Skip Navigation LinksHome > January 2014 - Volume 21 - Issue 1 > Innovative Therapies in Ewing Sarcoma
Advances in Anatomic Pathology:
doi: 10.1097/PAP.0000000000000003
Review Articles

Innovative Therapies in Ewing Sarcoma

Amaral, Ana Teresa BSc*; Ordóñez, José Luis PhD*; Otero-Motta, Ana Pastora PhD*; García-Domínguez, Daniel J. PhD*; Sevillano, María Victoria MLT*; de Álava, Enrique MD, PhD*,†

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*Molecular Pathology Program, Institute of Biomedical Research of Salamanca-Centro de Investigación del Cáncer, Centro de Investigación del Cancér (IBSAL-CIC), Campus Miguel de Unamuno S/N, Salamanca

Pathology Department, Hospital Universitario Virgen del Rocio-IBiS, Sevilla, Spain

All figures can be viewed online in color at http://www.anatomicpathology.com.

A.T.A. and J.L.O. contributed equally.

The Centro de Investigación del Cáncer participates in European Clinical trials in Rare Sarcomas within an integrated translational trial network (FP7-HEALTH-2011-2-stage, Project ID 278742 EUROSARC) European Commission and PROspective VAlidation of Biomarkers in Ewing Sarcoma for personalized translational medicine (PROVABES). Work in the Molecular Pathology laboratory is supported by the ISCIII (FIS-FEDER) (PI11/00018) and PROVABES (PI12/03102), CSIC (Contratos JAE-DOC to JLOG), the Fundação para a Ciência e Tecnologia, Ministério para a Investigaçãoe Tecnologia, Portugal (ATA fellowship SFRH/BD/69318/2010), Fundación Memoria de D. Manuel Solorzano Barruso, Fundación Cris contra el cancer, and Fundación María García Estrada.

The authors have no conflicts of interest to disclose.

Reprints: Enrique de Álava, MD, PhD, Molecular Pathology Program, Institute of Biomedical Research of Salamanca-Centro de Investigación del Cáncer (IBSAL-CIC), Centro de Investigación del Cáncer- Instituto de Biología Molecular y Celular del Cáncer (IBMCC) (IBSAL-CIC), Campus Miguel de Unamuno S/N, 37007 Salamanca, Spain (e-mail: edealava@usal.es).

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Abstract

Ewing Sarcoma is a developmental tumor characterized by balanced chromosomal translocations and formation of new fusion genes, which are the main hallmark of this rare entity. Despite the vast knowledge regarding the molecular aspects of this rare malignancy obtained in the last few years, including the discovery of new therapeutic targets, many questions still remain open. In this review we focus on the research on targeted therapies in this malignancy, and discussed some bottlenecks related to this such as the possible role of pathologists, the availability of samples, the lack of appropriate animal models, and the resources needed to carry out preclinical and clinical research.

Ewing Sarcoma (ES) is a rare malignancy that affects mainly children and young adults. ES is a small round cell sarcoma showing pathognomonic molecular findings, and varying degrees of neuroectodermal differentiation detected by light or electron microscopy, or immunohistochemistry. The term primitive neuroectodermal tumor (PNET) was classically used for ES with evidence of neuroectodermal differentiation.

ES is molecularly characterized by recurrent balanced translocations which lead to the formation of novel fusion oncogenes that are the key to pathogenesis. ES pathogenesis is driven by pathognomonic and etiologic EWSR1-ETS gene fusions, which, as described below, encode chimeric transcription factors that are expressed in these tumors. EWSR1-ETS fusion proteins activate or repress specific sets of target genes that, together with the right timing and cellular context, give rise to the transformed phenotype of ES. Recent data point either mesenchymal stem cells or neural crest–derived stem cells as the cell of origin of ES.1,2

In this review, we gather information on the latest preclinical and clinical evidence developed in ES therapy as schematically summarized in Figure 1. Information regarding recent, ongoing and completed clinical trials (CTs) was collected from http://www.clinicaltrials.gov, and is briefly described in Table 1.

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TABLE 1
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INCIDENCE AND PREVALENCE

ES is relatively uncommon, accounting for 6% to 8% of primary malignant bone tumors. However, it is the second most common malignant bone tumor in children and young adults after osteosarcoma.47 ES shows a slight predilection for male individuals, with the ratio of 1.4 to 1. Nearly 80% of patients are younger than 20 years, and the peak age at incidence is during the second decade of life. The incidence of ES in individuals older than 30 years is extremely uncommon.48 These tumors are virtually unknown in individuals of African or (to a lesser extent) Asian descent. One proposed explanation is the reduced size of EWSR1 intron 6, near the translocation breakpoint region, in the African population.49

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CLINICAL FEATURES

ES symptomatology is not very extensive and includes mostly pain severe enough to wake up patients (96%), with or without a mass in the involved area (61%). Moreover, intermittent fever (21%), anemia, and unspecific laboratory results are often seen. Pathologic fractures (16%) at diagnosis are uncommon. Radiographically, an ill-defined osteolytic lesion involving the diaphysis of a long tubular bone is the most common feature. Permeative or moth-eaten bone destruction often associated with “onion skin”–like multilayered periosteal reaction is also characteristic. A large, ill-defined soft tissue mass is a frequent association. Magnetic resonance imaging studies help demonstrate the extent of the tumor in the bone and soft tissue. The most prevalent sites are, in descending order: (i) the diaphysis or metaphyseal-diaphyseal portion of long bones (femur, tibia, humerus); (ii) the pelvis and ribs; and (iii) other bones, such as the skull, vertebra, scapula, and short tubular bones of hands and feet. About 10% of cases are extraskeletal.

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PROGNOSIS

The prognosis in ES has improved considerably with current therapy, and approximately two thirds of patients are cured of their disease.50 However, outcome for patients with disseminated disease or early relapse remains dismal, and the presence of metastatic disease appears to be the major prognostic factor.51,52

Important pathologic prognostic features include the stage, anatomic location, and the size of the tumor. Histopathologic assessment of tumor necrosis after induction chemotherapy has prognostic value. Tumors that are metastatic at the time of diagnosis, arise in the pelvis, and are large tend to do poorly. The extent of neural differentiation does not appear to predict outcome.53 Several molecular features were reported as having prognostic value, including p53 status, INK4A loss, telomerase expression, percentage of genome altered, and additional chromosomal aberrations such as gains of chromosome 1q, but practical application requires validation in prospective cooperative studies.54

EWSR1-ETS fusion status was initially reported as providing prognostic information; among localized tumors with EWSR1-FLI1 gene fusions, those with the most common type 1 EWSR1 exon 7/FLI1 exon 6 fusions had a better prognosis than cases with larger fusion types.54–60 However, the use of more intensive therapeutic regimens appears to have eliminated these differences.61,62

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STANDARD OF CARE: LOCALIZED AND METASTATIC/RELAPSED/REFRACTORY DISEASE

Local therapy supplemented with chemotherapy/radiotherapy is the current standard of care for a vast number of solid tumors, including ES. Accordingly, this type of treatment does not specifically target the pathogenetic mechanisms of ES cells, and therefore drug-related resistance frequently emerges. First-line therapy consists of neoadjuvant therapy, usually combining 4 to 6 chemotherapeutic agents among vincristine (V), doxorubicin (D), etoposide (E), cyclophosphamide (C), ifosfamide (I); and/or dactinomycin (D) also named actinomycin-D (A), followed by surgery and/or radiotherapy when appropriate.51 The most widely used combination in Europe is VIDE/VAI, whereas in the United States is VDC/IE.63 These multimodality treatments were able to increase overall survival up to 60% to 70% in localized disease. Nevertheless, in cases of multifocal disease, overall survival is <20%.63,64

Under the Euro-EWING99 CT (NCT00020566) patients presenting primary or disseminated multifocal ES, followed several cycles of multimodal therapeutic combinations of VIDE, VAI, radiotherapy, surgery, and high-dose chemotherapy (busulfan-melphalan) with autologous stem cell transplantation. Outcome from this trial revealed that the best approach resulted from the combination of local treatment with surgery and/or radiotherapy plus multimodal intensive therapeutic strategies.51,64 Interestingly, prospective studies from this CT determined a lack of prognostic benefit related to the molecular subtype of the fusion transcript EWSR1-FLI1, in contrast to previous studies including an inferior number of patients, which had showed a correlation between this transcript and better prognosis.60,61

Under the ongoing, phase III CT, EWING 2008 (NCT00987636), patients with localized and disseminated ES were randomized into 3 broad groups according to standard risk: (1) patients with localized disease with tumor volume <200 mL are being treated with zoledronic acid and/or the retinoid derivative fenretinide in addition to induction chemotherapy (VIDE); (2) patients with localized/metastasis disease with tumor volume >200 mL are being treated with high-dose chemotherapy (busulfan-melphalan) and autologous stem cell transplantation; (3) patients with primary disseminated disease are being treated with 8 cycles of standard adjuvant chemotherapy (VIDE/VAI), followed by high-dose chemotherapy (treosulfan-melphalan) plus autologous stem cell transplantation.

