[Time to Antibiotics (TTA) - Reassessment from the German Working Group for Fever and Neutropenia in Children and Adolescents (DGPI/GPOH)]. / Time to Antibiotics (TTA) ­ Überlegungen der Arbeitsgruppe Fieber bei Granulozytopenie im Kindes- und Jugendalter (GPOH/DGPI) zu einer Neubewertung.

BACKGROUND: The current German guidance from 2016 recommends a Time to Antibiotics (TTA) of<60 min in children and adolescents with febrile neutropenia (FN). METHODS: Critical analysis of available studies and recent meta-analyses, and discussion of the practical consequences in the FN working group of the German Societies for Paediatric Oncology and Haematology and Paediatric Infectious Diseases. RESULTS: The available evidence does not support a clinically significant outcome benefit of a TTA<60 min in all paediatric patients with FN. Studies suggesting such a benefit are biased (mainly triage bias), use different TTA definitions and display further methodical limitations. In any case, a TTA<60 min remains an essential component of the 1st hour-bundle in paediatric cancer patients with septic shock or sepsis with organ dysfunction. CONCLUSION: Provided that all paediatric FN patients receive a structured medical history and physical examination (including vital signs) by experienced and trained medical personnel in a timely fashion, and provided that a sepsis triage and management bundle is established and implemented, a TTA lower than 3 hours is sufficient and reasonable in stable paediatric cancer patients with FN.

Organizational characteristics of European pediatric onco-critical care: An international cross-sectional survey.

Background: Intensified treatment protocols have improved survival of pediatric oncology patients. However, these treatment protocols are associated with increased treatment-related morbidity requiring admission to pediatric intensive care unit (PICU). We aimed to describe the organizational characteristics and processes of care for this patient group across PICUs in Europe. Methods: A web-based survey was sent to PICU directors or representative physicians between February and June 2021. Results: Responses were obtained from 77 PICUs of 12 European countries. Organizational characteristics were similar across the different countries of Europe. The median number of PICU beds was 12 (IQR 8-16). The majority of the PICUs was staffed by pediatric intensivists and had a 24/7 intensivist coverage. Most PICUs had a nurse-to-patient ratio of 1:1 or 1:2. The median numbers of yearly planned and unplanned PICU admissions of pediatric cancer patients were 20 (IQR 10-45) and 10 (IQR 10-30, respectively. Oncology specific practices within PICU were less common in participating centres. This included implementation of oncology protocols in PICU (30%), daily rounds of PICU physicians on the wards (13%), joint mortality and morbidity meetings or complex patients' discussions (30% and 40%, respectively) and participation of parents during clinical rounds (40%). Conclusion: Our survey provides an overview on the delivery of critical care for oncology patients in PICU across European countries. Multidisciplinary care for these vulnerable and challenging patients remains complex and challenging. Future studies need to determine the effects of differences in PICU organization and processes of care on patients' outcome.

Pediatric ICU Admissions After Adolescent Suicide Attempts During the Pandemic.

BACKGROUND AND OBJECTIVES: The worldwide severe acute respiratory syndrome coronavirus 2 pandemic challenges adolescents' mental health. In this study, we aim to compare the number of pediatric ICU (PICU) admissions after suicide attempts during the first German lockdown and one year later during a second, prolonged lockdown with prepandemic years. METHODS: A retrospective multicenter study was conducted among 27 German PICUs. Cases <18 years admitted to the PICU because of accidents or injuries between March 16 and May 31 of 2017 to 2021 were identified based on International Classification of Diseases, 10th Revision codes (German modification) and patient data entered into a database. This study is a subset analysis on suicide attempts in adolescents aged 12 to 17.9 years. The Federal Statistics Office was queried for data on fatal suicides, which were available only for 2020 in adolescents aged 10 to 17.9 years. RESULTS: Total admissions and suicide attempts declined during the first lockdown in 2020 (standardized morbidity ratio 0.74 (95% confidence interval; 0.58-0.92) and 0.69 (0.43-1.04), respectively) and increased in 2021 (standardized morbidity ratio 2.14 [1.86-2.45] and 2.84 [2.29-3.49], respectively). Fatal suicide rates remained stable between 2017 to 2019 and 2020 (1.57 vs 1.48 per 100 000 adolescent years) with monthly numbers showing no clear trend during the course of 2020. CONCLUSIONS: This study shows a strong increase in serious suicide attempts among adolescents during the course of the pandemic in Germany. More research is needed to understand the relation between pandemic prevention measures and suicidal ideation to help implement mental health support for adolescents.

