Interdisciplinary approach to the management of Ewing sarcoma: A case report

Ewing sarcoma is a common primary bone malignancy occurring in childhood and adolescence. This case report describes a 4-year-old female patient who had Ewing sarcoma in the left clavicular region. The patient underwent total excision of the left clavicle and subsequently developed periodontitis and multiple carious lesions after chemotherapy. Caries risk and salivary flow rate tests were performed, followed by periodontal treatment, topical fluoride application, restoration of caries, and oral hygiene instruction. The care of this patient demonstrates that an interdisciplinary approach is essential to eliminate all foci of infection, minimize morbidity, and improve the patient's general health before, during, and after oncological treatment. © 2012 Special Care Dentistry Association and Wiley Periodicals, Inc.

Identificazione di fattori trascrizionali associati al differenziamento neurale in cellule di sarcoma di Ewing: ruolo di NF-kB

Il sarcoma di Ewing (ES) è un tumore maligno pediatrico dell’apparato scheletrico; è associato a una traslocazione specifica codificante la proteina di fusione EWS-FLI1 e all’alta espressione di CD99, una glicoproteina di membrana fisiologicamente coinvolta in diversi processi biologici. EWS-FLI1 e CD99, sono riportati avere ruoli divergenti nella modulazione della malignità e del differenziamento di ES. CD99 inoltre è riportato modulare il pathway di MAPK, il quale interagendo con molteplici fattori di trascrizione partecipa a processi di proliferazione e differenziamento. In questo studio abbiamo investigato in due linee cellulari di ES silenziate per CD99 (TC-71shCD99 e IOR/CARshCD99) l’attività basale di diversi fattori trascrizionali quali: NF-kBp65, AP1, Elk-1, E2F e CREB. L’unico fattore trascrizionale statisticamente significativo è risultato essere NF-kBp65 e abbiamo valutato il suo ruolo nel differenziamento neurale di cellule di ES e la relazione con EWS-FLI1 e CD99. L’attività trascrizionale di NF-kB è stata valutata attraverso gene reporter assay in linee cellulari di ES a diversa espressione di CD99, EWS-FLI1 e NF-kB stesso. Il differenziamento neurale è stato valutato come espressione di βIII-Tubulin in immunofluorescenza. Il silenziamento di CD99 induce una down-modulazione dell’attività trascrizionale di NF-kB, mentre il knockdown di EWS-FLI1 ne induce un’aumento. Inoltre, il silenziamento di EWS-FLI1 non è in grado di contrastare la riduzione dell’attività di NF-kB osservata dopo silenziamento di CD99, suggerendo un ruolo dominante del CD99 nel signaling di NF-kB. Cellule deprivate di CD99 ma non di EWS-FLI1, mostrano un fenotipo differenziato in senso neurale, fenotipo che viene perso quando le cellule sono indotte a sovraesprimere NF-kB. Inoltre, in cellule CD99 positive, il silenziamento di NF-kB induce un leggero differenziamento neurale. In conclusione, questi dati hanno evidenziato il ruolo di NF-kB nel differenziamento di cellule di ES e che potrebbe essere un potenziale target nel ridurre la progressione di questo tumore.

Cell death mechanisms triggered by monoclonal antibodies against CD99 in Ewing sarcoma: Cross-talk between MDM2, IGF-1R and Ras/MAPK

CD99 is a 32 kDa transmembrane protein whose high expression characterizes Ewing sarcoma (ES), a very aggressive pediatric bone tumor. In addition to its diagnostic value, CD99 has therapeutic potential since it leads to rapid and massive ES cell death when engaged with specific antibodies. Here a novel mechanism of cell death triggered via CD99 is shown, leading, ultimately, to the appearance of macropinocytotic vescicles. Anti-CD99 mAb 0662 induces MDM2 ubiquitination and degradation, which causes not only a p53 reactivation but also the IGF-1R induction and its subsequent internalization; CD99 results internalized together with IGF-1R inside endosomes, but then the two molecules display a different sorting: CD99 is degraded, while IGF-1R is recycled on the surface, causing, as a final step, the up-regulation of RAS-MAPK. High-expressing CD99 mesenchymal stem cells show mild Ras induction but no p53 activation and escape cell death, but in presence of EWS/FLI1 mesenchymal stem cells expressing CD99 show a stronger Ras induction and a p53 reactivation, leading to a significant cell death rate. We propose that CD99 triggering in a EWS/FLI1-driven oncogenetic context creates a synergy between RAS upregulation and p53 activation in ES cells, leading to cell death. Moreover, our data rule out possible concerns on toxicity related to the broad CD99 expression in normal tissues and provide the rationale for the therapeutic use of anti-CD99 MAbs in the clinic.

