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Drug resistance is one of the major factors associated with poor outcome of cancer patients. Treatment of Ewing sarcoma (EwS), an aggressive neoplasm mainly affecting children, adolescents and young adults, is associated with therapy failure and tumor relapse in 30-80% of the cases. Thus, it supports the need to explore the mechanisms modulating drug activity. Here, we describe a novel mechanism of drug sensitivity based on the role of EWS::FLI1 in R-loop metabolism. Our results demonstrate that EWS::FLI1 promotes R-loop formation favoring the interaction between DHX9 and elongating RNA polymerase II. In addition, we discovered that EWS::FLI1 kidnaps DHX9 preventing the resolution of TOP1 poisoning-associated R-loops. Our findings indicate that R-loops accumulation promotes replicative stress, genome instability and cell sensitivity to SN-38. Collectively, these results uncover a novel mechanism behind EwS sensitivity to genotoxic agents, with relevant implications for EwS treatment.

[Background] To assess the cost-effectiveness of using next-generation sequencing (NGS) compared to sequential single-testing (SST) for molecular diagnostic and treatment of patients with advanced non-small cell lung cancer (NSCLC) from a Spanish single-center perspective, the Hospital Universitario Virgen del Rocio (HUVR)., [Research design and methods] A decision-tree model was developed to assess the alterations detection alterations and diagnostic cost in patients with advanced NSCLC, comparing NGS versus SST. Model inputs such as testing, positivity rates, or treatment allocation were obtained from the literature and the clinical practice of HUVR experts through consultation. Several sensitivity analyses were performed to test the robustness of the model., [Results] Using NGS for molecular diagnosis of a 100-patients hypothetical cohort, 30 more alterations could be detected and 3 more patients could be enrolled in clinical-trials than using SST. On the other hand, diagnostic costs were increased up to €20,072 using NGS instead of SST. Using NGS time-to-results would be reduced from 16.7 to 9 days., [Conclusions] The implementation of NGS at HUVR for the diagnostic of patients with advanced NSCLC provides significant clinical benefits compared to SST in terms of alterations detected, treatment with targeted-therapies and clinical-trial enrollment, and could be considered a cost-effective strategy., This work was funded by Roche Farma S.A.

Supplementary Table 1. Primers sequences

Supplementary Figure S4. Trabectedin activity on neural differentiation in EWS.

Ewing Sarcoma (ES) shows several deregulated autocrine loops mediating cell survival and proliferation. Therefore, their blockade is a promising therapeutic approach. We previously reported the in vitro effect of insulin-like growth factor 1 receptor (IGF1R)/KIT pathway blockade on ES cell lines, and we now extend our observations to changes induced by this treatment in interacting proteins/networks. A proteomic analysis revealed that Heat Shock Protein (HSP)90 was differentially expressed between ES cell lines sensitive and resistant to specific IGF1R/KIT inhibitors. We therefore inhibited HSP90 with 17-allylamino-17-demethoxygeldanamycin (17-AAG) and siRNA, and observed that ES cell line growth and survival were reduced, especially in the resistant cell lines. Conversely, HSP90 induced–expression conferred resistance to anti-IGF1R/KIT treatment in the sensitive cell lines. 17-AAG treatment induced HSP90 client protein degradation, including AKT, KIT, or IGF1R, by inhibiting their physical interaction with HSP90. Xenograft models developed with A673 ES cell line confirmed that HSP90 inhibition, alone or combined with IGF1R inhibition, significantly reduced tumor growth and expression of client proteins. Remarkably, using two independent clinical sample sets, we have found that nearly half of IGF1R-positive tumors also show HSP90 overexpression. This delineates a subset of patients that could benefit from combination of anti-HSP90 agents when considering IGF1R-targeting therapies. Importantly, sensitivity to drugs such as ADW/IMA depends not only on the levels of expression and basal activation of IGF1R/KIT, but also, and for the first time reported in ES, on the development of the stress response mechanism. Accordingly, HSP90 expression could be a predictive factor of response to IGF1R-targeting therapies. [Cancer Res 2008;68(15):6260–70]

Supplementary Methods, Table 1, Figures 1-8 from A Pivotal Role for Heat Shock Protein 90 in Ewing Sarcoma Resistance to Anti-Insulin-like Growth Factor 1 Receptor Treatment: In vitro and In vivo Study

Background and objectives: Bone tumours are relatively rare and, as a consequence, treatment in a centre with expertise is required. Current treatment guidelines also recommend review by a specialised pathologist. Here we report on international consensus-based datasets for the pathology reporting of biopsy and resection specimens of bone sarcomas. The datasets were produced under the auspices of the International Collaboration on Cancer Reporting (ICCR), a global alliance of major (inter-)national pathology and cancer organisations. Methods and results: According to the ICCR's process for dataset development, an international expert panel consisting of pathologists, an oncologic orthopaedic surgeon, a medical oncologist, and a radiologist produced a set of core and noncore data items for biopsy and resection specimens based on a critical review and discussion of current evidence. All professionals involved were bone tumour experts affiliated with tertiary referral centres. Commentary was provided for each data item to explain the rationale for selecting it as a core or noncore element, its clinical relevance, and to highlight potential areas of disagreement or lack of evidence, in which case a consensus position was formulated. Following international public consultation, the documents were finalised and ratified, and the datasets, including a synoptic reporting guide, were published on the ICCR website. Conclusion:These first international datasets for bone sarcomas are intended to promote high-quality, standardised pathology reporting. Their widespread adoption will improve the consistency of reporting, facilitate multidisciplinary communication, and enhance comparability of data, all of which will help to improve management of bone sarcoma patients.