Regarding relapsed and metastatic disease, in which patients present much worse prognosis, there are several nonrandomized ongoing CTs at the moment. These studies include treatment with Trabectedin and its related compound Zalypsis (NCT01222767); Src family tyrosine kynase (TK) inhibitor Dasatinib (NCT01643278); PARPinh olaparib (NCT01583543) or proteasome inhibitor bortezomib (NCT00027716) among others, in monotherapy, as well as other combinatory regimens, which we will discuss later on.

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INNOVATIVE THERAPIES IN EWING SARCOMA

Targeting the Ewing Sarcoma Fusion

ES is characterized by the presence of aberrant chromosomal translocations giving rise to chimeric proteins, typically involving the EWSR1 gene and 1 member of the ETS family. The most common translocation is produced between EWSR1-FLI1 genes, t(11;22) (q24;q12), leading to the upregulation of direct and indirect targets, such as CD99, IGF1, TERT, HSP, CAV-1, TOPK, among others, and downregulation of IGF binding protein 3 (IGFBP3) or p21.32,65–70 Translocations between the EWSR1 gene and other ETS family genes, namely ERG, ETV1, ETV4, or FEV, are less frequent in ES. Rare cases, known as “Ewing-like sarcomas,” may present ESWR1 fused with non-ETS genes, namely NFATc2, POU5F1, SMARCA5, ZSG, SP3, as well as unrelated translocations to EWSR1, such as CIC-DUX4. In addition, very rarely we can find translocations involving genes with functional homology to either EWSR1 such as FUS.70

The expression of the EWSR1-FLI1 oncogenic fusion protein specifically in tumor cells makes it a very attractive candidate in terms of targeted therapy. Despite the idea that transcription factors are often considered undruggable, recent works have shown that certain compounds, either chemotherapeutic agents, small molecules, or peptides, present certain specificity toward EWSR1-FLI1 and result in cytotoxic activity.

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Trabectedin and Related Compounds

The marine-derived chemotherapeutic agent Trabectedin, ET-743, is an alkylator agent, which has been largely studied for its antitumoral activity toward several types of tumors. Trabectedin is particularly toxic in sarcomas bearing translocations.6,71–73

In fact, in myxoid liposarcoma, one example of translocation-related sarcoma (TRS), Trabectedin downregulates the binding of FUS-CHOP to promoter regions of genes regulated by this chimeric protein, thus supporting the particular specificity of this agent toward TRS.71 As ES cells are characterized by the presence of transcriptional factors with structural and functional homology to FUS-CHOP, a similar approach was performed. This study showed that Trabectedin also interferes with the transcriptional activity of EWSR1-FLI1.5 In addition, several studies report this agent as one of the most potent antitumoral agents toward ES, with IC50 of proliferation inhibition on the subnanomolar range.5,6,74,75 Trabectedin presents a unique mechanism of action, creating adducts with the guanine-rich regions of the minor groove of the DNA and then bending toward the minor groove finally disrupting the double helix.73,76–78 Trabectedin has already been through phases I and II in CTs (NCT01453583 and NCT00070109). In phase II, a study on refractory relapsed pediatric tumors including 16 ES patients showed insufficient activity of Trabectedin in monotherapy; however, it was safely tolerated in children.7,8 Given its tremendous in vivo and in vitro effects, the development of molecules structurally related to ET-743, such as Zalypsis or Lurbinectedin, in search of better pharmocokinetic/pharmacodynamic characteristics has been highly successful. These agents are related to Trabectedin mainly because of similarities in their chemical structure, which are very well illustrated and reviewed by Molinski et al.73 Briefly, the basic chemical structure includes 3 tetrahydroisoquinoline rings, A, B, and C, and differences in ring C confer distinct pharmacokinetic and pharmacodynamic characteristics to each of these molecules.73

One of these compounds is Zalypsis, recently described as one of the most toxic agents toward multiple myeloma and acute myeloid blasts.11,12 It is also highly cytotoxic against a variety of solid tumors.9,10 In ES, there is some preclinical evidence that Zalypsis might well be a promising therapeutic approach.9,74 In fact, this drug has completed a phase II CT (NCT01222767) in monotherapy in patients with unresectable locally advanced and/or metastatic ES who are progressing and have already undergone standard chemotherapy.9,74,75 More recently, a work published by Romano and colleagues described that Trabectedin, Zalypsis, and also Lurbinectedin, another Trabectedin-related compound, are very similar in terms of in vitro cytotoxicity; however, some differences were observed in terms of in vivo pharmacokinetics. This study included several cancer cell lines among which some were sarcoma cell lines: murine fibrosarcoma and murine reticulosarcoma. ES cell lines were not studied in this work.13

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Mithramycin

A high-throughput study that included 50,000 compounds has highlighted mithramycin for its specificity toward cells bearing EWSR1-FLI1 translocations.14 In vivo and in vitro studies unveiled that mithramycin inhibits EWSR1-FLI1 activity, observed through the deregulation of its target genes after treatment.14 Mithramycin has demonstrated antineoplastic antibiotic activity, and its security parameters (phase I CTs) have already been overcome in other neoplasias. Currently, ES patients are being recruited for a phase I/II CT with mithramycin in monotherapy for patients bearing the EWSR1-FLI1 transcript (NCT01610570), and other solid tumors in children and adults.

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YK-4-279

The small molecule YK-4-279, specifically the enantiomer (S)-YK-4.279, has also shown promising results in terms of EWSR1-FLI1 deregulation, mostly by disrupting the bond of this oncogenic protein with the RNA helicase A.15–17 This agent blocks the interaction between the transcription factor and RNA helicase A, which is apparently relevant in terms of enhancing the oncogenic activity of EWSR1-FLI1.79 Preclinical studies revealed that treatment with YK-4-279 led to apoptosis induction and tumor shrinkage in ES xenografts.15,17

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ARA-C

Cytosine arabinose, ARA-C, has also been identified through a high-throughput analysis based on gene expression. ARA-C is a compound able to attenuate the EWSR1-FLI1 signature.18 Accordingly, in vitro and in vivo assays showed that ARA-C induces cell death and diminishes tumor growth in xenografts. This fact is mainly justified by the reduction of the EWSR1-FLI1 chimeric protein in ES cells during treatment with ARA-C.18 Nevertheless, another work from Houghton and colleagues evaluated sensitivity to ARA-C on a large panel of pediatric tumor cell lines (n=23), followed by an in vivo assay in ES xenografts. Herein, they observed that in comparison with other tumors, ES cells were not particularly sensitive to cytarabine. In fact, the in vivo assays performed exclusively in 6 ES xenografts generated by the injection of 6 ES cell lines determined that only 1 cell line–generated tumor showed reduction, whereas the other 5 cell lines showed no reduction of the tumor growth rate at the studied dose.19 Meanwhile, clinical evaluation of ARA-C in ES patients was performed in a phase II CT including patients with relapsed or refractory ES (NCT00470275). Despite the particular interest of ARA-C, since it already passed safety trials in other cancer types, general outcome from this specific trial resulted in minimal activity in ES patients as well as hematological toxicity.20

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Targeting the IGF1-IGF1R Pathway

One of the mostly described downstream targets of the ES fusion is the IGF1-IGF1R pathway. Insulin-like growth factor receptor 1 (IGF1R) is a TK receptor located in the cell membrane, formed by 4 subunits, 2 units located on the extracellular part of the cell membrane and other 2 trespassing the membrane. In physiological conditions, TK receptor IGF1R and its ligand IGF1 are critical in terms of cell growth and accurate cell differentiation. In fact the whole system involves several TK receptors: IGF1R; insulin receptor (IR) (isoforms A and B); and hybrid receptors IGFIR and IGF2R as well as ligands: IGF1, IGF2, and insulin.80 In addition, the IGFBPs play an important role blocking IGF1 and IGF2 from activating the respective receptors, whenever suitable.

