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EURO EWING Consortium – International Clinical Trials to Improve Survival from Ewing Sarcoma

Final Report Summary - EEC (EURO EWING Consortium – International Clinical Trials to Improve Survival from Ewing Sarcoma)

Executive Summary:
Ewing sarcoma (ES) is a fatal, rare bone cancer particularly affecting young people. The EURO EWING Consortium (EEC) aimed to improve survival from ES by collaboration between clinicians, scientists and patients from across Europe to carry out investigator-driven, inclusive clinical trials underpinned by translational research. This was performed under strict ethical and governance procedures to protect the safety of all patients taking part.

The EEC has carried out a randomised phase III trial for newly-diagnosed ES patients, Euro Ewing 2012, and has shown that VDC/IE chemotherapy is superior to VIDE. These data have been shared with professional and patient experts and will positively influence the care and outcome for ES patients across Europe and beyond as it has provided robust evidence for the best standard of care for newly-diagnosed patients. The speed at which the results were obtained in such a rare disease, despite the challenges of bureaucratic hurdles, was due to the high level of collaboration and engagement across European institutions made possible by the establishment of the EEC.

The Consortium also carried out the first ever randomised phase II/III trial for ES patients with cancer that has recurred or not responded to treatment, the rEECur trial, which is comparing four different types of chemotherapy and using an innovative trial design. Targeted therapies are being planned to replace the other treatments once they have met their recruitment targets. This has demonstrated the flexibility and efficiency of the Multi-Arm, Multi-Stage (MAMS) trial design. The trial has facilitated new levels of engagement with pharma, especially valuable in such a rare disease. Funding has been awarded to continue recruitment beyond the period of EU funding.

Biospecimens from patients with ES are a valuable resource and necessary for carrying out research that will benefit future patients. The EEC has been extremely successful in both obtaining consent from patients to use their blood and tumour samples for future research and putting into place the procedures and resources to collect biospecimens. For example, the Consortium has exceeded typical levels of fresh frozen tumour collection (tissue is collected from less than 10% of patients in most studies) by collecting 182 fresh frozen tumour samples (29.5%). In addition, 11 tissue microarrays have been constructed from tumour samples of patients taking part in the Euro Ewing 2012 trial and these will be used by EEC Partners to carry out research projects directed towards identification of new treatments for ES.

Translational research activity has been very productive with 20 scientific publications arising from the EEC project. These papers include work on biomarkers with the potential to predict outcome for patients and to detect recurrence of the cancer. Studies on samples from patients have clarified the best ways in which to collect and store samples and the most appropriate sample types for measuring biomarkers.

Networking has been important to the success of the EEC in maintaining and building relationships and to develop collaboration both within and outside of the Consortium. This has been supported through well-attended networking meetings held twice a year, communication by email and social media and a dedicated website which provided up to date information for both Partners and the public. Partners have been very active in sharing information generated by the project through spoken presentations and posters at meetings and conferences, and through peer-reviewed publications.

Patient advocacy has been integral to the EEC, from the application stage and throughout the project. A core group of patient advocates have been regular attendees at EEC meetings and have made valuable contributions to discussions and decisions. They have provided highly-valued input into the early stage development of a clinical trial as well as the preparation of patient information on the closure of the Euro Ewing 2012 trial and reviewing applications for both translational research funding and for requests to access the Euro Ewing 2012 tissue microarrays

Project Context and Objectives:
The goal of the EURO EWING Consortium (EEC) is to improve survival from Ewing sarcoma (ES). ES are fatal, rare bone cancers particularly affecting young people. About 60% of patients achieve long-term survival with current treatment but there has been little improvement in this proportion for 25 years. Of the 600 new cases of ES occurring in the EU each year, less than half will receive treatment ‘appropriate’ to deliver the best outcome.

The EEC is a coalition of the most active clinicians and scientists working on ES in Europe. Improved survival will be achieved through an integrated programme of investigator-driven, inclusive clinical trials that are rigorously designed, conducted, analysed and reported, and underpinned by complementary embedded translational research. These include i) a first line randomised study which defines standards of care to prevent development of metastases and serves as a backbone for implementation of new agents and ii) a randomised study of current second line chemotherapy which will serve as a platform for testing of new agents. Translational research will be performed investigating tumour biology and underlying causes of differential response and toxicity. The project will be supported by new initiatives for the involvement of patients in research.