Impact of the First COVID Lockdown on Accident- and Injury-Related Pediatric Intensive Care Admissions in Germany-A Multicenter Study.

Children's and adolescents' lives drastically changed during COVID lockdowns worldwide. To compare accident- and injury-related admissions to pediatric intensive care units (PICU) during the first German COVID lockdown with previous years, we conducted a retrospective multicenter study among 37 PICUs (21.5% of German PICU capacities). A total of 1444 admissions after accidents or injuries during the first lockdown period and matched periods of 2017-2019 were reported and standardized morbidity ratios (SMR) were calculated. Total PICU admissions due to accidents/injuries declined from an average of 366 to 346 (SMR 0.95 (CI 0.85-1.05)). Admissions with trauma increased from 196 to 212 (1.07 (0.93-1.23). Traffic accidents and school/kindergarten accidents decreased (0.77 (0.57-1.02 and 0.26 (0.05-0.75)), whereas household and leisure accidents increased (1.33 (1.06-1.66) and 1.34 (1.06-1.67)). Less neurosurgeries and more visceral surgeries were performed (0.69 (0.38-1.16) and 2.09 (1.19-3.39)). Non-accidental non-suicidal injuries declined (0.73 (0.42-1.17)). Suicide attempts increased in adolescent boys (1.38 (0.51-3.02)), but decreased in adolescent girls (0.56 (0.32-0.79)). In summary, changed trauma mechanisms entailed different surgeries compared to previous years. We found no evidence for an increase in child abuse cases requiring intensive care. The increase in suicide attempts among boys demands investigation.

Role of extracorporeal membrane oxygenation in pediatric cancer patients: a systematic review and meta-analysis of observational studies.

BACKGROUND: The use of extracorporeal membrane oxygenation (ECMO) in pediatric patients with underlying malignancies remains controversial. However, in an era in which the survival rates for children with malignancies have increased significantly and several recent reports have demonstrated effective ECMO use in children with cancer, we aimed to estimate the outcome and complications of ECMO treatment in these children. METHODS: We searched MEDLINE, Embase and CINAHL databases for studies on the use ECMO in pediatric patients with an underlying malignancy from inception to September 2020. This review was conducted in adherence to Preferred Reporting Items for Systematic Review and Meta-Analysis statement. Study eligibility was independently assessed by two authors and disagreements resolved by a third author. Included studies were evaluated for quality using the Newcastle-Ottawa Scale (NOS). Random effects meta-analyses (DerSimonian and Laird) were performed. The primary outcomes were mortality during ECMO or hospital mortality. RESULTS: Thirteen retrospective, observational cohort studies were included, most of moderate quality (625 patients). The commonest indication for ECMO was severe respiratory failure (92%). Pooled mortality during ECMO was 55% (95% confidence interval [CI], 47-63%) and pooled hospital mortality was 60% (95% CI 54-67%). Although heterogeneity among the included studies was low, confidence intervals were large. In addition, the majority of the data were derived from registries with overlapping patients which were excluded for the meta-analyses to prevent resampling of the same participants across the included studies. Finally, there was a lack of consistent complications reporting among the studies. CONCLUSION: Significantly higher mortalities than in general PICU patients was reported with the use of ECMO in children with malignancies. Although these results need to be interpreted with caution due to the lack of granular data, they suggest that ECMO appears to represents a viable rescue option for selected patients with underlying malignancies. There is an urgent need for additional data to define patients for whom ECMO may provide benefit or harm.