Ewing Sarcoma (EWS) is an aggressive bone and soft tissue tumor that mainly affects children, adolescents, and young adults. The standard therapy, including chemotherapy, surgery, and radiotherapy, has substantially improved the survival of EWS patients with localized disease. Unfortunately, this multimodal treatment remains elusive in clinics for those patients with recurrent or metastatic disease who have an unfavorable prognosis. Consistently, there is an urgent need to find new strategies for patients that fail to respond to standard therapies. In this regard, in the last decade, treatments targeting epigenetic dependencies in tumor cells and the immune system have emerged into the clinical scenario. Additionally, recent advances in nanomedicine provide novel delivery drug systems, which may address challenges such as side effects and toxicity. Therefore, therapeutic strategies stemming from epigenetics, immunology, and nanomedicine yield promising alternatives for treating these patients. In this review, we highlight the most relevant EWS preclinical and clinical studies in epigenetics, immunotherapy, and nanotherapy conducted in the last five years., This work was supported by FIS-FEDER PI2000003 to E.d.Á.C.; L.H.-P. is supported by the Consejería de Salud y Familias, Junta de Andalucía (RH-0047-2021).

The effectiveness of targeted therapies with tyrosine kinase inhibitors in non-small-cell lung cancer (NSCLC) depends on the accurate determination of the genomic status of the tumour. For this reason, molecular analyses to detect genetic rearrangements in some genes (ie, ALK, ROS1, RET and NTRK) have become standard in patients with advanced disease. Since immunohistochemistry is easier to implement and interpret, it is normally used as the screening procedure, while fluorescence in situ hybridisation (FISH) is used to confirm the rearrangement and decide on ambiguous immunostainings. Although FISH is considered the most sensitive method for the detection of ALK and ROS1 rearrangements, the interpretation of results requires detailed guidelines. In this review, we discuss the various technologies available to evaluate ALK and ROS1 genomic rearrangements using these techniques. Other techniques such as real-time PCR and next-generation sequencing have been developed recently to evaluate ALK and ROS1 gene rearrangements, but some limitations prevent their full implementation in the clinical setting. Similarly, liquid biopsies have the potential to change the treatment of patients with advanced lung cancer, but further research is required before this technology can be applied in routine clinical practice. We discuss the technical requirements of laboratories in the light of quality assurance programmes. Finally, we review the recent updates made to the guidelines for the determination of molecular biomarkers in patients with NSCLC.

Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) shows potent preclinical anticancer activity in pediatric solid tumors such as Ewing sarcoma, rhabdomyosarcoma and neuroblastoma, but responses in clinical trials have been modest. In this work, we aimed to discover a rational biomarker-based approach to select the right candidate patients for this treatment. We assessed the efficacy of nab-paclitaxel in 27 patient-derived xenografts (PDX), including 14 Ewing sarcomas, five rhabdomyosarcomas and several other pediatric solid tumors. Response rate (partial or complete response) was remarkable in rhabdomyosarcomas (four of five) and Ewing sarcomas (four of 14). We addressed several predictive factors of response to nab-paclitaxel such as the expression of the secreted protein acidic and rich in cysteine (SPARC), chromosomal stability of cancer cells and expression of antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of proteins such as Bcl-2, Bcl-xL, Bcl-W and Mcl-1. Protein (immunoblotting) and gene expression of SPARC correlated positively, while immunoblotting and immunohistochemistry expression of Bcl-2 correlated negatively with the efficacy of nab-paclitaxel in Ewing sarcoma PDX. The negative correlation of Bcl-2 immunoblotting signal and activity was especially robust (r = 0.8352; P = 0.0007; Pearson correlation). Consequently, we evaluated pharmacological strategies to inhibit Bcl-2 during nab-paclitaxel treatment. We observed that the Bcl-2 inhibitor venetoclax improved the activity of nab-paclitaxel in highly resistant Bcl-2-expressing Ewing sarcoma PDX. Overall, our results suggest that low Bcl-2 expression could be used to select patients with Ewing sarcoma sensitive to nab-paclitaxel, and Bcl-2 inhibitors could improve the activity of this drug in Bcl-2-expressing Ewing sarcoma.

Nuestra hipótesis es que el aprendizaje de los alumnos mejorará si: i) se in- tegran mejor los contenidos procedimentales con los conceptuales a lo largo de Ciclos de Mejora en Aula (CIMA) que rompan la dicotomía tradicional entre clases teóricas y seminarios/prácticas, y si, además, ii) los alumnos desarrollan un papel activo en el aprendizaje de los contenidos estructurantes, mediante la realización de actividades en el aula, basadas en actividades que el estudiante ha trabajado de forma no presencial. Para llevar a cabo este proyecto hemos empleado la me- todología de CIMA, realizada en el Programa de Formación e Innovación Docente del Profesorado (FIDOP), pero la hemos combinado con el modelo de clase/aula inversa. La evaluación de la actividad, según la técnica de escaleras de aprendi- zaje y evaluación, opiniones de los alumnos, y autoevaluación, plantea algunos posibles puntos de mejora, pero sugiere que la intervención ha sido acertada en líneas generales. Palabras clave: Medicina, anatomía patológica, docencia universitaria, desa- rrollo profesional docente, Clase inversa.

Undifferentiated small round cell sarcomas (SRCSs) of bone and soft tissue comprise a heterogeneous group of highly aggressive tumours associated with a poor prognosis, especially in metastatic disease. SRCS entities mainly occur in the third decade of life and can exhibit striking disparities regarding preferentially affected sex and tumour localization. SRCSs comprise new entities defined by specific genetic abnormalities, namely EWSR1-non-ETS fusions, CIC-rearrangements or BCOR genetic alterations, as well as EWSR1-ETS fusions in the prototypic SRCS Ewing sarcoma. These gene fusions mainly encode aberrant oncogenic transcription factors that massively rewire the transcriptome and epigenome of the as yet unknown cell or cells of origin. Additional mutations or copy number variants are rare at diagnosis and, depending on the tumour entity, may involve TP53, CDKN2A and others. Histologically, these lesions consist of small round cells expressing variable levels of CD99 and specific marker proteins, including cyclin B3, ETV4, WT1, NKX3-1 and aggrecan, depending on the entity. Besides locoregional treatment that should follow standard protocols for sarcoma management, (neo)adjuvant treatment is as yet ill-defined but generally follows that of Ewing sarcoma and is associated with adverse effects that might compromise quality of life. Emerging studies on the molecular mechanisms of SRCSs and the development of genetically engineered animal models hold promise for improvements in early detection, disease monitoring, treatment-related toxicity, overall survival and quality of life., T.G.P.G. was supported by the Matthias-Lackas Foundation, the Dr. Leopold and Carmen Ellinger Foundation, the German Cancer Aid (DKH-70114278 and DKH-70114111), the Gert und Susanna Mayer Foundation, the Boehringer-Ingelheim Foundation, the Federal Ministry of Education and Research (BMBF: SMART-CARE; HEROES-AYA), the Deutsche Forschungsgemeinschaft (DFG-458891500), the SMARCB1 association, and the Barbara und Wilfried Mohr Foundation. F.C.-A. was supported by grants from the Barbara & Hubertus Trettner Foundation, and the Dr. Rolf M. Schwiete Foundation, and the German Cancer Aid.