IGF system, CD99 and tumor-specific chromosomal translocations: evaluation of their cross-talk and definition of their role in Ewing sarcoma and prostate cancer

Aberrant expression of ETS transcription factors, including FLI1 and ERG, due to chromosomal translocations has been described as a driver event in initiation and progression of different tumors. In this study, the impact of prostate cancer (PCa) fusion gene TMPRSS2-ERG was evaluated on components of the insulin-like growth factor (IGF) system and the CD99 molecule, two well documented targets of EWS-FLI1, the hallmark of Ewing sarcoma (ES). The aim of this study was to identify common or distinctive ETS-related mechanisms which could be exploited at biological and clinical level. The results demonstrate that IGF-1R represents a common target of ETS rearrangements as ERG and FLI1 bind IGF-1R gene promoter and their modulation causes alteration in IGF-1R protein levels. At clinical level, this mechanism provides basis for a more rationale use of anti-IGF-1R inhibitors as PCa cells expressing the fusion gene better respond to anti-IGF-1R agents. EWS-FLI1/IGF-1R axis provides rationale for combination of anti-IGF-1R agents with trabectedin, an alkylator agent causing enhanced EWS-FLI1 occupancy on the IGF-1R promoter. TMPRSS2-ERG also influences prognosis relevance of IGF system as high IGF-1R correlates with a better biochemical progression free survival (BPFS) in PCa patients negative for the fusion gene while marginal or no association was found in the total cases or TMPRSS2-ERG-positive cases, respectively. This study indicates CD99 is differentially regulated between ETS-related tumors as CD99 is not a target of ERG. In PCa, CD99 did not show differential expression between TMPRSS2-ERG-positive and –negative cells. A direct correlation was anyway found between ERG and CD99 proteins both in vitro and in patients putatively suggesting that ERG target genes comprehend regulators of CD99. Despite a little trend suggesting a correlation between CD99 expression and a better BPFS, no clinical relevance for CD99 was found in the field of prognostic biomarkers.

Loss of pulp sensitivity and pain as the first symptoms of a Ewing's sarcoma in the right maxillary sinus and alveolar process: report of a case

This case report describes the diagnosis and treatment of a Ewing's sarcoma in the right maxillary sinus and alveolar bone of a 19-year-old female patient. The first clinical symptoms were a loss of sensitivity of the premolars and first molar in the right maxilla and acute pain located in the area of these teeth. Initially, the referring dentist had treated these findings as an acute apical periodontitis with root canal medication. Because swellings on the palatal and buccal aspects of the teeth occurred and could not be treated with incision and drainage, the dentist referred the patient. Cone-beam computed tomography revealed a proliferation of soft tissue in the right maxillary sinus, with a radiopaque material at the tip of the mesiobuccal root of the first molar and resorptive signs of the mesiobuccal and distobuccal roots of the first molar. The palatal cortical bone of the right alveolar process seemed to be intact. After explorative surgery with biopsies from the buccal, palatal, and sinus proliferation areas, the pathologist diagnosed the lesion as a Ewing's sarcoma. Treatment of the patient consisted of initial chemotherapy, hemimaxillectomy, and postsurgical chemoradiotherapy.