A strategically-directed clinical trials programme is necessary given that ES is a very rare cancer of which 85% of cases occur before 30 years and is curable in some through delivery of highly specialised, complex and morbid multi-modality therapies. The age-standardised incidence from national population-based registries is approximately 2.5 per million, indicating about 600 new cases in the European Union annually, of whom no more than 30% are estimated to have been included at diagnosis in previous clinical trials. Population cancer registry data indicate that survival is around 55% at five years with considerable geographical variation within Europe. Survival of selected groups reported in clinical trials may considerably exceed this. For those with advanced metastatic disease, long-term survival is achieved in only a very small proportion.

There are numerous uncertainties surrounding the optimum clinical management of ES. Whether chemotherapy regimens are equal with regard to survival and toxicity is unclear due to the absence of comparative clinical trials. Studies evaluating new therapeutics have been few and none have yet been carried forward as components of standard adjuvant regimens. If ES recurs after adjuvant treatment, survival rates with further treatment are less than 20%. Again, few trials have been undertaken in this subgroup to inform about better approaches to management.

There is a strong record of conducting ES trials in Europe by individual clinical study groups. However, these have been limited to studies of adjuvant treatment, and accrual rates to randomised questions are slow so the overall impact on practice is insufficient to expect major advances in survival in acceptable timescales. The EEC represents a culmination of effort by several study groups who now embark on wider and more ambitious collaboration designed to deliver more rapid patient-centred outcomes from this often fatal disease.

3.2 Project objectives

The objectives of the EEC are to:
1. Undertake an investigator-initiated first line randomised trial in patients of all ages with ES which defines standards of care for adjuvant chemotherapy to prevent development of metastases and increase survival
2. Undertake an investigator-initiated randomised trial of current second line chemotherapy in patients of all ages with ES which establishes a standard and will serve as a platform for testing of new agents
3. Undertake comprehensive translational and other companion studies which investigate underlying causes of differential response and toxicity, as well as circulating biomarkers in conjunction with systemic treatments
4. Disseminate all emerging findings in conjunction with EEC partners and external agencies and to use these for development of further strategies to improve knowledge and outcomes from ES
5. Introduce public and patient involvement into the work of EEC

Project Results:
4.1 WP1 - Euro Ewing 2012 – First line randomised trial of adjuvant therapy
1.1 Objectives
To undertake an international randomised phase III trial of first line chemotherapy in newly diagnosed ES patients to determine the best standard of care. The Euro Ewing 2012 trial included two randomisations. Randomisation 1 (R1) compared the VIDE strategy (VIDE induction and VAI/VAC consolidation) with the VDC/IE strategy (compressed VDC/IE induction and IE/VC consolidation). Randomisation 2 (R2) aimed to determine whether the addition of zoledronic acid to consolidation chemotherapy, as assigned at R1, was associated with improved clinical outcome in good responders.

4.1.2 Task 1.1 Trial set up
A Trial Management Group (TMG) was set up and meetings have been held face to face twice a year at EURO EWING Consortium (EEC) meetings and via teleconference in between. The sponsor, UoB, provided the necessary trial documents (e.g. protocol, patient information sheets and consent forms) required to enable the National Coordinating Centres (NCCs) in Partner countries to obtain regulatory approvals. The NCCs then adapted the patient information sheet and consent forms according to local requirements (including translation) and developed country specific trial management plans for submissions to the applicable regulatory bodies. Each NCC identified suitable sites within its country or group to take part in the trial. UoB provided each NCC with example documents for use in producing an investigator site file and pharmacy file, including a pharmacy manual and laboratory manual. UoB provided training to the NCC main contacts and national coordinating investigators, before they were activated, on the trial design, trial procedures and their responsibilities. Each NCC subsequently trained their participating sites (site initiation) and entered into a Clinical Trial Agreement with them. The process of final site activation was controlled by UoB which provided access to the electronic remote data capture (eRDC) system for randomisation and data entry.