Nuestra hipótesis es que el aprendizaje de los alumnos mejoraría si se integrasen contenidos de diversas asignaturas y se adelantasen algunos contenidos clínicos al comienzo del Grado de Medicina. Para llevar a cabo este proyecto hemos empleado la metodología de ciclos de mejora docente, realizados en el Programa de Formación e Innovación Docente del Profesorado (FIDOP), pero la hemos empezado a aplicar al diseño de Ciclos de Mejora Docente Integrados (CMDI), que abarquen aspectos concretos de varias asignaturas. La evaluación de la actividad, según la técnica de escaleras de aprendizaje y evaluación, opiniones de los alumnos, y autoevaluación, ofrece algunos puntos de mejora, pero sugiere que la intervención ha sido acertada en líneas generales.

[Background] Detection of epidermal growth factor receptor (EGFR) mutations in exons 18–21 is recommended in all patients with advanced Non-small-cell lung carcinoma due to the demonstrated efficiency of the standard therapy with tyrosine kinase inhibitors in EGFR-mutated patients. Therefore, choosing a suitable technique to test EGFR mutational status is crucial to warrant a valid result in a short turnaround time using the lowest possible amount of tissue material. The Idylla™ EGFR Mutation Test is a simple, fast and reliable method designed for the detection of EGFR mutations from formalin-fixed paraffin-embedded samples. The aim of this study was the Clinical Performace Evaluation of the Idylla™ EGFR Mutation Test on the Idylla™ System., [Methods] EGFR mutational status was determined on 132 archived formalin-fixed paraffin-embedded tissue sections with Idylla™ technology. Results were compared with the results previously obtained by routine method in the reference lab (Therascreen® EGFR RGQ PCR v2, Qiagen in Molecular Pathology lab, Hospital Universitario Virgen del Rocío de Sevilla)., [Results] The overall agreement between results obtained with the Idylla™ EGFR Mutation Test and the Comparator test method was 95.38% (with 1-sided 95% lower limit of 91.7%) showing Positive Diagnostic Agreement of 93.22% and Negative Diagnostic Agreement of 97.18%, with a Limit Of Detection ≤5%., [Conclusions] The Idylla™ EGFR Mutation Test passed its clinical validity performance characteristics for accuracy., Supported by each clinical center. Biocartis provided free-of-charge Idylla™ EGFR Mutation Test cartridges.

Resumen El sarcoma histiocitico es un tumor maligno infrecuente, de patogenia incierta, caracterizado por una proliferacion de celulas neoplasicas con morfologia e inmunofenotipo de los histiocitos tisulares maduros. Aunque existen pocas referencias bibliograficas, afecta a pacientes de ambos sexos, de amplio rango de edad y afectacion nodal y/o extranodal. Se ha senalado la existencia en muchos casos de una estrecha relacion entre esta entidad y varias neoplasias hematolinfoides en un mismo paciente, lo que ha dado lugar a multiples estudios para esclarecer su etiologia y su patogenia. Su curso suele ser rapidamente progresivo y no se conoce una pauta terapeutica eficaz. Actualmente su diagnostico sigue siendo por exclusion. Presentamos el caso de un paciente de 82 anos con perforacion de un asa yeyunal con una tumoracion maligna que infiltra transmuralmente la pared y solo expresa CD68, CD45RO, CD163, lisozima y vimentina, junto con un reordenamiento clonal linfoide B con escasa amplificacion.

Background: Ewing sarcoma (ES) is a developmental bone/soft tissue neoplasia, for which mesenchymal stem cell (MSC) is the putative cell of origin. Although the outcome of ES patients bearing primary tumors has improved significantly, patients with metastatic ES still have a poor prognosis. The MSC marker endoglin/CD105 (ENG) and the ENG-shedding protease MMP14 have been, respectively, described as cell migration and invasion regulators. In our attempt to better understand the mechanism of ES progression, we are focusing on the role of ENG and MMP14 in ES cells. Methods: In vitro ENG-knockdown (ENG-KD; RM82 and SKNMC cell lines) and ENG-overexpressing (pENG; TC71 cell lines) models were generated by shRNA technology and pDisplay vectors, respectively, and confirmed by qRT-PCR, western blotting and flow cytometry. In vitro MMP14-knockout (MMP14-KO; RM82 and SKNMC) models were generated by CRISPR-Cas9 and confirmed by western blotting and Sanger sequencing. Transwell migration/invasion, adhesion, clonogenicity and proliferation assays were performed in vitro. Actin filament organization was evaluated by immunofluorescence. Extracellular vesicles (EVs) were isolated by ultracentrifugation and density gradient separation. Transcriptomic profiling was performed by Clarion S Human arrays followed by Gene Set Enrichment Analysis. HiPathia tool was used to recode the transcriptomic data into measurements of cell activity by applying mechanistic models. Reverse Phase Protein Array (RPPA) analysis was evaluated on the ENG-KD RM82 model. ENG-targeting immunoprecipitations (IP) from wild type RM82 protein extracts were analyzed by mass spectrometry (MS). Results: The impaired clonogenic potential of ENG-KD RM82 and SKNMC models suggests a role of ENG on the stemness capacity of ES, without affecting the proliferation rate. The reduced migratory and adhesive phenotype of the ENG-KD RM82 model and the transcriptomic profiling of both ENG-KD RM82 and SKNMC cells, suggest a role of ENG in ES cell migration and adhesion. The altered organization of actin filaments in ENG-KD and pENG models could support a role of ENG in actin filament remodeling. The transcriptomic profiling of ENG-KD models and the RPPA results underscore the regulation of FAK signaling by ENG. The IP-MS analysis of ENG partners identifies Integrin-Linked Kinase-Associated serine/threonine Phosphatase (ILKAP) as one of the main interactors of ENG in RM82 cells, suggesting its possible association with the focal adhesion member ILK. ENG is enriched in EVs from different ES cell lines. The transcriptomic profiling of MMP14-KO SKNMC cells highlights the involvement of MMP14 activity in the extracellular matrix organization of ES cells. Conclusion: ENG favors a pro-migratory phenotype in ES cells. These findings help to better understand the molecular landscape of tumoral cells from ES patients, and might have potential clinical implications. Citation Format: Pilar Puerto-Camacho, Ana Teresa Amaral, Juan Diaz-Martin, Alfonso Bolado-Carrancio, Carmen Jordan-Perez, Carmen Salguero-Aranda, Marina Esteban-Medina, Inmaculada Alamo-Alvarez, Joaquin Dopazo, Carmelo Bernabeu, Adam Byron, Valerie G. Brunton, Enrique de Álava. The relevance of endoglin and MMP14 to the metastatic potential of Ewing sarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6158.

