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Oncostatin M is a growth factor for ewing sarcoma: Potent role of the leukemia inhibitory factor receptor
S254
Abstracts / Bone 48 (2011) S251–S260
Abstract: Osteolytic bone disease is a major feature of multiple myeloma (MM), a malignant plasma cell tumour located in the bone marrow. We hypothesize that the interplay between bone marrow stroma and malignant plasma cells leads to functional changes in the stromal compartment that precede osteolysis. Our research aims to identify such pre-osteolytic deregulatory events at an early stage of tumour growth. We have employed the KMS12BM xenograft model of MM, together with in vivo live animal imaging to define spatial and temporal disease progression. This model recapitulates key features of MM associated bone disease: medullary engraftment of tumour cells, osteoblast suppression, osteoclast activation and lytic bone lesions, with disease developing over 8–10 weeks. 8-week old β2m/NOD/SCID animals (N=18) were transplanted with luciferase tagged KMS12BM cells by tail vein injection. Animals were then divided into two groups based on signal intensity. Animals with low signal intensities were defined as the early disease group (N=9) and were sacrificed at 3 weeks post-transplantation. Bone lining cells of femora were assessed for colony forming potential (CFU), and compared to age matched controls (N=9). Surprisingly, a significant decrease in CFU was already evident (diseased 38 ±12, control 67 ±33 colonies, p < 0.05). Additionally osteogenic potential was markedly decreased (diseased 14 ± 7, control 42± 26 ALP-positive colonies, p < 0.01) in diseased animals. Importantly micro-computed tomography analysis of femora of diseased animals revealed a decrease in bone volume (p< 0.05) and trabecular number (p < 0.01) with an increase in SMI (p< 0.05), although no osteolytic lesions were observed. Thus, significant deregulation of the stromal compartment may occur at an early disease stage despite low tumour burden. Changes become more marked with disease progression as analysis of diseased animals (N= 9) at 8 weeks revealed a more severe phenotype in all bone parameters, as well as the presence of osteolytic lesions. Collectively, our data provide preliminary evidence of early deregulation events. Further characterisation of such events will provide a platform for future mechanistic studies to identify key pathways leading to MM induced osteolytic bone disease. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.619
PP489-T/NIPP07 (recipient of a 2011 ECTS/IBMS New Investigator Award) Oncostatin M is a growth factor for ewing sarcoma: Potent role of the leukemia inhibitory factor receptor E. David a,⁎, P. Guihard a, F. Tirode b, K. Laud b, O. Delattre b, D. Heymann a, F. Redini a, F. Blanchard a a INSERM U957, Nantes, France b INSERM U830, Paris, France Abstract: Osteosarcoma (OS), Chondrosarcoma (CS) and Ewing sarcoma (ES) represent the majority of primary bone tumors and their treatment has to be improved. They all derive from bone mesenchymal stem cells (MSC) but have variable differentiation status. OS and CS are differentiated in the osteoblastic and chondrocytic lineages respectively whereas in ES, the fusion protein EWS-FLI1 inhibits differentiation. Oncostatin M (OSM), a cytokine from the IL-6 family, inhibits the proliferation of OS and CS through activation of the transcription factors STAT1/3 and induction of the cell cycle inhibitors p21WAF1 and p27KIP1. The aim of this study was to define the activity of OSM on ES in comparison to other IL-6 type cytokines. OSM induced the proliferation of 9 on 10 ES cell lines by inducing the quiescent cells in G0 (Ki67−) to enter into the cell cycle (Ki67+, cells mainly in S phase). Other IL-6 type cytokines appeared inactive except LIF (Leukemia Inhibitory