Anne Moreau

Receptor Activator of Nuclear Factor κB Ligand (RANKL)/Osteoprotegerin (OPG) Ratio Is Increased in Severe Osteolysis

Pathological osteolyses are considered a consequence of a disturbance in the mechanisms that govern the bone remodeling, mainly the communication between osteoclasts and osteoblasts. Osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) are newly discovered molecules that play a key role in these communications. RANKL is essential for osteoclast differentiation via its receptor RANK located on the osteoclast membrane. OPG is a soluble decoy receptor that inhibits osteoclast differentiation through its binding to RANKL. The aim of this study is the analysis of the RANKL/OPG balance by complementary methods (semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and enzyme-linked immunosorbent assay) in human osteolysis associated to various bone etiologies (n = 60), tumoral (primitive, secondary) or not, compared to healthy tissues (n = 16). Results demonstrated that RANKL/OPG ratio was significantly increased in patients suffering from severe osteolysis compared to the control group and that this imbalance is involved in bone resorption mechanisms. In this study, OPG expression appears to reflect a protective mechanism of the skeleton to compensate increased bone resorption by inhibiting osteoclast formation and bone resorbing activity. Moreover, as revealed by immunohistochemistry, RANKL and OPG were colocalized in all of the tissues analyzed. To define the veracity of RANKL/OPG index in assessing and managing patients with severe osteolysis, an extended population of patients suffering from severe osteolysis must be now monitored.

Interleukin‐34 is expressed by giant cell tumours of bone and plays a key role in RANKL‐induced osteoclastogenesis

Interleukin‐34 (IL‐34) is a newly discovered regulator of myeloid lineage differentiation, proliferation, and survival, acting via the macrophage‐colony stimulating factor receptor (M‐CSF receptor, c‐fms). M‐CSF, the main ligand for c‐fms, is required for osteoclastogenesis and has been already identified as a critical contributor of the pathogenesis of giant cell tumours of bone (GCTs), tumours rich in osteoclasts. According to the key role of M‐CSF in osteoclastogenesis and GCTs, the expression of IL‐34 in human GCTs was first assessed. Quantitative analysis of IL‐34 mRNA expression in 14 human GCTs revealed expression of this cytokine in GCTs as well as M‐CSF and c‐fms. Immunohistochemistry demonstrated that osteoclast‐like cells exhibited a huge immunostaining for IL‐34 and that mononuclear stromal cells were slightly positive for this protein. In contrast to osteoblasts, bone‐resorbing osteoclasts showed very strong staining for IL‐34, suggesting its potential role in the pathogenesis of GCTs by facilitating osteoclast formation. The role of IL‐34 in osteoclastogenesis was then studied in murine and human models. IL‐34 was able to support RANKL‐induced osteoclastogenesis in the absence of M‐CSF in all models. Multinucleated cells generated in the presence of IL‐34 and RANKL expressed specific osteoclastic markers and resorbed dentine. IL‐34 induced phosphorylation of ERK 1/2 and Akt through the activation of c‐fms, as revealed by the inhibition of signalling by a specific c‐fms tyrosine kinase inhibitor. Furthermore, IL‐34 stimulated RANKL‐induced osteoclastogenesis by promoting the adhesion and proliferation of osteoclast progenitors, and had no effect on osteoclast survival. Overall, these data reveal that IL‐34 can be entirely substituted for M‐CSF in RANKL‐induced osteoclastogenesis, thus identifying a new biological activity for this cytokine and a contribution to the pathogenesis of GCTs. Copyright

Human osteosarcoma cells express functional receptor activator of nuclear factor-kappa B.

RANK, RANK ligand (RANKL) and osteoprotegerin (OPG) are the key regulators of bone metabolism, both in normal and pathological conditions. Previous data have demonstrated that human osteosarcoma biopsies express RANKL as well as OPG, and functional RANK is expressed in a murine osteosarcoma cell line. As RANK expression in human osteosarcoma remains controversial, the aim of the present study was to analyse its expression in vitro in human osteosarcoma cell lines, ex vivo using pathological tissues, and then to determine its functionality in terms of signal transduction pathways modulated by RANKL. RT‐PCR analysis and immunohistochemistry experiments revealed that RANK is expressed at both transcriptional and protein levels in MNNG/HOS, Saos‐2 and MG‐63 human osteosarcoma cell lines, in contrast to the U‐2 OS osteosarcoma cell line and human osteoblasts, which were negative. RANK was also expressed in 57% of osteosarcoma biopsies. Furthermore, western blot experiments clearly demonstrated the functionality of RANK. Thus, RANKL significantly induced the phosphorylation of ERK1/2, p38 and IκB in RANK‐positive osteosarcoma cells. This study is the first report of functional RANK expression in human osteosarcoma cells: this strengthens the involvement of the RANK–RANKL–OPG axis in primary bone tumour biology and identifies novel therapeutic approaches targeting RANK‐positive osteosarcoma. Copyright