[Background] A solitary fibrous tumour is a rare soft-tissue tumour with three clinicopathological variants: typical, malignant, and dedifferentiated. Preclinical experiments and retrospective studies have shown different sensitivities of solitary fibrous tumour to chemotherapy and antiangiogenics. We therefore designed a trial to assess the activity of pazopanib in a cohort of patients with malignant or dedifferentiated solitary fibrous tumour. The clinical and translational results are presented here., [Methods] In this single-arm, phase 2 trial, adult patients (aged ≥ 18 years) with histologically confirmed metastatic or unresectable malignant or dedifferentiated solitary fibrous tumour at any location, who had progressed (by RECIST and Choi criteria) in the previous 6 months and had an ECOG performance status of 0–2, were enrolled at 16 third-level hospitals with expertise in sarcoma care in Spain, Italy, and France. Patients received pazopanib 800 mg once daily, taken orally without food, at least 1 h before or 2 h after a meal, until progression or intolerance. The primary endpoint of the study was overall response measured by Choi criteria in the subset of the intention-to-treat population (patients who received at least 1 month of treatment with at least one radiological assessment). All patients who received at least one dose of the study drug were included in the safety analyses. This study is registered with ClinicalTrials.gov, number NCT02066285, and with the European Clinical Trials Database, EudraCT number 2013-005456-15., [Findings] From June 26, 2014, to Nov 24, 2016, of 40 patients assessed, 36 were enrolled (34 with malignant solitary fibrous tumour and two with dedifferentiated solitary fibrous tumour). Median follow-up was 27 months (IQR 16–31). Based on central radiology review, 18 (51%) of 35 evaluable patients had partial responses, nine (26%) had stable disease, and eight (23%) had progressive disease according to Choi criteria. Further enrolment of patients with dedifferentiated solitary fibrous tumour was stopped after detection of early and fast progressions in a planned interim analysis. 51% (95% CI 34–69) of 35 patients achieved an overall response according to Choi criteria. Ten (29%) of 35 patients died. There were no deaths related to adverse events and the most frequent grade 3 or higher adverse events were hypertension (11 [31%] of 36 patients), neutropenia (four [11%]), increased concentrations of alanine aminotransferase (four [11%]), and increased concentrations of bilirubin (three [8%])., [Interpretation] To our knowledge, this is the first trial of pazopanib for treatment of malignant solitary fibrous tumour showing activity in this patient group. The manageable toxicity profile and the activity shown by pazopanib suggests that this drug could be an option for systemic treatment of advanced malignant solitary fibrous tumour, and provides a benchmark for future trials., Spanish Group for Research on Sarcomas (GEIS), Italian Sarcoma Group (ISG), French Sarcoma Group (FSG), GlaxoSmithKline, and Novartis.

ABSTRACT Desmoplastic small round cell tumor (DSRCT) was proposed as a distinct disease entity by William L Gerald and Juan Rosai in 1991. Over 850 patients have been reported in the medical literature. A specific translocation, t(11;22)(p13;q12), is seen in almost all cases, juxtaposing the EWS gene to the WT1 tumor suppressor gene. DSRCT is composed of nests of small round cells with polyphenotypic differentiation, typically a mixture of epithelial, mesenchymal and neural features, surrounded by a prominent desmoplastic stroma. DSRCT has a predilection for adolescent and young adult males, and primarily involves the abdominal cavity and pelvis. Survival is low despite their initial response to multimodal treatment. Most patients relapse with disseminated disease that is unresponsive to further therapy.

Resumen Introduccion La Anatomia Patologica sigue siendo desconocida, no solo para el publico en general, sino tambien para nuestros companeros de profesion. El uso generalizado de las redes sociales permite difundir nuestra labor. Por ello, se planteo generar un grupo de trabajo. Material y metodos Se emitio una convocatoria a los socios de la Sociedad Espanola de Anatomia Patologica a traves de su Vocalia de Comunicacion y Proyeccion Social titulada #IWantYouForSEAP, para conformar una red de trabajo en la red social Twitter. El periodo de reclutamiento fue de un mes (agosto del 2016). Se registro la actividad desarrollada durante el XXVIII Congreso de la SEAP-IAP de 2017 utilizando las herramientas analiticas Symplur y Tweet Binder. Resultados Se recibieron 32 solicitudes (29 patologos, 2 tecnicos de Anatomia Patologica, un administrativo), con una distribucion geografica dentro del territorio espanol que incluyo a participantes de 14 de las 17 comunidades autonomas, perteneciendo a 22 ciudades y 25 centros distintos. La actividad en relacion con el hashtag #SEAP2017V utilizado en el congreso incluyo a 685 participantes, 6.704 tweets y 8.837.435 impresiones; 28 de los 32 reclutados por la campana #IWantYouForSEAP participaron, contribuyendo con 2.410 tweets y generando 2.090.423 impresiones (el 36 y el 24% del total, respectivamente). Conclusiones Es posible promover y motivar el trabajo en grupo dentro de nuestra disciplina a traves de las redes sociales. Se trata de una fase inicial, pero los resultados experimentados son alentadores y existen grandes expectativas por parte de los participantes. Un uso adecuado de las redes sociales podria acercar la figura del patologo a la sociedad.

