone metastases.

Material and Methods: Bone resorption (TRAP 5b) and bone formation (BAP) markers were studied in 178 breast and 102 prostate cancer patients with and without bone involvement. Serum levels of TRAP 5b and BAP were measured using the enzyme immunoassay Bone TRAP («Medac Diagnostika») and BAP EIA kit («BCM Diagnostics»).

Results: Pre-treatment levels of TRAP 5b were significantly higher in breast and prostate cancer patients with bone meta-stases than in healthy women and men and patients without clinical signs of metastatic spread to bone. TRAP 5b elevation was associated with the extent of the metastatic bone disease in breast carcinoma patients (R=0.529; p = 0.00001). Serum levels of TRAP 5b gradually decreased in patients with positive clinical response to Bondronat therapy and in patients with progression of disease serial TRAP 5b values increased progressively. Diagnostic sensitivity of TRAP 5b for breast and prostate cancer patients was 78,9% and 64,6% (specificity 92,8% and 92,6%); sensitivity of BAP — 60,0% and 84,4% (specificity — 96,3% and 89,5%) respectively. Conclusions: Measurement of TRAP 5b may be used in the detection of bone metastases and for the evaluation of bisphosphonates treatment effects on bone in breast cancer patients and BAP in combination with TRAP 5b — in prostate cancer patients.

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