Metatarsal giant cell tumors and giant cell reparative granuloma are similar entities

Light and electron microscopic investigations and studies of the resorption ability in vitro of giant cells were done in two patients with giant cell osteolytic lesions of metatarsal bone. Giant cells harvested from both patients were similar in morphologic features and ability to resorb dentin. After other diagnoses of osteolytic lesions of metatarsal bone were ruled out, one lesion was considered a giant cell reparative granuloma and the other a true giant cell tumor of bone. Clinical, radiologic, ultrastructural, functional studies, and data in the literature, indicated that giant cell reparative granuloma only can be differentiated from giant cell tumor by younger age at diagnosis and the occurrence of giant cell clusters. All other features (cortical erosion, high rate of recurrence, hemorrhage areas, predominant intercellular collagenous substance) are characteristic of both lesions. If these two giant cell lesions are different entities, more accurate means are needed to distinguish them.

TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

Degenerative disc disease (DDD) primarily affects the central part of the intervertebral disc namely the nucleus pulposus (NP). DDD explains about 40% of low back pain and is characterized by massive cellular alterations that ultimately result in the disappearance of resident NP cells. Thus, repopulating the NP with regenerative cells is a promising therapeutic approach and remains a great challenge. The objectives of this study were to evaluate the potential of growth factor‐driven protocols to commit human adipose stromal cells (hASCs) toward NP‐like cell phenotype and the involvement of Smad proteins in this differentiation process. Here, we demonstrate that the transforming growth factor‐β1 and the growth differentiation factor 5 synergistically drive the nucleopulpogenic differentiation process. The commitment of the hASCs was robust and highly specific as attested by the expression of NP‐related genes characteristic of young healthy human NP cells. In addition, the engineered NP‐like cells secreted an abundant aggrecan and type II collagen rich extracellular matrix comparable with that of native NP. Furthermore, we demonstrate that these in vitro engineered cells survived, maintained their specialized phenotype and secretory activity after in vivo transplantation in nude mice subcutis. Finally, we provide evidence suggesting that the Smad 2/3 pathway mainly governed the acquisition of the NP cell molecular identity while the Smad1/5/8 pathway controlled the NP cell morphology. This study offers valuable insights for the development of biologically‐inspired treatments for DDD by generating adapted and exhaustively characterized autologous regenerative cells. Stem Cells 2016;34:653–667

Homozygous PTEN deletion in neuroblastoma arising in a child with Cowden syndrome.

Homozygous PTEN Deletion in Neuroblastoma Arising in a Child with Cowden Syndrome Franck Bourdeaut,* Bertrand Isidor, Sandrine Ferrand, Caroline Thomas, Anne Moreau, Marc-David Leclair, Albert David, Gaelle Pierron, Cedric Le Caignec, and Olivier Delattre CHU Nantes, service d’hemato-oncologie p ediatrique, Nantes, France Institut Curie, D epartement de p ediatrie, Paris, France INSERM U830, Laboratoire de G en etique et Biologie des cancers, Paris, France CHU Nantes, Service de g en etique m edicale, Nantes, France Institut Curie, Unit e de g en etique somatique, Paris, France CHU Nantes, service d’anatomie pathologique, Nantes, France Universit e de Nantes, Facult e de m edecine, Nantes, France CHU Nantes, Service de chirurgie infantile, Nantes, France INSERM, UMR915, Institut du thorax, Nantes, France