Activation of IGF1R occurs by reversible binding of its ligand, which can lead to receptor dimerization and activation of the nearby IGF1R, therefore enhancing pathway activation. Immediately, a cascade of downstream targets is activated. The IGF1R cascade can also be enhanced by internalization of activated TK receptors signaling through the cytoplasm in caveolin pits of clathrin-coated vesicles, as described below.31

IGF1R represents an attractive targeting strategy in ES as it is a known upregulated downstream target of EWSR1-FLI1. Particularly, it has been observed that, in vitro, ES cells present reduced expression of IGFBP3 with overexpression and constitutive activation of IGF1R downstream targets.81–84 Interestingly, ex vivo studies showed that among 290 samples, 70% to 80% of ES patients presented high expression of IGF1R and also of IR. A mere 10% were IGF1R negative, showing high IR expression. These results corroborated previous data, which determined that cells resistant to treatment with anti-IGF1 inhibitors were able to maintain their survival capacity by overexpressing IGF2/IR.85

Several efforts are currently being carried out, either through inhibition of IGF1R with small molecules or monoclonal antibodies (MAb), humanized or fully human. In ES, inhibition of the IGF1R pathway has without a doubt a demonstrated important preclinical value; nonetheless, up to today clinical outcome from IGF1R inhibition remains poor, thus stressing the need to develop distinct, or more suitable, therapeutic approaches to inhibit this pathway.

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Small Molecules

Various small molecules have been developed as a way to selectively inhibit IGF1R by ATP competition, namely Novartis compounds NVP-AEW541 and NVP-ADW742. Recently, a new small molecule named Linsitinib has demonstrated to inhibit both the IGF1R and IR, thus representing a clear benefit in terms of escaping drug resistance.23,24

AEW541 and ADW742 have demonstrated in vitro and in vivo activity toward ES.21,22,24 Our group previously confirmed the toxic activity of ADW742 and AEW451 in vitro and in vivo alone and in combination with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG).31

The new small molecule Linsitinib demonstrated in vitro and in vivo toxic activity in ES cells, with the particularity of inhibiting specifically IGF1R-IR, and thus blocking the autoloop previously described, in which ES cells switch from IGF1/IGF1R dependency to IGF2/IR dependency given the homology between both these receptors. In addition, Linsitinib activity will be evaluated under the phase I/II CT named LINES in ES patients with recurrent/disseminated disease under the EUROSARC project.

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Anti-IGF1R Monoclonal Antibodies

MAbs have been clinically developed in mice, initially humanized, IgG1 MAb (human-mouse-human), and recently fully human, IgG1, IgG2, or IgG4. MAbs are administered following high-safety measures to prevent immunogenic responses. Among MAbs, the fully human MAbs represent a safer approach in comparison with humanized MAbs, which increase the probability of side effects. Anti-IGF1R MAb mechanism is based not only on the obvious antagonistic effect with IGF1, inhibiting IGF1R, but also on the later downregulation of the IGF1R cascade. In fact, several anti-IGF1R MAbs, namely, AVE1642, Figitumumab, Cixutumumab, Robatumumab, have already shown preclinical and/or clinical efficacy in either ES or the broad group of sarcomas. These compounds have been brilliantly described in a recent review from Olmos et al86; therefore, here we will be describing the most representative MAbs with preclinical or clinical evidence in ES.

AVE1642 is a humanized IgG1, anti-IGF1R MAb, with demonstrated in vitro activity toward ES cells.23,24 The resistance of ES cells to this agent has been related to neural differentiation and angiogenesis.24 Outcome from phase I CT of AVE1642 in patients with advanced solid tumors, which did not include ES patients, determined good tolerability of this agent in combination with docetaxel and gemcitabine.25

Figitumumab (CP-751,871) fully human IgG2 anti-IGF1R MAb has shown preclinical evidence toward ES and has already passed phase I and II CT in patients with refractory ES (NCT00560235). According to preclinical evidence, resistance to Figitumumab is related to angiogenesis.24 Figitumumab entered phase I CT, which included 15 patients with refractory ES, and was safely evaluated; although the endpoint of this study was focused on security and not efficacy, some ES patients presented tumor shrinkage. The phase II study included 107 ES patients and showed that Figitumumab presented modest antitumor activity in monotherapy.26 In this set of patients, high IGF1 serum levels correlated with poor prognosis, and as both Figitumumab and IGF1 show very similar affinity toward IGF1R, the direct competition between high levels of ligand and MAb impaired figitumumab-IGF1R blockage.

Cixutumumab (IMC-A12) is also a fully human MAb, with demonstrated in vitro activity in various tumor types, namely head and neck squamous cell carcinoma and childhood malignancies including neuroblastoma, Wilms tumor, and sarcomas (rhabdomyosarcoma, ES, and osteosarcoma).27,87 Preclinical evidence reported by the Pediatric Preclinical Testing Program (PPTP) concluded that Cixutumumab was an active intermediate cytotoxic agent; however, only 1 of 5 ES xenografts was evaluable for its activity.27 In head and neck squamous cell carcinoma, cells present a coexpression of IGF1R and EGFR; therefore, treatment with Cixutumumab blocked IGF1R and resulted in the induction of mTOR and AKT expression. When combined with rapamycin and cetuximab, which caused inhibition of mTOR and EGFR, respectively, antitumor effects were promoted. These findings suggested that resistance to anti-IGF1R MAbs might be related to induction of AKT/mTOR.87 In fact, also under the PPTP, Cixutumumab and rapamycin alone/combined were studied in xenograft tumor models. Among the set of models studied here (ES, osteosarcoma, and rhabdomyosarcoma), ES showed the most dramatic response to the combinatory regimen, with a longer time to progression and clear synergistic effects.88 Moreover, IMC-A12 is currently in phase II CTs for advance sarcomas, in which metastatic/advanced ES represents one of the arms of this study (NCT00668148).

Robatumumab (R1507) is another anti-IGF1R MAb with preclinical and clinical evidence toward ES. ES cell lines showed sensitivity toward R1507; however, an siRNA targeting EWSR1-FLI1 fusion protein was able to induce higher levels of apoptosis compared with the MAb.28 In the clinic, a phase II study of Robatumumab in patients with recurrent or refractory ES included 132 patients and resulted in 10% of overall response for >6 months. Outcome from this study demonstrated that R1507 has modest activity as a single agent. This low activity could be explained by the lack of IGF1R:IR ratio prescreeening in the ES patiens enrrolled in this study. In addition, no drug combination was administrated.29

Regarding IGF1R inhibition, in vitro systems show that ES cells resistant to AVE1642, Figitumumab, and AEW541 were able to maintain their survival and proliferation ability by switching from the IGF1/IGF1R pathway to the IGF2/IR-A pathway.24 In fact, there is evidence that the IGF1R:IR ratio might very well be responsible for resistance to treatment with IGF1R inhibitors (MAbs or small molecules). This fact could justify why patients with higher IR levels do not respond to anti-IGF1R therapies, whereas patients with higher IGF1R respond better to these targeted therapies. Herein lies the peculiarity of the dual inhibitor Linsitinib, which might represent an advantage given that it indistinctively blocks both receptors.23 In addition, selection of patients according to the tumor IGF1R:IR ratio before treatment might be beneficial to further conclude whether an agent is really effective or simply that the tumor presents higher IR levels and therefore IGF1R inhibition accounts for a minor value on tumor shrinkage. In a very recent work O’Neill et al89 reflect over the realistic role of IGF1R in ES-targeted therapy. According to this work, IGF1R expression by flow cytometry determined that ES cell lines show reduced IGF1R expression when compared with a control cell line. In addition, the evaluation of mutations in tumor samples (n=47) and ES cell lines (n=8) resulted in the presence of only 1 mutation in a tumor sample. Detection of IGF1R by enzyme-linked immunosorbent assay in tumor samples showed low expression of this receptor. Despite these striking results, these last data did not fully correlate with the immunohistochemistry data. Hence, main conclusions from this work sustain that IGF1R expression is variable among ES tumors, and, once again, IGF1R-targeted therapies probably need to be performed in selected groups of patients to avoid the low response rates obtained in the CT previously described.89