Ewing sarcoma is an undifferentiated small-round-cell sarcoma. Although molecular detection of pathognomonic EWSR1-ETS fusions such as EWSR1-FLI1 enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics. To identify novel diagnostic immunohistochemical markers for Ewing sarcoma, we performed comparative expression analyses in 768 tumors representing 21 entities including Ewing-like sarcomas, which confirmed that CIC-DUX4-, BCOR-CCNB3-, EWSR1-NFATc2-, and EWSR1-ETS-translocated sarcomas are distinct entities, and revealed that ATP1A1, BCL11B, and GLG1 constitute specific markers for Ewing sarcoma. Their high expression was validated by immunohistochemistry and proved to depend on EWSR1-FLI1-binding to highly active proximal super-enhancers. Automated cut-off-finding and combination-testing in a tissue-microarray comprising 174 samples demonstrated that detection of high BCL11B and/or GLG1 expression is sufficient to reach 96% specificity for Ewing sarcoma. While 88% of tested Ewing-like sarcomas displayed strong CD99-immunoreactivity, none displayed combined strong BCL11B- and GLG1-immunoreactivity. Collectively, we show that ATP1A1, BCL11B, and GLG1 are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple, and cost-efficient way to diagnose Ewing sarcoma by immunohistochemistry. These markers may significantly reduce the number of misdiagnosed patients, and thus improve patient care., This work was supported by the Kind-Philipp-Foundation (to J.M., M.C.B., M.F.O., M.D., A.M, and G.S.), the ‘Deutsche Stiftung für Junge Erwachsene mit Krebs’ (to M.D.), the German National Academic Foundation (to M.C.B. and M.M.L.K.), the ‘Verein zur Förderung von Wissenschaft und Forschung an der Medizinischen Fakultät der LMU München’ (WiFoMed; to T.G.P.G.), the Daimler and Benz Foundation in cooperation with the Reinhard Frank Foundation (to T.G.P.G.), the Friedrich-Baur-Stiftung (to T.G.P.G.) by LMU Munich’s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative (to T.G.P.G.), the ‘Mehr LEBEN für krebskranke Kinder - Bettina-Bräu-Stiftung’ (to T.G.P.G.), the Fritz-Thyssen Foundation (FTF-40.15.0.030MN, to T.G.P.G. and G.S.), the Wilhelm-Sander-Foundation (2016.167.1 to T.G.P.G.), and the German Cancer Aid (DKH-111886 and DKH-70112257 to T.G.P.G.; DKH-108128 to U.D.), EU FP7 and TRANSCAN EraNet - PROVABES [01KT1310], EU-FP7 EEC [602856-2] to U.D.

11062 Background: Extraskeletal myxoid chondrosarcoma (EMC) is an exceedingly rare sarcoma, marked by a specific translocation involving the gene NR4A3 that can be rearranged with different partners. Preliminary retrospective data suggest that sunitinib is active, but no formal prospective studies are available. We report on a multicentric European prospective, investigator-driven, Phase 2 study on pazopanib (P) in NR4A3+ advanced EMC patients (pts), carried out by the Spanish, Italian and French Sarcoma groups. Methods: From June 2014 to November 2016, 24 advanced EMC pts entered this study (median age: 64 yrs - disease extent: metastatic 77%, locally advanced 23% - prior medical treatment: 18 (86%) naive; 2 (9%) 1 line, 1 (5%) > 1 line). Path diagnosis and NR4A3 rearrangement (FISH and/or real-time PCR analysis) were centrally confirmed. Pts received P 800 mg/day (relative dose intensity = 0,82%, 658 mg/day), until progression or toxicity. The primary study end-point was response rate as per RECIST 1.1. Secondary end-points were overall survival, progression-free survival (PFS), clinical benefit rate (CBR) (RECIST CR+PR+SD≥6mos). An exploratory evaluation of the correlation between the rearrangement subtype and the outcome is ongoing. Results: 20/24 pts were evaluable for response (1 early death; 3 too early). One patient (5%) had a partial response, 17 (75%) stable disease, 2 (10%) progression as their best RECIST responses. At the time of this analysis, 12 pts were still under treatment, while 12 interrupted P (10 progression, 1 toxicity, 1 other). At a 13-month median follow-up, the median PFS was 13 months (range 1.6-25.1), with 29% pts progression-free at 18 months and a 65% CBR. Median OS was not reached. Conclusions: This Phase 2 study is formally negative since the target of at least 3/21 RECIST responses was not reached. However, looking at PFS, P was associated with a prolonged disease stabilization in a significant proportion of pts. This suggests to further explore the use of P in EMC. Clinical trial information: NCT02066285.