Extracorporeal Membrane Oxygenation in Children With Cancer or Hematopoietic Cell Transplantation: Single-Center Experience in 20 Consecutive Patients.

Extracorporeal membrane oxygenation (ECMO) is a rescue therapy for severe respiratory and/or circulatory failure. Few data exist on the potential benefit of ECMO in immunocompromised pediatric patients with cancer and/or hematopoietic cell transplantation (HCT). Over a period of 12 years, eleven (1.9%) of 572 patients with new diagnosis of leukemia/lymphoma and nine (3.5%) of 257 patients post allogeneic HCT underwent ECMO at our center. Five (45%) and two (22%) patients, respectively, survived to hospital discharge with a median event-free survival of 4.2 years. Experiences and outcomes in this cohort may aid clinicians and families when considering ECMO for individual patients.

The Receptor Tyrosine Kinase RON and Its Isoforms as Therapeutic Targets in Ewing Sarcoma.

The receptor tyrosine kinase (RTK) RON is linked to an aggressive metastatic phenotype of carcinomas. While gaining interest as a therapeutic target, RON remains unstudied in sarcomas. In Ewing sarcoma, we identified RON among RTKs conferring resistance to insulin-like growth factor-1 receptor (IGF1R) targeting. Therefore, we explored RON in pediatric sarcoma cell lines and an embryonic Tg(kdrl:mCherry) zebrafish model, using an shRNA-based approach. To examine RON-IGF1R crosstalk, we employed the clinical-grade monoclonal antibody IMC-RON8, alone and together with the IGF1R-antibody IMC-A12. RON silencing demonstrated functions in vitro and in vivo, particularly within micrometastatic cellular capacities. Signaling studies revealed a unidirectional IGF1-mediated cross-activation of RON. Yet, IMC-A12 failed to sensitize cells to IMC-RON8, suggesting additional mechanisms of RON activation. Here, RT-PCR revealed that childhood sarcomas express short-form RON, an isoform resistant to antibody-mediated targeting. Interestingly, in contrast to carcinomas, treatment with DNA methyltransferase inhibitor did not diminish but increased short-form RON expression. Thus, this first report supports a role for RON in the metastatic progression of Ewing sarcoma. While principal molecular functions appear transferrable between carcinomas, Ewing sarcoma and possibly more common sarcoma subtypes, RON highlights that specific regulations of cellular networks and isoforms require better understanding to successfully transfer targeting strategies.

Treatment of Children With Cancer and/or Hematopoietic Stem Cell Transplantation in the Intensive Care Unit: Experience at a Large European Pediatric Cancer Center.

Pediatric cancer treatment and hematopoietic stem cell transplantation (HSCT) carry considerable risks of morbidity. We conducted a single-center retrospective analysis of intensive care unit (ICU) admissions in unselected children and adolescents treated for cancer or undergoing HSCT. In a 10-year time period, 140 patients had 188 ICU admissions for a life-threatening condition. Main reasons for ICU admission were respiratory or cardiovascular insufficiency and sepsis. Mortality in the ICU was 19.1% and related to organ failure or acute complications in 77.8% and progress of the underlying malignancy in 22.2%. Mortality rates at 30, 100, and 365 days after discharge from the ICU were 24.5%, 30.9%, and 39.9%. Kaplan-Meier survival probabilities at 5 and 10 years were 46.4% and 39.8%, respectively. Multivariable analysis revealed the number of failed organ systems, the number of prior ICU stays, and days spent in the ICU as parameters independently associated with death. Taken together, the outcome of pediatric cancer and/or HSCT patients admitted to the ICU for life-threatening conditions was not as dismal as reported elsewhere. Most patients benefitted from ICU care, and survival was predominantly compromised by the evolution of complications.

PICU mortality of children with cancer admitted to pediatric intensive care unit a systematic review and meta-analysis.