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Targeting Downstream IGF1R
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mTOR Inhibitors

The mammalian target of rapamycin (mTOR) is a molecule downstream of AKT-PI3K, activated by IGF1R signaling. Inhibition of mTOR results in decreased cell proliferation. This protein is a serine-threorine kynase participating in normal cell growth; however, in ES and several other cancer types, its signaling is deregulated and culminates in aberrant increased cell proliferation and even angiogenesis. Another important aspect regarding mTOR signaling concerns the negative feedback loops from mTOR to IGF1R, decreasing IGF1R activation in a physiological state. In cancer cells, this negative feedback appears to be absent or decreased, hence contributing to further deregulation of the IGF1R pathway. In fact, a recent phase I CT combined Figitumumab and mTOR inhibitor everolimus in advanced sarcomas (NCT00927966). Among the 21 patients, only 1 had ES. Outcome from this study revealed the safe combination of these agents. In particular, the ES patient presented disease stability, which, however, was not accompanied by tumor reduction.90 Moreover, Cixutumumab has been combined with mTOR inhibitor Temsirolimus in refractory ES, and the combination was well tolerable in ES patients.90

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HSP90 Inhibitors

Heat shock proteins (HSPs) are a functional class of chaperone proteins, which are transcriptionally upregulated in response to stress. These molecules help maintain the stability, activity, and maturation of proteins called “client proteins” and also target misfolded client proteins to be degraded by the ubiquitin-proteasome system (UPS).91 Among HSP, HSP90 is overexpressed in cancer cells, and its >200 client proteins are involved in oncogenic signaling pathways, angiogenesis metastasis, and resistance to apoptosis. Therefore, drug targeting of HSP90 is an indirect way of inhibiting these oncogenic mechanisms.22,91 Several HSP90 inhibitors are currently in clinical evaluation. Structurally, all of them, except SNX-5422, are derivatives of Geldanamycin or Resorcinol or purine and purine-like analogs.92 The Geldanamycin derivate named Tanespimycin or 17-AAG was the first HSP90 inhibitor to be evaluated in CTs with good results.91 In a proteomic analysis of ES cell lines treated with IGF1R inhibitor ADW742 and/or c-kit inhibitor Imatinib, our group found that HSP90 was the protein showing the highest expression changes. Functional in vitro and in vivo studies using siRNA or Tanespimycin demonstrated the role of HSP90 in ES resistance, raising the possibility that targeting HSP90 might be of therapeutic value in ES, especially in cases of previous resistance to IGF1R/KIT pathway blockade.22

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Caveolin-1 (Inhibition of IGF1R Internalization)

Caveolins are a group of molecules localized in the plasma membrane, which form vesicles, named caveolae, transporting portions of the plasma membrane incorporating active receptors throughout the cytoplasm. Endocytosis is considered the major counterpart of signaling by caveolins.93 Among the broad group of caveolins, caveolin-1 (CAV-1) represents its major component.

CAV-1 was initially described as a tumor suppressor in sarcomas. Expression of CAV-1 was observed in mesenchymal stem cells and benign tumors. In sarcomas such as leiomyosarcomas, fibrosarcomas, and synovial sarcomas CAV-1 showed reduced or even absent expression, suggesting that CAV-1 could act as a tumor-suppressor gene in sarcomas.94,95

However, in ES several groups have determined that ES cells overexpress CAV-1. In fact, CAV-1 was described as a direct target of the EWSR1-FLI1 fusion.32,96 Several manuscripts showed that overexpression of CAV-1 contributes to malignancy, chemotherapy resistance, capacity to metastasize, and even tumor progression by autostimulation of cell proliferation.32–34,94

Recently, our group showed evidence that the inhibition of IGF1R internalization by CAV-1 either with CAV-1-siRNA or drug treatment with methyl-B-cyclodextrin, alone and especially in combination with TK inhibitors, results in reduced cell proliferation and increased apoptosis.31

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Targeting CD99 in Ewing Sarcoma

CD99 is a cell membrane receptor overexpressed in almost all of ES cells. Several investigators have demonstrated that, together with the ES fusion gene, CD99 represents a hallmark on ES biology, despite the fact that CD99 functionality is yet to be described. Accordingly, CD99 is responsible for maintaining the oncogenic phenotype and differentiation of ES.97

Together with the presence of the ES fusion gene, overexpression of CD99 in >90% of ES cells is the most characteristic marker, although very unspecific because of its common presence in other neoplasias.97 CD99 has been considered a key cause of ES malignancy and therefore a potential therapeutic target. Nevertheless, the function of CD99 in ES remains unknown despite the extensive research described below.

Recent studies have shown that there is a connection between the chimeric protein EWSR1-FLI1 and CD99, mainly because of the presence of miRNA, which suggests that CD99 is regulated by the EWSR1-FLI1.98 Targeting CD99 represents an attractive treatment strategy as it would target almost all of the tumor cells. Evidence from Scotlandi and colleagues shows that the inhibition of CD99 leads to cell death through apoptosis.35,99 In fact, the combination of Doxorubicin and the monoclonal antibody anti-CD99 0662 not only increased apoptosis but, most importantly, reduced the formation of tumor in mice xenografts.100 A new anti-CD99 MAb was recently described, scFvC7, which shows benefits regarding the previous one based on its particular specificity to ES cells.101

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Other Promising Drugs in Ewing Sarcoma
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PARP Inhibitors

An enzyme of the family of the poly(ADP-ribose) polymerases (PARP), named PARP1, is a very promising target for cancer therapy. This gene appears upregulated in some human cancers and its overexpression is related to chemoresistance. Studies carried out in animal models of cancer, such as skin cancer, showed an active role of PARP1 in terms of tumor formation.102 In addition, the role of PARP1 in tumors with ETS-fusions, including ES, has been recently shown.36,37 PARP family of proteins includes several members, namely, PARP1, PARP2, PARP3, PARP4, PARP5a, and PARP5b. Among them, PARP1 and PARP2 physically interact with each other and show redundant functions. They are the only known members of the PARP family that are activated by union to DNA.102,103 PARP proteins catalyze a type of posttransductional modification that entails the addition of ADP-ribose groups to the acceptor proteins using NAD+ as a donor substrate of ADP-ribose groups and formation of poly-ADP-ribose biopolymers (PAR), the poly-ADP-ribosylation. In response to stress situations, entailing DNA damage, PARP proteins are upregulated and the generated biopolymer PAR recruits other members of DNA damage repair (DDR) such as the XRCC1 factor and the DNA-ligase III to the damaged site. In turn, these are poly-ADP-ribosylated and consequently activated favoring the signaling and activation of DDR.102 Therefore, PARP family proteins play a relevant role in the mechanisms of DNA repair. In addition, it has been demonstrated that they also contribute to: (i) the maintenance of the genomic stability; (ii) chromatin remodeling; (iii) transcription; and (iv) cell cycle/cell death regulation. Hence, they are excellent candidates for cancer therapy.