11003 Background: SFT is a rare soft tissue tumor. In advanced SFT chemotherapy has only limited activity. With the rationale of a rich vascular network & VEGF (tumor cells and endothelium) and VEGFR1/2 (endothelial cells) expression in SFT, we designed an international, single-arm phase II trial to test pazopanib (P) in advanced SFT. Clinical and preclinical evidence suggesting that antiangiogenics was less effective in more aggressive compared with less aggressive SFT (Stacchiotti et al), led us to conduct the trial on two different cohorts: typical and malignant (M)/dedifferentiated (DD) SFT. Here we present the outcome of the latter cohort. Methods: Most relevant inclusion criteria were: unresectable or metastatic, M/DD SFT confirmed by central pathologic review with evidence of STAT6 (IHC and /or FISH or RT-PCR), ≥ 18 years, ECOG 0-2, progressive and measurable disease. Main endpoint was response rate (RR) according Choi criteria. Central radiological assessment was mandatory. P was administered at 800 mg/d continuously until progression or toxicity. Results: From June 2014 to November 2016, 34 patients (pts) were enrolled with a median age of 61 y (23-87). Median tumor size and mitosis at diagnosis were 77 mm and 8x10 HPF. Most relevant grade 3-4 toxicity were neutropenia (9%) and hypertension (12%). At the time of the present analysis, 31 pts are evaluable for response. RR according to Choi and RECIST were: PR 16 (52%), SD 7 (22%), PD 8 (26%) and PR 1 (3%), SD 19 (61%), PD 11 (35%) respectively. With a median follow-up of 15 months, the median PFS was 5.53 months (4.24-6.82), while 72% survived at 18 months. Size > 5 cm, mitosis > 8 and DD subtype showed significantly worse PFS. The 18-month OS was 90% for those with SD and PR and 25% for PD according to Choi (p < 0.001), while 94% for SD and PR and 45% for PD according RECIST (p = 0.002). In multivariate analysis, only Choi was an independent prognostic factor for OS with PD showing a HR of 11.9 (2.3-63.1), p = 0.003 for the risk of death. Conclusions: Pazopanib showed activity in malignant SFT. Choi criteria exhibited a more accurate assessment of response than RECIST. Clinical trial information: NCT02066285.

[EN]: Soft tissue myoepithelial carcinomas (STMM) are clinically aggressive tumors that arise in the extremities and limb girdles of young patients. Histologically, they show moderate to severe atypia and infiltrative margins. EWSR1-gene rearrangements are present in 45% of cases and are associated with increased malignancy.A large mass in the right hip of a 26-year-old male that had been surgically removed showed strands of cells embedded in a hyalin-myxoid matrix without ductal differentiation. Cells expressed cytokeratins, EMA, S-100 protein and calponin. STMM was diagnosed. Although the tumor did not have an EWSR1 gene rearrangement, it behaved aggressively and the patient died from multiple metastases 18 months after diagnosis. © 2011 SEAP y SEC., [ES]: Los carcinomas mioepiteliales de partes blandas (STMM), afectan a las extremidades o cinturas de pacientes jóvenes y evolucionan agresivamente. Muestran una marcada atipia y bordes infiltrantes y su pronóstico empeora con el reordenamiento del gen EWSR1, presente en el 45% de los casos. Presentamos el caso de un varón de 26 años al que se le extirpó un STMM situado en la cadera derecha, compuesto por cordones y nidos sólidos de células atípicas rodeadas de un estroma mixoide-hialino, sin diferenciación ductal. Expresaban citoqueratinas, EMA, S100 y calponina. La hibridación in situ fluorescente (FISH) no reveló reordenamientos del gen EWSR1. Tras recibir cuidados paliativos, el paciente murió por múltiples metástasis a los 18 meses del diagnóstico. Se trata de un caso de STMM sin reordenamiento del gen EWRS1 pero inusualmente agresivo., Partially supported by the Ministry of Science and Innovation of Spain-FEDER (PI081828, RD06/0020/0059).

Ewing sarcoma (ES) and primitive neuroectodermal tumor (PNET) are round-cell sarcomas with varying degrees of neuroectodermal differentiation; they are considered members of the same family of tumors based on their common genetic profile. ES/PNET are very infrequent in the female genital tract, the vagina being the rarest gynecological location, with 7 cases reported to date. The authors present a case of vaginal ES/PNET with typical morphological and immunohistochemical features. EWS gene rearrangement was demonstrated by fluorescent in situ hybridization analysis. Of the 7 previously reported cases, only 2 have molecular confirmation. The age at presentation for vaginal ES/PNET is slightly higher than that for these neoplasms in bone and soft tissues, with only 1 reported patient younger than 30 years. The outcome seems to be similar to that of other superficially sited ES/PNET and more favorable than for those observed in the more typical locations. However, the currently available data are limited, and therefore, a larger number of cases is necessary to draw any firm conclusions.

Carcinoma in situ (CIS) is a non-papillary high-grade, potentially aggressive, and unpredictable manifestation of bladder urothelial carcinoma. The aim of this study was to assess patterns of Cyclin D3 gene amplification in Bacillus Calmette-Guerin (BCG)-treated CIS and correlate gene status with recurrence-free and progression-free survival. A sequential cohort series of 28 primary (isolated) or secondary (concomitant) bladder CIS samples in which there was enough tissue material to assess Cyclin D3 gene status by fluorescent in situ hybridization was the study group. Cyclin D3 gene amplification was present in 29% of secondary CIS; none of primary CIS samples had Cyclin D3 gene amplification. Cyclin D3 amplification was related to recurrence- (p=0.046) and progression-free survival (p=0.002). Type of bladder CIS (primary vs. secondary) was unrelated to recurrence- or progression-free survival in the current series. Cox's regression analysis selected Cyclin D3 as an independent predictor of progression-free survival (p=0.041, relative risk=61.503, 95% confidence interval=1.1-274.710). None of primary CIS cases recurred on follow-up; nine secondary CIS recurred and four of them progressed to invasive bladder carcinoma HG T1 (n=1), T2b N0M0 (n=1), T3b N1M0 (n=1) and T4aN1M1 (n=1). Mean recurrence±SD (months) occurred at 19.5±2.06 (95% (confidence interval (CI)), 15.5-23.6); mean progression (months) occurred at 23.8±1.46 (95% (CI), 20.9-26.7). Our study suggests that Cyclin D3 gene amplification might be a predictor of aggressiveness in BCG-treated CIS. © 2010 Springer-Verlag., Supported by the grants SAF2007-64942 (Ministry of Education and Research, Madrid, Spain), P07-CVI-02974 and PI0003/2007 (Junta de Andalucia, Seville, Spain). Work at CIC is also funded by Instituto de Salud Carlos III (PI081828, RD06/020/0059). JLO is supported by ISCIII (Contratos Postdoctorales Sara Borrell CD6/00001).