UoB developed and maintain the eRDC and deal with any problems or queries from sites or NCCs regarding this system. UoB also developed other trial documentation and procedures, including the pharmacovigilance processes, data management and quality assurance documents, and distributed them to the NCCs, as detailed further below in tasks 1.3 and 1.4.

Euro Ewing 2012 opened in all of the three planned NCCs. Aside from those included in this grant, one additional NCC with one site in Dublin, Ireland also opened. Ten countries in total are active (Belgium, Czech Republic, Denmark, France, Hungary, Netherlands, Republic of Ireland, Spain, Switzerland and UK) (Figure 1) and the trial was opened in 112 sites.
Many challenges were encountered during set up resulting in delays to the opening of the trial in countries. Despite the EU Clinical Directive, there is still a lack of uniformity in the process to open trials across Europe and this caused delays in opening Euro Ewing 2012 across the partner countries.

It took longer than anticipated for each country to open due to the complex nature of activating a single trial across multiple countries in which each has separate ethical and competent authorities and country-specific regulations/processes. The delays in set-up were firstly due to the time taken to negotiate contracts between sponsor and NCC, and secondly the length of time taken to prepare the documents for submission in each country. These delays in set-up resulted in delays in gaining ethical and competent authority approvals across all countries.

The EEC has learned from the experience of setting up the Euro Ewing 2012 trial. The NCC model is preferred by most participating countries over the co-sponsorship model. Draft protocols and documents should be sent to partner countries while waiting for approval in the sponsor country so that work can be begun on processes such as translating documents and checking feasibility of the protocol in that country. For future trials, in addition to feasibility studies, discussions could be held at an early stage with key members of clinical and trial administration teams for each country to identify potential issues, such as sponsorship models or funding for drugs. While finding the person with suitable knowledge within each country is a challenge, this should be easier now that collaboration is in place through Euro Ewing 2012 and rEECur.

There is a need to plan realistic recruitment targets and to recognise how long it takes to set trials up to work within established networks and understand the processes within them e.g. regulatory idiosyncrasies. Other important measures are to understand national patient pathways, to pre-identify disease-specific national investigators and to pre-agree contract clauses. The European Cancer Drug Development Network work for Childhood Cancer, part of the ITCC, is developing standardised practices for trial set up and management. They have already generated sponsor-NCC agreement templates, protocol guidelines, national pharmacovigilance information and national specific-insurance requirements. In addition, a process map for sponsor-NCC sites has been developed. The EEC is now a well-established trials network with relevant and experienced sites (across age groups), clinical investigators expertise, leadership in innovative trial design, sponsor/CTU expertise in international trial delivery and multi-stakeholder engagement. It is hoped that all of these measures will help future international ES trials to open more quickly and efficiently.

4.1.3 Task 1.2: Recruitment
The target was to recruit at least 600 patients to R1. Euro Ewing 2012 was activated in the UK in December 2013 and the first patient was recruited in March 2014. The planned recruitment period was five years and, at R1 closure on 3 May 2019, a total of 640 patients had been recruited, and this was both on time and to target (Table 2). Recruitment to R2 is still continuing for patients on R1. R2 recruitment is currently at 97% of target for this stage, with 273 patients randomised to date. Figure 2 and Figure 3 below show R1 and R2 cumulative recruitment respectively. Recruitment to R1 exceeded the monthly target for all but two months over the previous two and a half years.

4.1.4 Task 1.3 Data management
UoB has overall responsibility for the data quality and creates data clarification forms to query missing, discrepant or outlying data and generates reminders for outstanding case report forms which are sent to sites on a monthly basis. Data return overall is currently at 92% which UoB consider to be very good.

4.1.5 Task 1.4 Regulatory compliance
UoB is responsible for the pharmacovigilance reporting and has developed the relevant documentation for this (i.e. Serious Adverse Event (SAE) forms and instructions).

4.1.6 Task 1.5 Interim analysis
Interim analyses of accrual, safety and efficacy have been conducted annually by UoB and supplied in confidence to the independent data monitoring committee (DMC). R1 closed on the 3 May 2019 after the Data Monitoring Committee (DMC) had reviewed the interim analyses and recommended stopping recruitment. This was because the results showed with a high level of certainty that Arm B (VDC/IE chemotherapy) has improved event-free survival and overall survival compared to Arm A (VIDE/VAI/VAC chemotherapy). The decision was communicated to investigators at participating sites via a webinar, and UoB trials unit, with the assistance of the Work Package 1 patient representative, produced letters to patients to inform them of the closure of the trial.