Factor). In comparison to OS or CS, ES cell lines expressed higher levels of LIF receptor (LIFR or type I OSM receptor) and lower levels of OSMR (or type II OSM receptor). In ES cells, OSM treatment resulted in higher STAT activation, induction of c-myc (implicated in G0–G1 transition) but not C/EBPb (implicated in growth inhibition by OSM in OS and CS). Knock down of EWS-FLI1 with doxycycline-inducible shRNA induced osteoblastic, chondrocytic and adipocytic differentiation, reduced the LIFR/OSMR ratio, OSM becoming a growth inhibitor as observed in OS and CS. Finally, a retrospective clinical study confirmed the high LIFR/OSMR ratio in ES patients and suggested that the LIFR is a new prognostic marker linked to metastatic relapse (p= 0.066, Logrank test). This study shows that within primary bone sarcomas, OSM has opposite effects on tumor cell proliferation depending on the LIFR/OSMR ratio. The EWS-FLI1 oncogene could directly control expression of these receptors but the stemness of ES cells could be also implicated. On differentiated mesenchymal bone cells (osteoblasts / osteosarcoma and chondrocytes / chondrosarcoma), this cytokine has a growth inhibitory activity in vitro and in vivo that could be used to improve current anti-cancer treatments. In contrast on less differentiated cells (mesenchymal stem cells / Ewing sarcoma), OSM behaves as a growth factor. Therefore, neutralization of the LIFR appears as a new potent therapeutic strategy for Ewing sarcoma. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: E. DAVID Grant / Research Support from Ligue contre le Cancer, P. GUIHARD: none declared, F. TIRODE: none declared, K. LAUD: none declared, O. DELATTRE: none declared, D. HEYMANN: none declared, F. REDINI: none declared, and F. BLANCHARD: none declared.
doi:10.1016/j.bone.2011.03.620
PP490-S Dickkopf-1 and sclerostin in different phases of multiple myeloma; The effect of lenalidomide and dexamethasone treatment with or without bortezomib E. Terpos a,⁎, D. Christoulas a, M. Gkotzamanidou a, C. Bratengeier b, M. Gavriatopoulou a, W. Woloszczuk c, E. Kastritis a, M.A. Dimopoulos a a Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece b Biomarker Design Forschungs GmbH, Vienna, Austria c Biomedica Medizinprodukte GmbH & Co KG, Vienna, Austria Abstract: The aim of this study was to evaluate the circulating levels of the Wnt inhibitors dickkopf-1 (Dkk-1) and sclerostin in different phases of multiple myeloma (MM) and their alterations post therapies with novel anti-myeloma agents. We studied 284 MM patients (153M/131F, median age 66 years): 167 consecutive patients were newly diagnosed (20 had asymptomatic MM and 147 symptomatic MM), 29 patients were at the plateau phase of MM and 88 patients had relapsed/refractory MM and received therapy with the combination of lenalidomide plus dexamethasone with or without bortezomib (VRD or RD). Circulating levels of Dkk-1 and sclerostin were measured using ELISA methodology (R&D Systems & Biomedica Medizinprodukte) in all patients and in 20 healthy controls. Circulating Dkk-1 and sclerostin concentrations of newly diagnosed symptomatic MM were increased compared to controls (p < 0.001 and p = 0.03, respectively) and to asymptomatic MM at diagnosis (p < 0.001 and p = 0.001, respectively). Patients at plateau phase had increased circulating sclerostin compared to controls (p = 0.002) but also compared to MM patients at diagnosis (p = 0.02). In contrast, they had lower serum Dkk-1 compared to MM patients at diagnosis (p < 0.001) and no difference compared to controls. Patients with ISS-3 stage had higher Dkk-1 and sclerostin than ISS-1 and ISS-2 (p = 0.031 and p = 0.001, respectively). Patients with lytic disease at diagnosis (n = 116) had increased Dkk-1 compared to patients with no lytic disease (n = 51; p = 0.002). Sclerostin showed only borderline