Desmoplastic spindle cell tumor of the liver is a recently described and extremely unusual neoplasm that affects children and young adults. We report 1 case in a 33-year-old man. The patient had abdominal pain and dyspepsia. Abdominal examination showed that the liver was enlarged and palpable until umbilical region. Laboratory studies demonstrated positive serologic markers to hepatitis B virus. All other analytical studies were irrelevant. Computed tomography revealed a large tumor mass in left hepatic lobe showing heterogeneous densities, with hyperdense peripheral areas, as multiple nodular calcifications of less than 1 cm. In the central part of the mass, a big hypodense area was observed. There was no evidence of extrahepatic disease. Grossly, the tumor was well circumscribed with multiple nodular calcifications. The tumor was characterized by the presence of cohesive nests of bland spindle cells arranged in short fascicles and surrounded by desmoplastic stroma, intermixed with epithelioid cells. Mitotic activity was very low. Extensive osteoid formation was seen inside the cell nests. The tumor cells showed cytoplasmic reactivity for vimentin and pan-cytokeratin. The cells of desmoplastic stroma were immunoreactive for actin. The biologic behavior is still uncertain with only 5 published cases, but current information suggests that they are low-grade tumors with an indolent course. The clinical and morphologic features of this tumor are very similar to those of tumors previously reported as >nested stromal-epithelial tumor of liver> and >ossifying stromal-epithelial tumor of liver.> We describe the histologic, immunohistochemical, and molecular genetic features of a case of desmoplastic spindle cell tumor of the liver and review the literature. © 2010.

Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subclones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subclones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p16) protein. Ten cell lines showed missense mutations of the TP53 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53wt cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53wt osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable.

Sarcomas and Gastrointestinal Stromal Tumors: Presentations From the 2008 European Society for Medical Oncology International Symposium.-- et al., Despite complex genetics, aneuploid tumors like dedifferentiated liposarcoma have specific and reproducible chromosomal changes such as amplification of HDM2 and CDK4 that represent potential targets for systemic therapy. In addition, there are cancer cell survival pathways that may not be the target of chromosomal translocations or mutations that are still estimable targets for new systemic therapeutics, be it pathways involved in angiogenesis or apoptosis. In this review, we examine target selection for specific sarcoma subtypes, and demonstrate with a few examples new techniques being used to delineate novel therapeutic inroads for patients with sarcoma. © 2009 Elsevier Inc. All rights reserved., Enrique de Alava is supported by the European Commission (NoE EuroBoNet), Ministry of Science and Innovation of Spain-FEDER (PI052524, RD06/0020/0059). Silvana Pilotti is supported by Associazione Italiana per la Ricerca sul Cancro (AIRC). Robert G. Maki is supported by Program Project Grant No. P01-CA47179; Cycle for Survival. David M. Thomas is supported by the Victorian Cancer Agency Clinician Researcher fellowship, and Cancer Australia.

To assess the effect of long-term alfa interferon therapy (12 months) on liver histology of chronic hepatitis C, we studied 61 treated patients, and compared their outcome with 28 untreated cases followed as controls. A liver biopsy was taken from all patients, before (month 0) and after the completion of the treatment or the control period (month 12). A third liver specimen taken at month 24 was available in 29 treated cases. Liver biopsies were blindly graded following Knodell's method. In 33 out of the 61 treated patients (54.1%), aminotransferase levels became normal shortly after starting therapy and remained within normal values until the end of treatment (sustained response). Nine (27%) sustained responders relapsed after interferon discontinuation, while the remaining 24 (73%) continued with normal aminotransferase values during follow-up (16.8 +/- 9.9 months). All histological parameters, except fibrosis, improved significantly after 12 months of therapy (periportal necrosis, month 0: 2.7 +/- 1.0, month 12: 1.6 +/- 1.1, p < 0.0001; lobular damage, month 0: 2.5 +/- 1.1, month 12: 1.4 +/- 0.9, p < 0.0001; portal inflammation, month 0: 3.6 +/- 0.5, month 12: 3.0 +/- 0.9, p < 0.0001). Histological improvement was especially marked in patients who did not relapse, although those who relapsed and partial responders also improved. Overall histological diagnoses improved in most patients. A sustained response to interferon was predicted by high periportal and lobular scores, and by a low fibrosis score on the pretreatment liver biopsy. At 24 months, histological improvement persisted in patients without posttreatment relapse, while liver inflammation had returned to pretreatment levels in the remaining cases.

Chromosome 14 loss in meningiomas are associated with more aggressive tumour behaviour. To date, no studies have been reported in which the entire chromosome 14q of meningioma tumour cells has been studied by high-resolution array comparative genomic hybridization (a-CGH). Here, we used a high-resolution a-CGH to define the exact localization and extent of numerical changes of chromosome 14 in meningioma patients. An array containing 807 bacterial artificial chromosome clones specific for chromosome 14q (average resolution of ∼130Kb) was constructed and applied to the study of 25 meningiomas in parallel to the confirmatory interphase fluorescence in situ hybridization (iFISH) analyses. Overall, abnormalities of chromosome 14q were detected in 10/25 cases (40%). Interestingly, in seven of these cases, loss of chromosome 14q32.3 was detected by iFISH and confirmed to correspond to monosomy 14 by a-CGH. In contrast, discrepant results were found between iFISH and a-CGH in the other three altered cases. In one patient, a diploid background was observed by iFISH, while monosomy 14 was identified by a-CGH. In the remaining two cases, which showed gains of the IGH gene by iFISH, a-CGH did not detected copy number changes in one case showing a tetraploid karyotype, while in the other tumour, varying genetic imbalances along the long arm of chromosome 14 were detected. In summary, here, we report for the first time, the high-resolution a-CGH profiles of chromosome 14q in meningiomas, confirming that monosomy 14 is the most frequent alteration associated with this chromosome; other numerical abnormalities being only sporadically detected., This work has been partially supported by grants from Consejería de Sanidad (44-05) Junta de Castilla y León (Valladolid, Spain; Fondo de Investigaciones Sanitarias (FIS/FEDER 06/0312 and RTICC RD06/0020/0035 from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain) and Fundación MMA (Madrid, Spain). MD Tabernero is supported by IECSCYL. AB Espinosa is supported by grant FI05/00266 from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Madrid, Spain).