BACKGROUND: Outcomes for children diagnosed with cancer have improved dramatically over the past 20 years. However, although 40% of pediatric cancer patients require at least one intensive care admission throughout their disease course, PICU outcomes and resource utilization by this population have not been rigorously studied in this specific group. METHODS: Using a systematic strategy, we searched Medline, Embase, and CINAHL databases for articles describing PICU mortality of pediatric cancer patients admitted to PICU. Two investigators independently applied eligibility criteria, assessed data quality, and extracted data. We pooled PICU mortality estimates using random-effects models and examined mortality trends over time using meta-regression models. RESULTS: Out of 1218 identified manuscripts, 31 studies were included covering 16,853 PICU admissions with the majority being retrospective in nature. Overall pooled weighted mortality was 27.8% (95% confidence interval (CI), 23.7-31.9%). Mortality decreased slightly over time when post-operative patients were excluded. The use of mechanical ventilation (odds ratio (OR): 18.49 [95% CI 13.79-24.78], p < 0.001), inotropic support (OR: 14.05 [95% CI 9.16-21.57], p < 0.001), or continuous renal replacement therapy (OR: 3.24 [95% CI 1.31-8.04], p = 0.01) was significantly associated with PICU mortality. CONCLUSIONS: PICU mortality rates of pediatric cancer patients are far higher when compared to current mortality rates of the general PICU population. PICU mortality has remained relatively unchanged over the past decades, a slight decrease was only seen when post-operative patients were excluded. This compared infavorably with the improved mortality seen in adults with cancer admitted to ICU, where research-led improvements have led to the paradigm of unlimited, aggressive ICU management without any limitations on resuscitations status, for a time-limited trial.

The CXCR4 antagonist plerixafor (AMD3100) promotes proliferation of Ewing sarcoma cell lines in vitro and activates receptor tyrosine kinase signaling.

BACKGROUND: The CXCR4 receptor antagonist plerixafor (AMD3100) is raising interest as an anti-cancer agent that disrupts the CXCL12-CXCR4 chemokine - receptor interaction between neoplastic cells and their microenvironment in tumor progression and metastasis. Here, we investigated plerixafor for anti-cancer activity in Ewing sarcoma, a rare and aggressive cancer of bone and soft tissues. METHODS: We used a variety of methods such as cell viability and migration assays, flow cytometry, phospho-tyrosine arrays and western blotting to determine plerixafor effects on five characterized Ewing sarcoma cell lines and a low-passage culture in vitro. RESULTS: Unexpectedly, plerixafor led to an increase in cell viability and proliferation in standard cell growth conditions, and to chemotactic migration towards plerixafor. Exploring potential molecular mechanisms underlying this effect, we found that Ewing sarcoma cell lines divided into classes of high- and low-level CXCR4 surface expression. Proliferative plerixafor responses were observed in both groups, maintained despite significant CXCR4 down-regulation or inhibition of Gαi-protein signal transduction, and involved activation of multiple receptor tyrosine kinases (DDR2, MERTK, MST1R, NTRK1, RET), the most prominent being platelet-derived growth factor receptor beta (PDGFRB). PDGFRB was activated in response to inhibition of the CXCL12-CXCR4 axis by plerixafor and/or pertussis toxin (Gαi-protein inhibitor). Dasatinib, a multi-kinase inhibitor of both PDGFRB and the CXCR4 downstream kinase SRC, counteracted this activation in some but not all cell lines. CONCLUSION: These data suggest a feedback interaction between the CXCR4 chemokine receptor and RTK signaling cascades that elicits compensatory cell survival signaling and can shift the net effect of plerixafor towards proliferation. PDGFRB was identified as a candidate driver RTK and potential therapeutic co-target for CXCR4 in Ewing sarcoma. Although as yet limited to in vitro studies, these findings call for further investigation in the cancer - microenvironment interplay in vivo.

Target discovery screens using pooled shRNA libraries and next-generation sequencing: A model workflow and analytical algorithm.