25 Prévalence et facteurs de risque de résection inadéquate dans la chirurgie des sarcomes des tissus mous

Introduction La planification des marges de resection dans la chirurgie des sarcomes des tissus mous resulte d’un compromis entre sacrifice fonctionnel et securite therapeutique. En pratique, l’analyse histologique des marges de resection montre souvent que l’objectif preoperatoire n’est pas obtenu. Nous avons defini ceci comme une « discordance anatomo-chirurgicale » et en avons etudie la prevalence et les facteurs de risque. Materiel et methode Il s’agit d’une serie prospective monocentrique de 133 patients. Les objectifs de resection, les resultats de l’examen anatomopathologique, ainsi qu’une fiche de transmission peroperatoire ont ete colliges. Les marges ont ete classees selon l’UICC (R0, R1, R2). Les donnees ont ete incluses dans une grille regroupant egalement les informations preoperatoires caracterisant les patients et les tumeurs. Les cas de discordance definis comme R0 planifie pour R1 ou R2 obtenu, ont ete identifies et etudies a l’aide d’un logiciel statistique (Statview 5.0). Resultats La prevalence des discordances anatomo-chirurgicales etait de 25,2 %. Parmi les facteurs analyses, l’aspect des limites etait significativement lie au mauvais resultat chirurgical (Odds Ratio = 2,85 [1,47-5,52] (p = 0,0031)). L’etude des differentes variables n’a pas permis la mise en evidence d’autres facteurs de risque significatif. Au-dela de 2 mm de marge, la chirurgie etait consideree comme adequate dans 100 % des cas. Discussion Notre etude n’a pas permis d’identifier de facteur predictif preoperatoire de marges de resection inadequates. En postoperatoire, l’aspect microscopique des limites de la proliferation lors de l’examen anatomopathologique final est pour nous significativement associe aux cas de resection inadequate. Or, la classification actuelle des marges (entre R0 et R1) reste peu precise dans les cas de resection marginale (dans notre etude, inferieurs a 2 mm), particulierement pour les tumeurs mal limitees. Cela semble etre la source de difficultes d’interpretation pour le pathologiste et pose des problemes de prise en charge pour le chirurgien. Seul le recul permettant d’evaluer le devenir de ces patients nous permettra d’eclaircir ce point. Conclusion Nous n’avons pas mis en evidence de facteur predictif de resection inadequate accessible en preoperatoire. Un aspect microscopique mal limite de la tumeur est significativement associe aux resections inadequates. Cependant, la classification actuelle pose des problemes d’interpretation pour les exereses avec marges inferieures a 2 mm. Concernant ces cas, il reste necessaire de clarifier la definition des marges afin d’optimiser la prise en charge des patients.

Expression of Leukemia Inhibitory Factor by Cartilage-forming Tumors of Bone: An Immunohistochemical Study

Recent studies have implicated leukemia inhibitory factor in connective-tissue metabolism involving the remodeling of bone and the destruction of cartilage tissue. This cytokine, which has also been implicated in the proliferation of solid tumor, is expressed by osteotropic tumor cell lines. The present study investigated the presence of leukemia inhibitory factor in cartilage tissue harvested from cartilage-forming bone tumors. Immunohistochemical study showed that it was present in all benign enchondromas (n = 8) and malignant chondrosarcomas (n = 6) but not in control tissue (n = 3). The cytokine was localized in only cytoplasmic areas of cartilage cells. The number of stained cells ranged from less than 5% in enchondroma of the hand to more than 70% in grade-III chondrosarcoma. Moreover, high levels of leukemia inhibitory factor were found in the primary culture of tumor tissues (n = 7). These results question the significance of leukemia inhibitory factor in tumor-associated bone resorption and the potential role of this cytokine as a prognostic marker.

Sur les inclusions paracristallines des cellules nourricières de la galle provoquée par Diplolepis rosae L. sur Rosa canina L.

Numerous crystalline inclusions are found in the nutritive cells lining the larval cavities of the galls induced by Diplolepis rosae L. on Rosa canina L. Electron microscopic investigations show that these intracytoplasmic inclusions consist of staggered, closely parallel, and slightly wavy filaments. In each filament three fibrils can be distinguished inside a less electron-dense matrix. The different aspects that the filaments may present according to the section plans are studied with an electron microscope equipped with a goniometric stage. Enzymatic digestion of ultrathin sections show that the paracrystals are essentially composed of proteins. This conclusion is sustained by the results of an ultrastructural autoradiographic study using tritiated aminoacids. The physiological significance of these paracrystals is discussed: their presence is probably related to a larval action which stimulates proteosynthesis in the cells surrounding the consumed ones.