The first concept to introduce regarding PARP and PARPinh is that of synthetic lethality. Synthetic lethality arises when a combination of mutations in 2 or more genes leads to cell death or apoptosis, whereas a mutation in only 1 of these genes does not, and by itself is said to be viable. In a synthetic lethal genetic screen, it is necessary to begin with a mutation that, although unable to kill the cell, confers a particular phenotype (such as slow growth) and then to systematically test other mutations at additional loci to determine which confer lethality. For instance, it happens with BRCA genes, which usually regulate the homologous recombination (HR) mechanisms, and PARP1, which plays a main role in base excision repair. There are several known PARPinh, such as olaparib and veliparib. Most of them compete with NAD+ to bind to the active site of the enzyme, a highly conserved region among the different members of the PARP family.102–104 The use of PARPinh in monotherapy for breast cancer in BRCA1-defective and/or BRCA2-defective tumors is probably the best example of synthetic lethality in the clinic.102 The loss of heterozygosis in BRCA1 or BRCA2 tumor-suppressor genes provokes a strong deficiency in the HR machinery, inducing genomic instability, which leads to the development of tumors. Given that this HR loss of function is not observed in normal cells, PARPinh can be used as a selective cytotoxic molecule in BRCA1-defective and/or BRCA2-defective tumor cells through the following chain of events. Administration of PARPinh leads to increasingly higher number of irreparable single-stranded breaks (SSBs), as PARP is not active. Consequently, the replication fork collapses leading to double-stranded breaks (DSBs). In turn, these DSBs, which should be repaired by HR effectors BRCA1/2, can only be repaired by the nonhomologous end joining system, which also induces genomic instability and finally cell death.102 Therefore, the possibility of using PARPinh in tumors with serious defects in HR components is of obvious great interest.102,105 However, more recently, the mechanism of PARP/BRCA synthetic lethality described above has been challenged, stressing the role of PARP1 in replication fork stability.106 Furthermore, it has been published that deficiencies in PTEN sensitize tumor cells to PARPinh.103 Apart from monotherapy, PARPinh can also play the role of an adjuvant, sensitizing tumor cells to chemotherapeutic agents inducing SSBs and DSBs, particularly in tumors harboring mutant genes in the DNA repair machinery (DDR). In ES, chemotherapeutic agents such as Doxorubicin have been largely used as potent alkylator agents in combination therapies for first-line treatments. Combination of PARPinh with alkylator agents, namely Trabectedin or its related compounds could be an interesting approach in ES treatment. In fact, Trabectedin and PARPinh show synergism in ES cell lines (Ordoñez JL and Amaral AT, 2013; unpublised results). Furthermore, it is also interesting to highlight that PARP1 seems to play a relevant role in angiogenesis. Preclinical studies suggest a promising role of PARPinh in cancer therapy by reducing the vascularity of the tumor. In particular, these agents seem to reduce the induction of the hypoxia-inducible factor 1α, which regulates some key genes in angiogenesis, metabolism, cellular proliferation, and apoptosis.102

In ES, recent studies have shown that inhibition of PARP may also be a promising therapeutic strategy.36,37 In fact, in vitro and in vivo studies demonstrated that ES cells are sensitive to treatment with olaparib especially when combined with the alkylator agent temozolomide.36,37 This drug combination practically eliminated tumor growth in a xenograft model of ES.36 These works suggested that ES cells are sensitive to olaparib, by the interaction of PARP and the ES transcript.36,37 Given the importance of these preclinical results, a phase II CT was initiated in metastatic/recurrent ES patients as a second-line treatment (NCT01583543). This study was stopped recently as preliminary data suggest that olaparib appears to be insufficient in monotherapy, hence combined therapy is highly suggested. Furthermore, patients included in this study were not selected on the basis of tumor-specific mutations. To this regard, recently, synthetic lethal interactions in vitro between PARP and cohesins have been shown.106,107 Therefore, PARPinh could be effective in those particular ES cases in which cohesins are already mutated.

In conclusion, the use of PARPinh in ES treatment is now on vogue, as, despite its apparent failure in monotherapy, in vivo and in vitro studies suggest that the combination with alkylator agents could be a powerful tool in the clinic.

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Zoledronic Acid

Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, is a third-generation bisphosphonate that inhibits the farnesyl diphosphate synthase, which causes a loss of osteoclast activity and apoptosis, consequently inhibiting bone resorption. In fact, bisphosphonates are frequently used to prevent side effects of cancer treatments on bone health. As a result, this drug has been used in the treatment of cancer-induced bone disease or osteoporosis to prevent skeletal fractures and to ease bone pain.108 Interestingly, ZOL also acts as an antitumoral agent preventing cancer bone metastasis of solid tumors and inhibiting the growth of tumor cells such as chondrosarcoma, osteosarcoma, or fibrosarcoma, showing in some cases synergism with other drugs.109

The action mechanism of ZOL involves the inhibition of the farnesyl diphosphate synthase, which in turn alters the prenylation of small GTPases and other proteins.109 Cenp-F/mitosin, a kinetochore-associated protein taking part in the segregation of chromosomes during mitosis, can be posttranslationally prenylated. The inhibition of Cenp-F farnesylation by ZOL affects the adequate kinetochore assembly, which in turn alters cell cycle and provokes inhibition of tumor cell proliferation.110 Some resistance to the ZOL therapy has been found in osteosarcoma cells related to the expression of HSP70.111 However, promising data have been found in ES preclinical studies.38,39 An intraosseous model of ES was developed in mice by injecting TC71 cells intratibially and then treating them with ZOL and/or Paclitaxel. Animals treated with ZOL, developed 40% and 50% less tumors than the Paclitaxel and control groups, respectively, after 5 weeks of treatment. When both drugs were combined, incidence of tumors was reduced by 75% with respect to the control group.39

In a recent preclinical study, ZOL induced cell cycle arrest in the S phase and inhibited the growth of ES tumors cells growing in the medullar cavity of the tibia of mice (intraosseous model), but it did not affect the growth of ES when cells were implanted in the tibia muscle (soft tissue model) of immunosuppressed mice. Interestingly, when ZOL was combined with ifosfamide, synergistic effects were observed in the soft tissue xenograft model, which shows promise in patients who could benefit from a reduction in chemotherapy.38 In contrast, Mueller et al112 published a study in which ZOL impaired the antitumoral activity in vitro of natural killer cells in ES patients.

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NAE Inhibitor: MLN4924

NEDD8-activating enzyme (NAE) activates the complex E3 ligases, cullin-RING ligases (CRLs). CRLs are protein complexes involved in the UPS. In higher eukaryotes, the UPS comprises 2 discrete steps: the covalent attachment of multiple ubiquitin molecules to the protein substrate and degradation of the polyubiquitylated protein by the proteasome 26S complex. This system allows the regulation of many processes such as cell cycle, homeostasis, cell division, and responses to the environment.

The NAE enzyme participates in the process of neddylation. Neddylation is similar to a route that allows ubiquitination activation of some of the components of the complex CRL, the cullins.113 The cullins are scaffold proteins that enable physical alignment between the substrate (to degrade protein) and E3 ubiquitin ligases . To be active, cullins should be attached to the protein NEDD8, through the neddylation system. Joining NEDD8 allows assembly of the other components of the complex CRL. It also prevents the binding of other proteins, such as CAND1, which block the cullin.113,114 The NAE enzyme is responsible for activating NEDD8 by expenditure of ATP for attaching to cullin.

There is an investigational novel antitumoral agent that inhibits this activation—MLN4924 (Millennium Pharmaceuticals Inc., Takeda Oncology Company, Cambridge, MA).115 This drug is covalently attached to the nucleotide-binding site of NAE. In this way the CRL assembled is interrupted and the degradation pathway of the target protein is inhibited. Consequently MLN4924 is more specific and less toxic than other full-scale proteasome inhibitors such as Bortezomib.116

MLN4924 has undergone preclinical studies in different tumor cells. Despite the high specificity in inhibiting its target, the molecular mechanism triggered by the drug that also triggers the phenotype resulting in different tumor cells is heterogenous.115 It has been reported that in HTC116 (colorectal cancer cell), MLN4924 inhibits CUL1 and causes the accumulation of target Cdt1; this causes rereplication, DNA damage, and apoptosis.117 However, subsequent study of liver cancer cells revealed that the drug caused autophagy and apoptosis.118 In preclinical models of activated B cell–like diffuse large B-cell lymphoma, MLN4924 induces a rapid accumulation of plκBα, a decrease in nuclear p65 content, a reduction of nuclear factor-κB (NF-κB) transcriptional activity, and G1 arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-κB pathway inhibition.117 Something similar occurs in myeloid leukemia.119 The ability to induce senescence through p21 is also shown.120 Another peculiarity is the radiosensitization of human pancreatic cancer cells by MLN4924.121 CTs with favorable results for the treatment of melanoma, large B-cell lymphoma, and other nonhematologic malignancies are currently being conducted.