In the search for novel therapeutic targets, RNA interference screening has become a valuable tool. High-throughput technologies are now broadly accessible but their assay development from baseline remains resource-intensive and challenging. Focusing on this assay development process, we here describe a target discovery screen using pooled shRNA libraries and next-generation sequencing (NGS) deconvolution in a cell line model of Ewing sarcoma. In a strategy designed for comparative and synthetic lethal studies, we screened for targets specific to the A673 Ewing sarcoma cell line. Methods, results and pitfalls are described for the entire multi-step screening procedure, from lentiviral shRNA delivery to bioinformatics analysis, illustrating a complete model workflow. We demonstrate that successful studies are feasible from the first assay performance and independent of specialized screening units. Furthermore, we show that a resource-saving screen depth of 100-fold average shRNA representation can suffice to generate reproducible target hits despite heterogeneity in the derived datasets. Because statistical analysis methods are debatable for such datasets, we created ProFED, an analysis package designed to facilitate descriptive data analysis and hit calling using an aim-oriented profile filtering approach. In its versatile design, this open-source online tool provides fast and easy analysis of shRNA and other count-based datasets to complement other analytical algorithms.

Successful Extracorporeal Life Support in a Pediatric Hematopoietic Stem Cell Transplant Recipient With Periengraftment Respiratory Failure.

The use of extracorporeal life support (ECLS) as ultimate salvage therapy for hematopoietic stem cell transplant recipients remains controversial among oncologists and critical care specialists. Prognosis is poor, particularly after allogeneic transplantation, and literature to guide clinical decision-making is scarce. Our report describes successful ECLS in a pediatric patient undergoing allogeneic hematopoietic stem cell transplantation, who developed acute respiratory failure during severe neutropenia, followed by immediate neutrophil engraftment. This unique case highlights periengraftment respiratory failure as a possible patient subgroup that could benefit from ECLS; and illustrates that the distinct etiologies of respiratory failure and the patients' immune status deserve closer consideration in future studies evaluating ECLS in this high-risk population.

Suberanilohydroxamic acid (vorinostat) synergistically enhances the cytotoxicity of doxorubicin and cisplatin in osteosarcoma cell lines.

Osteosarcoma is the most common primary bone cancer in children and is a highly malignant disease, in which 25% of patients present with metastasis at diagnosis. Considerable advances in the treatment of localized disease have been achieved since the introduction of combined modality treatment, increasing the prognosis of overall survival to 70%. Yet, established therapies have only limited success in treating both metastatic disease and nonresponders to primary chemotherapy. Therefore, new therapeutic approaches are required, particularly for the control of osteosarcoma in these patient groups. Epigenetically modifying substances are a class of emerging drugs that have shown therapeutic potential in various hematological and solid cancers. We examined the cytotoxic effects of 5-azacitidine, 3-deazaneplanocin A, and suberanilohydroxamic acid (SAHA) on osteosarcoma cell lines HOS, MG-63, MNNG, and ZK-58. SAHA was the only chemical agent that exerted a strong, growth-limiting effect in all cell lines tested. The growth-limiting effect of SAHA was accompanied by features characteristic of apoptotic death. We found that cotreatment with SAHA and cisplatin showed strong synergism in all cell lines. The effect of cotreatment with SAHA and doxorubicin was cell line dependent. In the cell lines HOS, MG-63, and MNNG, the combined effect was synergistic, whereas in the cell line ZK-58, SAHA antagonized doxorubicin. The strong synergism of SAHA indicated that in combination with cisplatin, it might enable a promising add-on to current therapy regimens. However, considering the cell line-dependent effect that was found when SAHA was combined with doxorubicin, further experimentation is needed.

The ENCCA-WP7/EuroSarc/EEC/PROVABES/EURAMOS 3rd European Bone Sarcoma Networking Meeting/Joint Workshop of EU Bone Sarcoma Translational Research Networks; Vienna, Austria, September 24-25, 2015. Workshop Report.