Resumen Caso clinico Los autores presentan un sarcoma de partes blandas cervicales que por su forma de presentacion clinica, localizacion yuxtatiroidea y aspecto histologico simulaba un tumor maligno de glandula tiroides. Resultados Aunque la histologia podia ser compatible con varias formas de carcinoma tiroideo, especialmente con un carcinoma medular, los resultados del estudio inmunohistoquimico y las alteraciones moleculares del gen EWS mediante FISH en parafina correspondieron a un tumor maligno de celulas redondas de estirpe mesenquimal, concordante con sarcoma de Ewing/ PNET. Conclusion Este caso, de gran dificultad diagnostica, sirve para recordar que en el diagnostico diferencial de tumores tiroideos pobremente diferenciados hemos de considerar la posibilidad de afectacion secundaria del mismo por tumores de estructuras vecinas, ya que los sarcomas primarios de la glandula tiroides son muy infrecuentes y en su mayoria corresponden a formas sarcomatoides de carcinomas tiroideos.

Neuroblastoma (NB) is a pediatric neoplasia that shows complex combinations of acquired genetic aberrations. The specific genes and the molecular mechanisms responsible for development and progression of NB remain poorly understood. Our main objective is to compare the results obtained with different techniques for the detection of genomic data in 20 patients with NB using the information obtained to select the appropriate technique in routine analysis for the therapeutic stratification. The genetic methods used in this study are multiprobe fluorescence in situ hybridization (FISH) assay, metaphasic comparative genomic hybridization (mCGH), array comparative genomic hybridization (aCGH), and the multiplex ligation-dependent probe amplification (MLPA). Genomic copy number abnormalities were used to group the cases in four categories: MYCN amplification cases; 11q deletion tumors; cases with partial chromosome gains or losses and samples with entire chromosome alterations. The data obtained from the multigenomic techniques showed a high degree of concordance and our findings support the hypothesis that NB consists of biologically distinct subgroups that differ by genetic characteristics of prognostic relevance. FISH will be essential for the mandatory study of MYCN status. The use of MLPA as routine technique is an advantage procedure for detecting the implication of the common genetic alterations in NB. © 2008 Springer-Verlag., Supported by the grants FIS RD06/0020/0102 (Instituto de Salud Carlos III; Ministerio de Sanidad y Consumo, Spain), RD06/0020/0059, and PI06/1576; PI4/07-36 (Fundación Sociedad Española de Oncología Pediátrica).

Clinical Trial, Phase III; Comparative Study; Journal Article; Randomized Controlled Trial; Research Support, Non-U.S. Gov't; BACKGROUND Taxanes are among the most active drugs for the treatment of metastatic breast cancer, and, as a consequence, they have also been studied in the adjuvant setting. METHODS After breast cancer surgery, women with lymph node-positive disease were randomly assigned to treatment with fluorouracil, epirubicin, and cyclophosphamide (FEC) or with FEC followed by weekly paclitaxel (FEC-P). The primary endpoint of study-5-year disease-free survival (DFS)-was assessed by Kaplan-Meier analysis. Secondary endpoints included overall survival and analysis of the prognostic and predictive value of clinical and molecular (hormone receptors by immunohistochemistry and HER2 by fluorescence in situ hybridization) markers. Associations and interactions were assessed with a multivariable Cox proportional hazards model for DFS for the following covariates: age, menopausal status, tumor size, lymph node status, type of chemotherapy, tumor size, positive lymph nodes, HER2 status, and hormone receptor status. All statistical tests were two-sided. RESULTS Among the 1246 eligible patients, estimated rates of DFS at 5 years were 78.5% in the FEC-P arm and 72.1% in the FEC arm (difference = 6.4%, 95% confidence interval [CI] = 1.6% to 11.2%; P = .006). FEC-P treatment was associated with a 23% reduction in the risk of relapse compared with FEC treatment (146 relapses in the 614 patients in the FEC-P arm vs 193 relapses in the 632 patients in the FEC arm, hazard ratio [HR] = 0.77, 95% CI = 0.62 to 0.95; P = .022) and a 22% reduction in the risk of death (73 and 95 deaths, respectively, HR = 0.78, 95% CI = 0.57 to 1.06; P = .110). Among the 928 patients for whom tumor samples were centrally analyzed, type of chemotherapy (FEC vs FEC-P) (P = .017), number of involved axillary lymph nodes (P < .001), tumor size (P = .020), hormone receptor status (P = .004), and HER2 status (P = .006) were all associated with DFS. We found no statistically significant interaction between HER2 status and paclitaxel treatment or between hormone receptor status and paclitaxel treatment. CONCLUSIONS Among patients with operable breast cancer, FEC-P treatment statistically significantly reduced the risk of relapse compared with FEC as adjuvant therapy. Yes

A clear cell sarcoma-like tumor with osteoclast-like giant cells of the gastrointestinal tract without immunoexpression of CD117 was recently proposed as a new tumor entity. In this article, a case of a 37-year-old man with a neoplasm of the jejunum composed of polygonal cells with clear to eosinophilic cytoplasm forming nests and fascicles is reported. Giant cells were not identified. Immunohistochemically, the tumor cells expressed strongly S100 protein, human melanoma black 45, platelet-derived growth factor receptor α, B-cell lymphoma 2, p53, and to a lesser extent vimentin, neuron-specific enolase, and epithelial membrane antigen. Mindbomb homolog-1 index was 35%. Immunoreactivity for CD34 and CD117 was negative. The fluorescence in situ hybridization analysis showed a translocation involving chromosome 22q12, the diagnostic hallmark of clear cell sarcoma of soft tissues. This case indicates a close histogenetic relationship with the recently reported clear cell sarcoma with osteoclast-like giant cells of the gastrointestinal tract, as well as with the clear cell sarcoma of soft tissues.