The collaboration of Partners within the Consortium enabled a phase III trial in ES to be carried out in a timely manner and has demonstrated the value of central support for academic international trials in rare cancers. Patients across Europe will benefit from the results of the trial as the best standard treatment for newly-diagnosed ES has been defined. The speed at which the results were obtained in such a rare disease, despite the challenges of bureaucratic hurdles, was due to the high level of collaboration and engagement across European institutions and would not have been possible without EU funding.

4.2 WP2 - Randomised trial of second line chemotherapy – rEECur

4.2.1 Objectives
To undertake the first randomised phase II/III study, rEECur, in patients with recurrent ES or ES that has not responded to treatment, using a multi arm, multi stage (MAMS) trial design, comparing treatments at relapse of ES to evaluate efficacy. The primary aim is to compare the objective response rate and progression free survival of the treatments. The trial will also compare overall survival, toxicity, quality of life and patient satisfaction and time spent in hospital, between the different treatments.

4.2.2. Task 2.1: Trial set-up
The University of Birmingham (UoB) is the Sponsor of the rEECur trial and is responsible for the overall coordination of both the UK and international NCCs. UoB provided the necessary trial documents (e.g. the protocol, patient information sheets and consent forms) in order to enable the NCCs to obtain regulatory approvals in their countries. The NCCs adapted the patient information sheets and consent forms in order to comply with the local requirements, (including translation). UoB provided each NCC with example documents for use in producing investigator site files and pharmacy files, including a pharmacy manual.

UoB provided training on trial design, trial procedures and their responsibilities to the NCC main contacts and national coordinating investigators before they were activated. These initiation sessions offered an opportunity for the NCC personnel to discuss the protocol and trial processes and ask any questions before opening the trial. Each NCC trained their participating sites, (site initiation), and entered into a Clinical Trial Agreement with them.

UoB also oversaw the process of site activation and provided access to the electronic remote data capture (eRDC) system for randomisation and data entry. UoB developed trial documentation and procedures, including the pharmacovigilance processes, data management and quality assurance documents and distributed them to the NCCs.

There was a total of 176 sites planned to open the rEECur trial. All NCCs have received required approvals and been activated. To date 136 sites have opened to recruitment, across 16 countries and eight NCCs (Table 3). There are sites in Netherlands, Sweden, Germany, Australia and New Zealand still in the process of set-up.

As described for the Euro Ewing 2012 clinical trial, it has taken longer than anticipated for each country to open due to the complex nature of activating a single trial across multiple countries in which each has separate ethical and competent authorities and country specific regulations/processes. In addition to the initial time taken to agree a final version of the protocol, the delays in set-up have been due to three principal factors: i) time taken to negotiate contracts between Sponsor and NCC; ii) time taken to prepare the documents for submission in each country; and iii) some national regulatory bodies have identified issues which have delayed approval (these issues have been different in each country and none have fundamentally altered the trial protocol).

A major achievement of the past year has been opening rEECur in Germany. The situation in Germany has been complex due to prolonged issues with gaining ethical approval. After initial submission in July 2015, the German competent authority issued approval, but the ethics committee raised queries. There was a long period of dialogue between the ethics committee and the sponsor. Some of the issues were based on statistical queries that required further statistical modelling to be carried out. The outcome of this correspondence was that the ethics committee were not satisfied and issued a final rejection letter in December 2016. The Chief Investigator contacted the ethics committee in January 2017 to discuss the matter further. An amended protocol was agreed and received approval from the German ethics committee in April 2018.

4.2.3 Task 2.2 Recruitment
The initial aim of the trial, assuming that two arms were dropped and two taken forward to a phase III study, was to recruit a minimum of 525 patients. Recruitment is behind target due to the challenges described in Task 2.1 but has improved as additional countries and sites open. 342 patients have been randomised to the trial (Table 4). Although this is behind the recruitment target (Figure 4), rEECur is the first and largest international, randomised trial in recurrent and refractory ES. As there has never been a randomised trial in recurrent Ewing sarcoma, an accurate target accrual figure was not possible to predict. Monthly recruitment has been variable, but on average, seven patients have been recruited per month for the last two years. Opening new centres is crucial to achieving stable, high recruitment and this remains a priority and recruitment is expected to improve as the remaining international sites open.