increases in patients with advanced lytic disease (>3 lesions and/or fractures; p = 0.072). Relapsed patients had increased Dkk-1 and sclerostin levels compared to controls and to asymptomatic MM at diagnosis (p < 0.001 for all comparisons). In patients who received RD, Dkk-1 was increased and sclerostin was decreased after 6 cycles of therapy. Responders to RD had a median increase of 9% in Dkk-1 serum levels after 6 cycles of therapy, while nonresponders had a median increase of 91% compared to baseline values (p < 0.01). Patients who did not respond to RD showed an increase in CTX (p = 0.021) after 6 cycles of therapy. VRD administration resulted in a significant reduction of sRANKL (p = 0.024) and increase of osteocalcin (p = 0.01) after 6 cycles, but showed only minimal reduction of Dkk-1 (p = 0.08) and no alterations on sclerostin. In conclusion, these results further support the rationale for the use of drugs targeting Dkk-1 and sclerostin in MM. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.621
PP491-M Low bone mineral density and high bone turnover in patients with non-Hodgkin's lymphoma (NHL) who receive frontline therapy: Results of a multicenter prospective study K. Anargyrou a, T.P. Vassilakopoulos b, K. Tsionos a, P. Kokkoris c, G. Boutsikas a, M.K. Angelopoulou b, D. Christoulas d, S. Masouridou b, M. Gkotzamanidou d, M. Dimou b, A. Papatheodorou e, D.N. Chatzifotiadis f, P. Panayiotidis b, G.A. Pangalis g, J. Meletis b, E. Terpos d,⁎ a Department of Hematology, 251 General Air Force Hospital, Greece b Department of Hematology, University of Athens School of Medicine, Greece c Department of Endocrinology, 251 General Air Force Hospital, Greece d Department of Clinical Therapeutics, University of Athens School of Medicine, Greece e Department of Medical Research, Greece f Department of Nuclear Medicine, 251 General Air Force Hospital, Greece g Department of Hematology, Iatriko Athinon, Psychikon Branch, Athens, Greece Abstract: There is limited information regarding the effects of chemotherapy on bone metabolism of adult patients with non-Hodgkin's lymphoma (NHL). To elucidate this issue we scheduled a prospective study in which patients with newly-diagnosed NHL had a
Abstracts / Bone 48 (2011) S251–S260
Abstract: Osteolytic bone disease is a major feature of multiple myeloma (MM), a malignant plasma cell tumour located in the bone marrow. We hypothesize that the interplay between bone marrow stroma and malignant plasma cells leads to functional changes in the stromal compartment that precede osteolysis. Our research aims to identify such pre-osteolytic deregulatory events at an early stage of tumour growth. We have employed the KMS12BM xenograft model of MM, together with in vivo live animal imaging to define spatial and temporal disease progression. This model recapitulates key features of MM associated bone disease: medullary engraftment of tumour cells, osteoblast suppression, osteoclast activation and lytic bone lesions, with disease developing over 8–10 weeks. 8-week old β2m/NOD/SCID animals (N=18) were transplanted with luciferase tagged KMS12BM cells by tail vein injection. Animals were then divided into two groups based on signal intensity. Animals with low signal intensities were defined as the early disease group (N=9) and were sacrificed at 3 weeks post-transplantation. Bone lining cells of femora were assessed for colony forming potential (CFU), and compared to age matched controls (N=9). Surprisingly, a significant decrease in CFU was already evident (diseased 38 ±12, control 67 ±33 colonies, p < 0.05). Additionally osteogenic potential was markedly decreased (diseased 14 ± 7, control 42± 26 ALP-positive colonies, p < 0.01) in diseased animals. Importantly micro-computed tomography analysis of femora of diseased animals revealed a decrease in bone volume (p< 0.05) and trabecular number (p < 0.01) with an increase in SMI (p< 0.05), although no osteolytic lesions were observed. Thus, significant deregulation of the stromal compartment may occur at an early disease stage despite low tumour burden. Changes become more marked with disease progression as analysis of diseased animals (N= 9) at 8 weeks revealed a more severe phenotype in all bone parameters, as well as the presence of osteolytic lesions. Collectively, our data provide preliminary evidence of early deregulation events. Further characterisation of such events will provide a platform for future mechanistic studies to identify key pathways leading to MM induced osteolytic bone disease. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.619
PP489-T/NIPP07 (recipient of a 2011 ECTS/IBMS New Investigator Award) Oncostatin M is a growth factor for ewing sarcoma: Potent role of the leukemia inhibitory factor receptor E. David a,⁎, P. Guihard a, F. Tirode b, K. Laud b, O. Delattre b, D. Heymann a, F. Redini a, F. Blanchard a a INSERM U957, Nantes, France b INSERM U830, Paris, France Abstract: Osteosarcoma (OS), Chondrosarcoma (CS) and Ewing sarcoma (ES) represent the majority of primary bone tumors and their treatment has to be improved. They all derive from bone mesenchymal stem cells (MSC) but have variable differentiation status. OS and CS are differentiated in the osteoblastic and chondrocytic lineages respectively whereas in ES, the fusion protein EWS-FLI1 inhibits differentiation. Oncostatin M (OSM), a cytokine from the IL-6 family, inhibits the proliferation of OS and CS through activation of the transcription factors STAT1/3 and induction of the cell cycle inhibitors p21WAF1 and p27KIP1. The aim of this study was to define the activity of OSM on ES in comparison to other IL-6 type cytokines. OSM induced the proliferation of 9 on 10 ES cell lines by inducing the quiescent cells in G0 (Ki67−) to enter into the cell cycle (Ki67+, cells mainly in S phase). Other IL-6 type cytokines appeared inactive except LIF (Leukemia Inhibitory Factor). In comparison to OS or CS, ES cell lines expressed higher levels of LIF receptor (LIFR or type I OSM receptor) and lower levels of OSMR (or type II OSM receptor). In ES cells, OSM treatment resulted in higher STAT activation, induction of c-myc (implicated in G0–G1 transition) but not C/EBPb (implicated in growth inhibition by OSM in OS and CS). Knock down of EWS-FLI1 with doxycycline-inducible shRNA induced osteoblastic, chondrocytic and adipocytic differentiation, reduced the LIFR/OSMR ratio, OSM becoming a growth inhibitor as observed in OS and CS. Finally, a retrospective clinical study confirmed the high LIFR/OSMR ratio in ES patients and suggested that the LIFR is a new prognostic marker linked to metastatic relapse (p= 0.066, Logrank test). This study shows that within primary bone sarcomas, OSM has opposite effects on tumor cell proliferation depending on the LIFR/OSMR ratio. The EWS-FLI1 oncogene could directly control expression of these receptors but the stemness of ES cells could be also implicated. On differentiated mesenchymal bone cells (osteoblasts / osteosarcoma and chondrocytes / chondrosarcoma), this cytokine has a growth inhibitory activity in vitro and in vivo that could be used to improve current anti-cancer treatments. In contrast on less differentiated cells (mesenchymal stem cells / Ewing sarcoma), OSM behaves as a growth factor. Therefore, neutralization of the LIFR appears as a new potent therapeutic strategy for Ewing sarcoma. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: E. DAVID Grant / Research Support from Ligue contre le Cancer, P. GUIHARD: none declared, F. TIRODE: none declared, K. LAUD: none declared, O. DELATTRE: none declared, D. HEYMANN: none declared, F. REDINI: none declared, and F. BLANCHARD: none declared.