In ES, a total of 14 ES cell lines were treated with MLN4924 to estimate its ability to impair the in vitro growth of ES cell cultures. The median IC50 (the concentration that reduces cell population by 50%) obtained (81 nM) was noticeably lower than that previously obtained for 10 cell lines belonging to 7 different tumor entities (200 nM) and similar to that of diffuse large B-cell lymphoma cell lines, which are the most sensitive to MLN4924 treatment as reported to date (76 nM).115,117 Interestingly, a recently published study has surveyed the in vitro sensitivity of a panel of pediatric cancer cell lines to MLN4924, finding ES cells to be the most sensitive ones to this compound, among those tested.122

Surprisingly, the molecular mechanism of MLN4924 in ES is not characterized by the accumulation of Cdt1 or NF-κB pathway impairment unlike that described in previous preclinical studies. In ES, Wee1, which participates in the regulation of Cdc2 by means of Y15 phosphorylation, causes G2/M arrest and is accumulated by inhibiting activation of CUL1. MLN4924 has a dual role: (A) low doses cause G2/M arrest and can be reversed by inhibiting Wee1 by using specific drugs (PD0166285) or shRNA against the target; (B) at higher doses, MLN4924 causes delay in phase S progression, DNA damage, and apoptosis. In ES, the accumulation of cyclin E by MLN4924 alters cyclin A-Cdk2 union in late phase S, which in turn affects the phosphorylation of CDK2-cyclin A targets. This is the case of Cdc6, which is accumulated in the nucleus of the cell during the late phase S, which in turn alters the normal progression of the replication process.40 The promising results of the preclinical study of MLN4924 support future clinical studies toward ES.

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EPIGENETICS IN EWING SARCOMA

Epigenetics is a major topic in cancer research, especially given the relevance of recent data from the ENCODE project (http://genome.ucsc.edu/ENCODE/). In particular, the pathogenesis of ES can be explained in part as caused by epigenetics mechanisms such as methylation or histone modification. Several polycomb proteins that are highly expressed in ES seem to regulate differentiation processes by regulation of DNA methylation and histone modification of genes.123–125 Furthermore, EWSR1-FLI1 fusion protein upregulates some genes such as NKX2.2, which seems to play a key role in repressing transcription through histone modification by recruiting TLE corepressor proteins.44,126

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Demethylating Agents

It is well known that ES cell lines are sensitive to demethylating agents such as azacitidine (5-AZA) and histone deacetylase (HDAC) inhibitors.127,128 In a recently published work, Patel and colleagues studied the DNA methylation profile of 52 ES primary tumors using a bead array platform to identify the methylation status of 503 genes using DNA obtained from formalin-fixed paraffin-embedded primary ES. Herein, they found that 129 genes were in fact hypermethylated, and, more importantly, among these, 36 genes were found to be highly hypermethylated. The expression of 19 of these genes was restored after treatment with 5-AZA. Moreover, 6 of those hypermethylated genes [SERPINE1, Versican (VCAN), AXL, COL1A1, CYP1B1, and LYN] were also found to be downregulated in an independent cohort of tumors.41 In another recently published paper, a histone demethylase named lysine-specific demethylase 1 (LSD1/KDM1A/AOF2/BHC110) was pointed out as a new target in ES, chondrosarcoma, and osteosarcoma. It is localized in cell nuclei and acts as an epigenetic coregulator of transcription. In fact, Bennani-Baiti et al42 used a specific inhibitor of LSD1, approved by the Food and Drug Administration, and observed in vitro ES cell growth inhibition.

The role of epigenetics may also be critical in the early initiating events in ES tumorigenesis. In normal stem cells, developmental genes are regulated by polycomb proteins, which act as transcriptional repressors of developmental genes by means of histone modification and chromatin remodeling.129 As recently shown, transduction of EWSR1-FLI1 in the neural crest stem cell, which is one of the cell types proposed as the cell of origin of ES, induced the expression of polycomb proteins BMI-1 and EZH2 (a well-known direct transcriptional target of EWSR1-FLI1). In addition, upregulation of BMI-1 was associated with reversible epigenetic silencing of p16 and cellular senescence escape.2 In contrast, KIT expression and methylation were studied in 2 PNET cell lines and 1 PNET tumor sample. Both cell lines displayed partial methylation of the promoter region of c-KIT. Despite this, methylation does not seem like a plausible epigenetic regulatory mechanism regarding KIT inhibition in these tumors. However, more studies are needed to confirm or reject any implication of KIT methylation in these tumors.130

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Histone Deacetylase Inhibitors

Acetylation/deacetylation of histones (H2A, H2B, H3, and H4) is an epigenetic mechanism of regulation of transcription. HDACs repress transcription by the removal of the acetyl groups. By contrast, transcriptional activation is achieved when H3 and H4 are acetylated by histone acetyl-transferases (HATs). Moreover, HDACs and HATs not only modify histones but also transcription factors. Therefore alteration of the acetylation/deacetylation process favors tumorigenesis. Interestingly, in ES the EWSR1-FLI1 fusion protein targets the transcriptional cofactor CBP/p300 repressing its HAT activity.128,131 Furthermore, EWSR1-FLI1 and normal FLI1 bind to the p21 promoter, which induces the inhibition of p300 HAT activity, eventually downregulating p21.128 Interestingly, EWSR1-FLI1 and ERG, which is a member of the ETS family, share the same p21 target sequences. In prostate cancer ERG interferes with acetylation of p53 in 2 distinct ways: (i) by binding and inhibiting CBP/p300 histone acetyltransferase activity; or (ii) by inducing the expression of HDACs. As Fortson et al demonstrated,43 the use of HDAC inhibitors on ERG-positive prostate cancer cells recovers HAT activity, which could be of interest ES as well.

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THE MICROENVIRONMENT IN EWING SARCOMA

Tumor formation accounts for several changes on the surrounding tissue, from activation of the immune system to neoangiogenesis. In fact, interaction between tumor cells and the surrounding microenvironment has been largely studied in terms of targeted therapy.

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Developments in Immunotherapy in Ewing Sarcoma

Interaction between tumor cells and the immune system has always been an attractive field in the design of new therapies in cancer. Moreover, in ES, this field has recently experienced substantial advances. Multimodal therapeutic approaches fail to significantly improve outcome in ES patients with metastatic, refractory disease or in relapse. Immunologic elimination of cancer cells in solid tumors requires a complex response, which culminates with the detection of tumor cells by the innate immunologic system, followed by their elimination. Nevertheless, it is well established that cancer cells can “disguise” to escape and proliferate undetected by the immune system.132,133 Altogether, these facts stress the immediate need to invest in more effective treatments, including immunologically based therapies. The innate immunosystem consists of natural killer cells (NK), macrophages, T lymphocytes, and dendritic cells, among others. Cytotoxic effector cells such as NK are able to recognize abnormal cells and eliminate them. The human major histocompatibility complex encoding HLA class I and II molecules is responsible for the recognition step. HLA class I molecules (HLA-A/B/C and β2-microglobulin) are classically present in nucleated cells, whereas HLA class II molecules (HLA-DR/DP/DQ) are constitutively expressed by antigen-presenting cells.134 In tumors, evidence demonstrates the expression of aberrant or impaired HLA class I molecules.135,136

The elimination process may occur either through the death receptor pathway or the calcium-dependent granule exocytosis pathway. Shortly, NK cells recognize tumor cells and cross-link members of the necrosis factor receptor family such as FasL with target cells. A complex system is generated, and the recruitment of procaspase-8 and subsequent activation of the cascade of caspases culminates with apoptosis. In contrast, NK cells also secrete cytotoxic granules (perforin and granzyme B) when binding the target cell, which penetrate the tumor cell and initiate a complex process that results in apoptosis. Hereby, the main step is the recognition and binding of tumor cells by the effector cells.132 NK cell–based therapies represent some advantages compared with T lymphocytes and dendritic cells. NK cells do not cause graft versus host disease, and also these cells do not require identification of tumor antigens. Receptors such as NKG2D have been described as one of the most important receptors in terms of NK-cytotoxic response. NKG2D is an activating receptor expressed by NK cells, upregulated upon cytokine stimulation. NKG2D ligands include MICA, MICB, ULBP-1/2/3/4, which are upregulated in stress situations and very often in cancer cells. Other NK receptors associated with tumor cytotoxicity are DNAM-1 and TRAIL.