High expression of neuropeptide Y1 receptors in ewing sarcoma tumors

PURPOSE: Peptide receptors are frequently overexpressed in human tumors, allowing receptor-targeted scintigraphic imaging and therapy with radiolabeled peptide analogues. Neuropeptide Y (NPY) receptors are new candidates for these applications, based on their high expression in specific cancers. Because NPY receptors are expressed in selected sarcoma cell lines and because novel treatment options are needed for sarcomas, this study assessed the NPY receptor in primary human sarcomas. EXPERIMENTAL DESIGN: Tumor tissues of 88 cases, including Ewing sarcoma family of tumors (ESFT), synovial sarcomas, osteosarcomas, chondrosarcomas, liposarcomas, angiosarcomas, rhabdomyosarcomas, leiomyosarcomas, and desmoid tumors, were investigated for NPY receptor protein with in vitro receptor autoradiography using (125)I-labeled NPY receptor ligands and for NPY receptor mRNA expression with in situ hybridization. RESULTS: ESFT expressed the NPY receptor subtype Y1 on tumor cells in remarkably high incidence (84%) and density (mean, 5,314 dpm/mg tissue). Likewise, synovial sarcomas expressed Y1 on tumor cells in high density (mean, 7,497 dpm/mg; incidence, 40%). The remaining tumors expressed NPY receptor subtypes Y1 or Y2 at lower levels. Moreover, many of the sarcomas showed Y1 expression on intratumoral blood vessels. In situ hybridization for Y1 mRNA confirmed the autoradiography results. CONCLUSIONS: NPY receptors are novel molecular markers for human sarcomas. Y1 may inhibit growth of specific sarcomas, as previously shown in an in vivo mouse model of human ESFT. The high Y1 expression on tumor cells of ESFT and synovial sarcomas and on blood vessels in many other sarcomas represents an attractive basis for an in vivo tumor targeting.

Miss Nettie Spencer [Illustration]

Rudolf Marcuse

Vasculogenesis: A process of vessel formation and its role during Ewing's sarcoma development

Vasculogenesis is the process by which Endothelial Precursor Cells (EPCs) form a vasculature. This process has been traditionally regarded as an embryological process of vessel formation. However, as early as in the 60's the concept of postnatal vasculogenesis was introduced, with a strong resurface of this idea in recent years. Similarly, previous work on a mouse skin tumor model provided us with the grounds to consider the role of vasculogenesis during tumor formation. ^ We examined the contribution of donor bone marrow (BM)-derived cells to neovascularization in recipient nude mice with Ewing's sarcoma. Ewing's sarcoma is a primitive neuroectodermal tumor that most often affects children and young adults between 5 and 30 years of age. Despite multiple attempts to improve the efficacy of chemotherapy for the disease, the 2-year metastases-free survival rate for patients with Ewing's sarcoma has not improved over the past 15 years. New therapeutic approaches are therefore needed to reduce the mortality rate. ^ The contribution of BM endothelial precursor cells in the development of Ewing's sarcoma was examined using different strategies to track the donor-derived cells. Using a BMT model that takes advantage of MHC differences between donor and recipient mice, we have found that donor BM cells were involved in the formation of Ewing's sarcoma vasculature. ^ Cells responsible for this vasculogenesis activity may be located within the stem cell population of the murine BM. These stem cells would not only generate the hematopoietic lineage but they would also generate ECs. Bone marrow SP (Side Population) cells pertain to a subpopulation that can be identified using flow cytometric analysis of Hoechst 33342-stained BM. This population of cells has HSC activity. We have tested the ability of BM SP cells to contribute to vasculogenesis in Ewing's sarcoma using our MHC mismatched transplant model. Mice transplanted with SP cells developed tumor neovessels that were derived from the donor SP cells. Thus, SP cells not only replenished the hematopoietic system of the lethally irradiated mice, but also differentiated into a non-hematopoietic cell lineage and contributed to the formation of the tumor vasculature. ^ In summary, we have demonstrated that BM-derived cells are involved in the generation of the new vasculature during the growth of Ewing's sarcoma. The finding that vasculogenesis plays a role in Ewing's sarcoma development opens the possibility of using genetically modified BM-derived cells for the treatment of Ewing's sarcomas. ^