doi:10.1016/j.bone.2011.03.620
PP490-S Dickkopf-1 and sclerostin in different phases of multiple myeloma; The effect of lenalidomide and dexamethasone treatment with or without bortezomib E. Terpos a,⁎, D. Christoulas a, M. Gkotzamanidou a, C. Bratengeier b, M. Gavriatopoulou a, W. Woloszczuk c, E. Kastritis a, M.A. Dimopoulos a a Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece b Biomarker Design Forschungs GmbH, Vienna, Austria c Biomedica Medizinprodukte GmbH & Co KG, Vienna, Austria Abstract: The aim of this study was to evaluate the circulating levels of the Wnt inhibitors dickkopf-1 (Dkk-1) and sclerostin in different phases of multiple myeloma (MM) and their alterations post therapies with novel anti-myeloma agents. We studied 284 MM patients (153M/131F, median age 66 years): 167 consecutive patients were newly diagnosed (20 had asymptomatic MM and 147 symptomatic MM), 29 patients were at the plateau phase of MM and 88 patients had relapsed/refractory MM and received therapy with the combination of lenalidomide plus dexamethasone with or without bortezomib (VRD or RD). Circulating levels of Dkk-1 and sclerostin were measured using ELISA methodology (R&D Systems & Biomedica Medizinprodukte) in all patients and in 20 healthy controls. Circulating Dkk-1 and sclerostin concentrations of newly diagnosed symptomatic MM were increased compared to controls (p < 0.001 and p = 0.03, respectively) and to asymptomatic MM at diagnosis (p < 0.001 and p = 0.001, respectively). Patients at plateau phase had increased circulating sclerostin compared to controls (p = 0.002) but also compared to MM patients at diagnosis (p = 0.02). In contrast, they had lower serum Dkk-1 compared to MM patients at diagnosis (p < 0.001) and no difference compared to controls. Patients with ISS-3 stage had higher Dkk-1 and sclerostin than ISS-1 and ISS-2 (p = 0.031 and p = 0.001, respectively). Patients with lytic disease at diagnosis (n = 116) had increased Dkk-1 compared to patients with no lytic disease (n = 51; p = 0.002). Sclerostin showed only borderline increases in patients with advanced lytic disease (>3 lesions and/or fractures; p = 0.072). Relapsed patients had increased Dkk-1 and sclerostin levels compared to controls and to asymptomatic MM at diagnosis (p < 0.001 for all comparisons). In patients who received RD, Dkk-1 was increased and sclerostin was decreased after 6 cycles of therapy. Responders to RD had a median increase of 9% in Dkk-1 serum levels after 6 cycles of therapy, while nonresponders had a median increase of 91% compared to baseline values (p < 0.01). Patients who did not respond to RD showed an increase in CTX (p = 0.021) after 6 cycles of therapy. VRD administration resulted in a significant reduction of sRANKL (p = 0.024) and increase of osteocalcin (p = 0.01) after 6 cycles, but showed only minimal reduction of Dkk-1 (p = 0.08) and no alterations on sclerostin. In conclusion, these results further support the rationale for the use of drugs targeting Dkk-1 and sclerostin in MM. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.621
PP491-M Low bone mineral density and high bone turnover in patients with non-Hodgkin's lymphoma (NHL) who receive frontline therapy: Results of a multicenter prospective study K. Anargyrou a, T.P. Vassilakopoulos b, K. Tsionos a, P. Kokkoris c, G. Boutsikas a, M.K. Angelopoulou b, D. Christoulas d, S. Masouridou b, M. Gkotzamanidou d, M. Dimou b, A. Papatheodorou e, D.N. Chatzifotiadis f, P. Panayiotidis b, G.A. Pangalis g, J. Meletis b, E. Terpos d,⁎ a Department of Hematology, 251 General Air Force Hospital, Greece b Department of Hematology, University of Athens School of Medicine, Greece c Department of Endocrinology, 251 General Air Force Hospital, Greece d Department of Clinical Therapeutics, University of Athens School of Medicine, Greece e Department of Medical Research, Greece f Department of Nuclear Medicine, 251 General Air Force Hospital, Greece g Department of Hematology, Iatriko Athinon, Psychikon Branch, Athens, Greece Abstract: There is limited information regarding the effects of chemotherapy on bone metabolism of adult patients with non-Hodgkin's lymphoma (NHL). To elucidate this issue we scheduled a prospective study in which patients with newly-diagnosed NHL had a