ES cells are sensitive to NK cell cytotoxicity, mainly by receptors NKG2D and DNAM-1. In fact, Cho and colleagues reported a method to expand NK cells extracted from peripheral blood from a donor, for 2 to 3 weeks, using an IL-15-expressing cell line and low dose of IL-2. The ES patient, after local irradiation, will increase the expression of NKG2D ligands, which will be easily recognized by the posteriorly expanded NK cells, increasing tumor cell death in a semitargeted manner.137,138 This work shows a clinical application of irradiation, sensitizing ES cells to NK cells, combined with NK cell transfusion, and also proves that traditional chemotherapy fails to increase NKG2D ligands in ES cells, and therefore combination of CT with NK transfusion would not represent a benefit in treatment. Moreover, regarding NK cells, a pilot study performed in children bearing metastatic/refractory solid tumors, including ES (n=2), reported that haplo-stem cell transplantation might represent a promising resource. Overexpression of NKG2D ligands was observed in most tumors after transplantation; however, in ES there was a low intensity of MICA and no expression of ULBP2. Outcome from this study resulted in clinical response from all of the enrolled patients.139

A set of manuscripts published by Berghuis et al140–142 also elucidated the role of immunotherapy in ES. In vivo studies carried out in chemotherapy-resistant ES cells, expressing NK ligands, determined that these were practically unsusceptible to resting NK cells. However, after activation of NK cells with IL-15, this resistance was shut down. In fact, HDAC inhibitors induced NKG2D ligand expression and increased sensitivity of CT-resistant ES cells to NK cells. Altogether, these data suggest that in cases of resistance to treatment, immunotherapy appears as a beneficial option in ES treatment.142 Consistently, a recent in vitro study demonstrated that in a set of 14 ES cell lines, at least 1 NKG2D ligand (among MICA, MICB, and ULBP-1/2/3) was expressed by each cell line. Here, NKG2D ligands were exploited as therapeutic targets to obtain ES cell death by activated T cells and CD4-positive and CD8-positive cells. Results obtained from this study confirmed that NKG2D ligands are plausible targets in immunotherapy, via CAR-mediated T cells, in ES cell lines, including those with low expression of NKG2D.143 Another recent work, this time in an in vitro model of ES, described how ZOL acts in the immune system inhibition. Herein, expanded NK cells treated with ZOL not only showed impaired growth but also a reduction in terms of cytolytic activity toward ES cells.112

Interestingly, regarding Trabectedin, a very recent work observed that tumor-associated macrophages are selectively targeted by Trabectedin. Actually, a particular fraction of tumor-associated macrophages, the Ly6-high, known to be the most inflammatory, is selectively affected by the drug treatment, decreasing the recruitment of more macrophages to the tumor site and the immune response. This study, carried out in mice models from fibrosarcoma and ovarian cancer, also showed that Trabectedin reduces angiogenesis and therefore the routes of action of the immune system toward the cancer cells. This study, however, did not include ES mice models.3

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Antiangiogenesis in Ewing Sarcoma

Solid tumors require a constant mode of metabolite release and a constant supply of oxygen and nutrients. In ES, these mechanisms are directly related to angiogenesis and tumor cell vascular mimicry.144,145 Briefly, the production of the soluble protein vascular endothelial growth factor (VEGF) stimulates neoangiogenesis—the formation of new vascular endothelium within the tumor mass and also its surroundings. In vitro evidence suggests that VEGF is crucial for ES proliferation, as its lack of expression leads to formation of smaller tumors.146–149 In particular, one isoform termed as VEGF165 seems to play an important role in stimulating vasculogenesis, a process characterized by the neoformation of endothelial cells and pericytes within the tumor and its surroundings, after recruitment and posterior differentiation of bone marrow–derived cells. Therefore, to inhibit angiogenesis, VEGF165 could be a good therapeutic target.147,149,150

Among the preclinical strategies studied so far, we would like to focus on bevacizumab, an MAb targeting all VEGF isoforms. In fact, in vitro and in vivo experiments performed on ES determined that the inhibition of VEGF either with bevacizumab or VEGF-trap resulted in delayed tumor growth.151 Other agents such as TK inhibitor sunitinib are also known to inhibit VEGF in ES in vivo models, namely by the PPTP.45

Anti-VEGF therapies have already been tested in the clinic and are currently undergoing CT. MAb bevacizumab has already been engaged in phase II CT (NCT00516295) in young patients with refractory or first recurrent extracranial ES in comparison with traditional chemotherapy, including cyclophosphamide, topotecan, and vincristine. A new phase II study NCT01492673 combining cyclophosphamide, topotecan, and bevacizumab exclusively in patients with relapsed/refractory ES and neuroblastoma is also currently ongoing. Sunitinib has also been engaged in CT NCT00474994. A phase II study was held in patients with metastatic locally advanced or recurrent sarcomas.46 This study included ES patients undergoing standard neoadjuvant therapy. In addition, regarding Trabectedin, very recent evidence associated Trabectedin treatment with VEGF expression inhibition correlated with reduction of vessels in vivo (NCT00474994).3,4 Regarding the process known as vascular mimicry, present in several solid tumors, it acquired a particular relevance in ES because of its role in supporting tumor growth in an independent way from VEGF. Briefly, ES cells form a mass with small spaces between them, these spaces simulate vascular spaces. These spaces can be filled with blood and are formed by ES cells and not by differentiated endothelial cells, creating what Van der Shaft et al145 called an “alternative circulatory system.” This in vitro and in vivo evidence support the need to explore the angiogenesis pathway, dependent and independent of VEGF, as a therapeutic target in ES. In fact, some studies have been carried out to this end and were brilliantly summarized in a recent review by DuBois et al.152

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RESEARCH MODELS

Animal Models in Ewing Sarcoma

Some researchers have tried to develop genetically engineered mouse models of ES,153,154 but none has completely succeeded so far (reviewed in Ordonez et al70,155). Thus, in vivo ES drug tests have been performed mostly in xenograft models generated by subcutaneous injection of tumor cells into immunodepressed mice.22,69 Following this approach, the PPTP, supported by the National Cancer Institute, systematically used xenograft models generated by implantation of cancer cells into the severe combined immunodeficiency (SCID) mice to test the effects of new drugs against ES and other childhood cancers.122,155 Our group studied the role of HSP90 as a response mechanism to IGF1R inhibition by using in vitro studies and a xenograft model of ES generated by subcutaneous injection of cells from the A673 cell line. In this model, 17-AAG plus AEW541 treatment reduced tumor growth, which confirmed the role of HSP90 in the resistance mechanism to anti-IFG1R therapy.22 In another study, inhibition of EWSR1-FLI1 fusion by stable RNA interference reduced the tumor growth in a model generated by implantation of TC71 into NOD/SCID mice.69 More recently, doses of 60 and 90 mg/kg of MLN4924 caused regression of tumor growth in a xenograft model generated after implantation of RDES and RM82 cells, respectively, into the SCID mice.40 In contrast, interestingly, individualized therapeutic protocols in xenograft models generated by subcutaneous implantation of tumor material from patients into immunodepressed mice has been proposed recently as an efficient way to test specific treatments in patients with relapse or nonresponding tumors.156,157 Furthermore, as tumor biopsy from patients and early-passage tumor xenograft generated in mice from the tumor biopsy show great genomic similarity, the serial transfer of tumors in mice from a patient biopsy is an excellent way of increasing the amount of tumor material to help carry out new molecular studies.155–157 In contrast, in the study of ES, some other research groups have used orthotopic models generated by implantation of tumor material into places (bones or muscles) usually affected by ES in humans patients.158 Following this approach, Zhou et al39 demonstrated the efficacy of combining ZOL with Paclitaxel in a model generated after injection of TC71 cells into the tibia of athymic nude mice. Odri et al38 more recently found synergistic effects when ZOL and ifosfamide were administered in athymic nude mice injected with A673 or TC71 cells into the tibial muscle.