This report summarizes the results of the 3rd Joint ENCCA-WP7, EuroSarc, EEC, PROVABES, and EURAMOS European Bone Sarcoma Network Meeting, which was held at the Children's Cancer Research Institute in Vienna, Austria on September 24-25, 2015. The joint bone sarcoma network meetings bring together European bone sarcoma researchers to present and discuss current knowledge on bone sarcoma biology, genetics, immunology, as well as results from preclinical investigations and clinical trials, to generate novel hypotheses for collaborative biological and clinical investigations. The ultimate goal is to further improve therapy and outcome in patients with bone sarcomas.

Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal ROR1 as a potential therapeutic target in metastatic disease.

Receptor tyrosine kinases (RTKs) have provided molecular targets for the development of novel, prognosis-improving agents in many cancers; however, resistances to these therapies occur. On the cellular level, one resistance mechanism is attributed to functional RTK redundancies and compensatory cross-signaling, leading to perception of RTKs as signaling and target networks. To provide a basis for better exploitation of this network in Ewing sarcoma, we generated comprehensive qPCR gene expression profiles of RTKs in Ewing sarcoma cell lines and 21 untreated primary tumors. Key findings confirm broad-spectrum RTK expressions with potential for signaling redundancy. Profile analyses with regard to patient risk-group further revealed several individual RTKs of interest. Among them, VEGFR3 and TIE1 showed high-level expressions and also were suggestive of poor prognosis in localized tumors; underscoring the relevance of angiogenic signaling pathways and tumor-stroma interactions in Ewing sarcoma. Of note, compared to localized disease, tumors derived from metastatic disease were marked by global high-level RTK expressions. Nine individual RTKs were significantly over-expressed, suggesting contributions to molecular mechanisms of metastasis. Of these, ROR1 is being pursued as therapeutic target in leukemias and carcinomas, but un-characterized in sarcomas. We demonstrate expression of ROR1 and its putative ligand Wnt5a in Ewing sarcomas, and of an active ROR1 protein variant in cell lines. ROR1 silencing impaired cell migration in vitro. Therefore, ROR1 calls for further evaluation as a therapeutic target in metastatic Ewing sarcoma; and described as a pseudo-kinase with several isoforms, underlines these additional complexities arising in our understanding of RTK signaling networks.

Gene Expression Profiling of Ewing Sarcoma Tumors Reveals the Prognostic Importance of Tumor-Stromal Interactions: A Report from the Children's Oncology Group.

Relapse of Ewing sarcoma (ES) can occur months or years after initial remission and salvage therapy for relapsed disease is usually ineffective. Thus, there is great need to develop biomarkers that can predict which patients are at risk for relapse so that therapy and post-therapy evaluation can be adjusted accordingly. For the current study we performed whole genome expression profiling on two independent cohorts of clinically annotated ES tumors in an effort to identify and validate prognostic gene signatures. ES specimens were obtained from the Children's Oncology Group (COG) and whole genome expression profiling performed using Affymetrix Human Exon 1.0 ST arrays. Lists of differentially expressed genes between survivors and non-survivors were used to identify prognostic gene signatures. An independent cohort of tumors from the Euro-Ewing cooperative group was similarly analyzed as a validation cohort. Unsupervised clustering of gene expression data failed to segregate tumors based on outcome. Supervised analysis of survivors vs. non-survivors revealed a small number of differentially expressed genes and several statistically significant gene signatures. Gene specific enrichment analysis (GSEA) demonstrated that integrin and chemokine genes were associated with survival in tumors where stromal contamination was present. Tumors that did not harbor stromal contamination showed no association of any genes or pathways with clinical outcome. Our results reflect the challenges of performing RNA-based assays on archived bone tumor specimens. In addition, they reveal a key role for tumor stroma in determining ES prognosis. Future biologic and clinical investigations should focus on elucidating the contribution of tumor:microenvironment interactions on ES progression and response to therapy. Key words: ES, gene expression profiling, prognostic signature.

Suberoylanilide hydroxamic acid synergistically enhances the antitumor activity of etoposide in Ewing sarcoma cell lines.