p16-induced apoptosis in Ewing's sarcoma

The tumor suppressor p16 is a negative regulator of the cell cycle, and acts by preventing the phosphorylation of RB, which in turn prevents the progression from G1 to S phase of the cell cycle. In addition to its role in the cell cycle, p16 may also be able to induce apoptosis in some tumors. Ewing's sarcoma, a pediatric cancer of the bone and soft tissue, was used to study the ability of p16 to induce apoptosis due to the fact that p16 is often deleted in Ewing's sarcoma tumors and may play a role in the oncogenesis or progression of this disease. The purpose of these studies was to determine whether introduction of p16 into Ewing's sarcoma cells would induce apoptosis. We infected the Ewing's sarcoma cell line TC71, which does not express p16, with adenovirus- p16 (Ad-p16). Ad-p16 infection led to the production of functional p16 as measured by the induction of G1 arrest. Ad-p16 infection induced as much as a 100% increase in G1 arrest compared to untreated cells. As measured by propidium iodide (PI) and Annexin V staining, Ad-p16 was able to induce apoptosis to levels 20–30 fold higher than controls. Furthermore, Ad-p16 infection led to loss of RB protein before apoptosis could be detected. The loss of RB protein was due to post-translational degradation of RB, which was inhibited by the addition of the proteasome inhibitors PS-341 and NPI-0052. Downregulation of RB with si-RNA sensitized cells to Ad-p16-induced apoptosis, indicating that RB protects from apoptosis in this model. This study shows that p16 leads to the degradation of RB by the ubiquitin/proteasome pathway, and that this degradation may be important for the induction of apoptosis. Given that RB may protect from apoptosis in some tumors, apoptosis-inducing therapies may be enhanced in tumors which have lost RB expression, or in which RB is artificially inactivated. ^

Bone marrow-derived stem/progenitor cells play an important role in the growth of Ewing's sarcoma tumors

Both angiogenesis and vasculogenesis contribute to the formation and expansion of tumor neovasculature. We demonstrated that bone marrow (BM)-derived cells migrated to TC71 Ewing's tumors and differentiated into endothelial cells lining perfused, functional tumor neovessels. In addition, a substantial fraction of recruited, BM-derived cells resided in the vessel vicinity but did not demonstrate endothelial differentiation. Rather, these perivascular cells expressed desmin and PDGFR-β, implying pericyte-like/vascular smooth muscle cell differentiation. No defined, consensus set of markers exists for endothelial progenitor cells (EPCs) and the specific subsets of BM cells that participate in vessel formation are poorly understood. We used a functional in vivo assay to investigate the roles performed by specific human- and murine-derived stem/progenitor subpopulations within Ewing's sarcoma tumors. CD34 +45+, CD34+38-, VEGFR2 + and Sca1+Gr1+ cells were demonstrated to establish residence within the expanding tumor vascular network and differentiate into endothelial cells and pericytes. By constrast, CD34-45 + and Sca1-Gr1+ cells predominantly localized to sites outside the Ewing's tumor vasculature, and differentiated into macrophages. Cytokines, such as VEGF, influence the recruitment of BM cells and their incorporation into the tumor vasculature. VEGF165-inhibited TC/siVEGF7-1 Ewing's tumors showed delayed in vivo tumor growth, decreased vessel density, and reduced infiltration of BM progenitor cells. We tested whether another chemoattractant, Stromal Cell-Derived Factor-1 (SDF-1), could augment the growth of these VEGF165-inhibited TC/siVEGF 7-1 tumors by enhancing the recruitment of BM cells and stimulating neovasculature expansion. SDF-1 promoted progenitor cell chemotaxis and retainment of BM-derived pericyte precursors in close association with functional, perfused tumor blood vessels. Treatment of TC/siVEGF7-1 tumors with adenovirus-SDF-1α resulted in augmented tumor size, enhanced pericyte coverage of tumor neovessels, remodeling of vascular endothelium into larger, functional structures, and upregulation of PDGF-BB, with no effect on VEGF165. Taken together, these findings suggest that the recruitment of BM stem/progenitor cells plays an important role in the growth of Ewing's tumors. ^