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Preclinical Massive Drug Research

In a recent work, Garnett et al37 give details of a study aimed at identifying new biomarkers of response to a broad panel of cancer drugs. To achieve this, they thoroughly studied the genomic and gene expression data of 639 human tumor cell lines representing a broad spectrum of tumors. More than 48,000 drug-cell line combinations were tested. Contrary to what was expected, in most cases the sensitivity of cell lines to drugs was not dependent on the cancer tissue type. A high number of associations between individual mutated cancer genes mostly oncogenes and drug sensitivity were found. Furthermore, associations between inactivating mutations in tumor-suppressor genes and drugs were found. For instance, mutation of p53, which usually occurs in 13.3% of ES patients,56 provides resistance to nutlin-3a, an inhibitor of the MDM2 E3 ligase. Interestingly, some novel gene-drug associations were also found. For sensitivity to 17-AAG, a known inhibitor of HSP90 involved in resistance to IGFIR treatment in ES22 was associated with inactivation of STK11, which could mitigate the repression of mTOR. Interestingly, in most cases, the sensitivity of cancer cell lines to specific drugs does not depend on only one unique variable, but is explained by many genomic and epigenetic factors. Thus, some drugs did not correlate properly with a mutational event but with transcriptional features. This is the case of the correlation between sensitivity to 17-AAG and expression of NQO1, a member of the NADPH dehydrogenase family.159

Garnett and colleagues also identified a highly significant association between EWSR1-FLI1 and sensitivity to PARPinh [olaparib (AZD2281) and AG-014699)]. ES cells were more sensitive to olaparib compared with other tumor cells. Sensitivity to PARPinh in ES seems to be related to the EWSR1-FLI1 transcriptional profile, whereas no association with BRCA1 or BRCA2 mutations or DNA damage was found.37

Another completely different approach is used by the PPTP supported by the National Cancer Institute from United States (http://pptp.nchresearch.org/index.html). This program is designed to evaluate new drugs active against childhood cancers. The program tests nearly 12 new drugs per year using both in vitro and in vivo models. In vivo models include a panel of 61 childhood tumor xenografts. Four of them are ES xenografts.122,155

In contrast, as many of the pathways altered in cancer are conserved between different species, large-scale genetic interaction screens in yeast can be useful to identify not only genes but also processes that can be susceptible to synthetic lethality in humans. By using this technology, synthetic lethal interactions were found between replication fork mediators and cohesins.106

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Deep Sequencing

Nowadays, only few profiling studies have been carried out on ES samples. In a recent work, the Ladanyi group used a Sequenom MassARRAY platform to identify novel mutations in 4 pediatric tumors including 75 tumor samples from ES. This platform allows the use of formalin-fixed paraffin-embedded material being more sensitive than Sanger sequencing.160 By using this methodology after studying the status of 29 oncogenes interrogating 275 point mutations, they identified gene mutations in only 3 of 75 (4%) of the ES samples studied. Specifically, a RAS family mutation, an activating BRAFV600E mutation, and a CTNNB1 mutation were found. The frequency of mutations found in ES in this study was lower than that for adult carcinomas in other pediatric tumors such as neuroblastoma (13%) and rhabdomyosarcoma (21%) but higher than that for desmoplastic small round cell tumor (0%). This low frequency of mutations found in ES in this study should be confirmed by whole-exome sequencing and whole-genome sequencing in the near future.161

Although only few mutations have been found in ES, some of them could be of interest to be targeted by drugs. To this regard, the use of MEK inhibitors has been proved of great therapeutic value in BRAF-mutated melanoma patients.162,163

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FUTURE PERSPECTIVES

ES is a relatively uncommon disease (in Europe about 3 new cases/million a year). This brings several problems in terms of research on new therapies.

FIGURE 2
FIGURE 2
Image Tools
  • First, the pharmaceutical industry is not particularly interested in research on new therapies in pediatrics, especially those with a low incidence such as ES.
  • From the biological point of view, ES lacks preneoplastic lesions. Moreover, there is no normal tissue counterpart. This is seen in most sarcomas, hindering not only prevention but also research on sarcomagenesis and tumor progression.
  • Translocations and gene fusions of ES seem at least in theory to be the ideal drug target. Nevertheless, subcellular location of these molecules is the nucleus, which might not be easily reachable for many drugs. In addition, translocation-derived gene fusions in most sarcomas correspond to chimeric transcription factors. They are actually involved in a complex mesh of protein-protein, protein-DNA, and epigenetic interactions, which represent the actual target. Therefore, the activity of most chimeric proteins cannot be regulated allosterically.
  • As previously expressed in this review, we lack genetically modified models of ES, in sharp contrast to other TRSs such as synovial sarcoma or myxoid liposarcoma. Needless to say that such a model would be extremely valuable to find pharmacodynamic markers of response to new therapies.
  • Clinical samples of ES are very scarce. Samples required include not only primary tumor but also relapsed/metastatic tumor samples, as well as body fluids (ie, plasma for proteomics and circulating biomarkers). A strong need for the development of biobanks with well-annotated samples exists that allows for adequate clinical validation of preclinical studies.
  • In fact, another major problem for biomarker validation in ES is that most studies assessing the prognostic value of biomarkers have been carried out using small retrospective series of patients. A number of promising biomarkers that have shown prognostic impact in retrospective studies but require careful validation in prospective joint investigation have recently been described by the ES scientific community. A higher degree of clinical evidence would be attainable through the use of prospective studies. Prospectively validated prognostic and/or predictive biomarkers are thus needed to better stratify patients and to provide personalized risk-adapted therapeutic approaches. PROVABES, a joint European proposal that aims to cover this need (http://provabes.uni-muenster.de/), has been recently launched. This consortium will focus on the prospective clinical validation of relevant ES biomarkers using samples and data from the EuroEwing trials.
  • Many coordinated resources are therefore needed to overcome the gap between basic research and clinical validation in ES. Figure 2 aims to represent them graphically.

The role of pathologists in ES research cannot be underemphasized. Their involvement in ES research is important at least for the following reasons:

  • Correct diagnosis/molecular characterization is a mandatory prerequisite to provide tumor samples with a precise histopathologic and molecular diagnostic characterization. This is required for further comprehensive characterization of the patient-derived biomaterials. Reference pathology panels should review cases to confirm the diagnosis and molecular characteristics of patient samples. The histopathologic review provides an accurate diagnosis based on the 2013 World Health Organization classification of ES and, together with strict adherence to SOPs, ensures the success of the in-depth molecular characterization.
  • Pathologists are also required for a careful and comprehensive collection of biomaterials. This is a mandatory backbone for any translational research approach. New sequencing techniques allow a genome-wide assessment of cancer-related genes and pathways. In addition to genetic parameters that may be associated with age, localization, and tumor phenotype, a particular interest focuses on parameters that may be associated with the aggressiveness of the disease. An in-depth characterization of tumor material obtained from clinically well-annotated patients will allow the identification and validation of novel prognostic or predictive biomarkers as well as druggable pathways. The comprehensive characterization of tumor material is necessary to understand the spectrum of distinct alterations in ES to identify patients who are likely to respond to particular therapies, and to facilitate the selection of treatment modalities. It should include identification and characterization of small noncoding RNAs present in tumor tissue by high-throughput sequencing, characterization of transcriptome signatures, and genomic and epigenomic variations in ES cells compared with the germline counterpart. SOPs for ES sample collection and banking should therefore be available in the pathology departments of all major sarcoma reference centers. Prognostic biomarker studies traditionally focus on investigations on primary tumors, although disseminated disease and genetic variation in drug metabolism and transport will contribute to a patients’ response to treatment and outcome. Furthermore, for real-time monitoring of patient response and disease course, clinically informative biomarkers that can be detected in blood are particularly attractive, as blood is a minimally invasive compartment for sampling. In cases in which such changes predict event-free survival or overall survival, circulating biomarkers may be useful to inform the selection of optimal therapy for individual patients and are especially attractive in cancer types such as ES in which viable tumor at diagnosis can be limited.
  • Prospective tissue microarray (TMA) sets should be performed by the reference pathology centers. The use of TMAs allows the processing of hundreds of tumor samples with a variety of techniques, that is, immunohistochemistry and fluorescent in situ hybridization, at one time. The TMA collections established within the biobank are one of the most crucial resources for discovering and validating potential biomarkers. Because representativeness of the tissue cores may be a disadvantage compared with full sections, a careful production of TMA by experienced reference pathologists is mandatory to achieve representative results. The TMA panels are used to prospectively validate markers that have been previously described and for future studies on markers that may be identified within these tasks.
  • Biomarkers include pharmacodynamic markers, which allow the assessment of the actual response to drugs by in situ evaluation of the response of its targets. Some of them are actually detected by immunohistochemistry, a robust approach familiar to almost all pathologists. Image analysis can be applied at pathology departments to provide an objective measure and quantification of response to drugs.
  • Pathologists can also play an important role in animal model characterization. This includes processing and characterization of tumor xenografts, including TMA construction and pharmacodynamics, as described above, and morphologic and molecular characterization of genetically modified animal models.
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Keywords:

Ewing sarcoma; therapy; clinical trials; animal model

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