Ewing sarcomas (ES) are highly malignant tumors arising in bone and soft tissues. Given the poor outcome of affected patients with primary disseminated disease or at relapse, there is a clear need for new targeted therapies. The HDAC inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA, Vorinostat) inhibits ES tumor growth and induces apoptosis in vitro and in vivo. Thus, SAHA may be considered a novel treatment. However, it is most likely that not a single agent but a combination of agents with synergistic mechanisms will help improve the prognosis in high-risk ES patients. Therefore, the aim of the present study was to assess a putative synergistic effect of SAHA in combination with conventional chemotherapeutic agents. The antitumor activity of SAHA in combination with conventional chemotherapeutics (doxorubicin, etoposide, rapamycin, topotecan) was assessed using an MTT cell proliferation assay on five well-characterized ES cell lines (CADO-ES-1, RD-ES, TC-71, SK-ES-1, SK-N-MC) and a newly established ES cell line (DC-ES-15). SAHA antagonistically affected the antiproliferative effect of doxorubicin and topotecan in the majority of the ES cell lines, but synergistically enhanced the antiproliferative activity of etoposide. In functional analyses, pretreatment with SAHA significantly increased the effects of etoposide on apoptosis and clonogenicity. The in-vitro analyses presented in this work show that SAHA synergistically enhances the antitumor activity of etoposide in ES cells. Sequential treatment with etoposide combined with SAHA may represent a new therapeutic approach in ES.

Anchorage-independent growth of Ewing sarcoma cells under serum-free conditions is not associated with stem-cell like phenotype and function.

Novel treatment strategies for Ewing sarcoma aim to eliminate residual tumor cells that have maintained the capacity to reinitiate tumor growth after intensive conventional therapy. Preclinical models that more closely mimic in vivo tumor growth than standard monolayer cultures are needed. Sphere formation under anchorage-independent, serum-free conditions has been proposed to enrich for cells with tumor-initiating, stem cell-like properties in various solid cancers. In the present study, we assessed the phenotype and functional stem cell characteristics of Ewing sarcoma spheres. Spheres were generated under serum-free culture conditions from four Ewing sarcoma cell lines and four relapse tumor biopsies. Standard monolayer cultures were established as controls. Median levels of surface expression of the Ewing sarcoma marker CD99 as well as the supposed stem cell marker CD133 and the neural crest marker CD57 were comparable between spheres and monolayers. Ewing sarcoma spheres from individual tumors failed to continuously self-renew by secondary sphere formation. They contained variable proportions of side populations (SPs). Sphere culture did not enhance the in vivo tumorigenicity of Ewing sarcoma cells in a murine xenograft model. We conclude that sphere formation under serum-free conditions is not a reliable tool to enrich for cells with stem cell characteristics in Ewing sarcoma. By mimicking the anchorage-independent, multicellular growth of Ewing sarcoma micrometastases, in vitro sphere growth may still add value as a preclinical tool to evaluate the efficacy of novel therapeutics.

The first European interdisciplinary ewing sarcoma research summit.

The European Network for Cancer Research in Children and Adolescents (ENCCA) provides an interaction platform for stakeholders in research and care of children with cancer. Among ENCCA objectives is the establishment of biology-based prioritization mechanisms for the selection of innovative targets, drugs, and prognostic markers for validation in clinical trials. Specifically for sarcomas, there is a burning need for novel treatment options, since current chemotherapeutic treatment protocols have met their limits. This is most obvious for metastatic Ewing sarcoma (ES), where long term survival rates are still below 20%. Despite significant progress in our understanding of ES biology, clinical translation of promising laboratory results has not yet taken place due to fragmentation of research and lack of an institutionalized discussion forum. To fill this gap, ENCCA assembled 30 European expert scientists and five North American opinion leaders in December 2011 to exchange thoughts and discuss the state of the art in ES research and latest results from the bench, and to propose biological studies and novel promising therapeutics for the upcoming European EWING2008 and EWING2012 clinical trials.