4.2.4 Task 2.3: Data management
UoB has overall responsibility for the data quality and creates data clarification forms to query missing, discrepant or outlying data and generates reminders for outstanding case report forms which are sent to sites on a monthly basis. Data return overall is currently at 93% which UoB consider to be very good.

4.2.5 Task 2.4 Regulatory compliance
UoB is responsible for the pharmacovigilance reporting and has developed the relevant documentation for this (i.e. Serious Adverse Event (SAE) forms and instructions). UoB and the NCCs ensure regulatory approval (competent authority and ethical) for the trial is obtained and maintained in the relevant countries. In total four protocol amendments have been submitted, the most recent being protocol version 5.0 that is approved for use at all sites. Amongst other changes, protocol 5.0 includes optional consent for circulating biomarker and tumour biological studies on patient samples, developed in conjunction with WP3 and WP4.

4.2.6 Task 2.5: Interim analysis
Interim analyses of accrual, safety and efficacy are conducted annually by UoB and supplied in confidence to the independent Data Monitoring Committee (DMC). The reports are presented by UoB and the Chief Investigator at these meetings and so far the DMC has recommended continuation of the trial at every meeting.

The first interim analysis, which was due once 50 patients had been recruited to each arm and had been assessed for the phase II primary outcome, concluded on 14 November 2018. One of the four arms (Ifosfamide) had not yet recruited 50 patients at that point. However, in accordance with the trial protocol, analysis was undertaken when the remaining three arms had recruited at least 50 evaluable patients. Based on the data provided in closed sessions by the trial statistician, the DMC made the recommendation to cease recruitment to the Gemcitabine and Docetaxel (GD) arm and this was communicated with the Trial Steering Committee (TSC). The TSC agreed with this recommendation and communication was sent out to all sites from the Chief Investigator that the GD arm was closed to recruitment. There were no safety concerns. More detailed data will be presented when all patients recruited to the GD arm have been followed up and their data analysed. The data was presented to an international audience by Dr Martin McCabe at the ASCO conference in Chicago in June 2019.

The second interim assessment took place in July 2019. The DMC was unable to make a recommendation that one arm was sufficiently worse than the other two arms, judged by either RECIST response (the primary outcome) or progression-free survival (a secondary outcome). Recruitment to the three arms therefore continues.

The EEC has demonstrated that it is possible to set up and recruit to an international randomised trial for patients with relapsed or recurrent disease in a rare cancer. This is a group of patients for whom the best standard of care is not yet known and the rEECur trial is making progress towards finding an answer to this question.

4.3 WP3 - Biobanking, reference pathology and molecular characterisation

4.3.1 Objectives
The objectives of WP3 were to collect high quality biospecimens from patients recruited to the Euro Ewing 2012 trial through standardised procedures established by the EEC, to carry out pathology central review on tumour samples, to construct tissue microarrays (TMAs) and to perform molecular characterisation.

4.3.2 Task 3.1: Harmonisation of biospecimen collection, storage, distribution and virtual tumour bank
Novel Standard Operation Procedures (SOP) updated for specific needs within the Consortium were established and distributed amongst the participating centres to enable prospective biobanking, not only for use in the Euro Ewing 2012, rEECur and EWING 2008 studies but also for future applications. They provide guidance on the collection of the following sample types: (i) frozen tumour material, (ii) blood in PAX-gene™ tubes and (ii) bone marrow in PAX-gene™ tubes.

Sample collection from Euro Ewing 2012 and latterly rEECur trial participants has been very successful with 5047 and 849 biospecimens collected respectively. Information from previous projects indicates that less than 10% of patients had frozen tumour collected and available for research. The EEC project has been successful in significantly exceeding this level to 30% (182 fresh frozen samples collected from 616 patients). Collection of other biospecimen types has also been very effective (Table 5 Number and types of samples recorded as being collected by randomisation stage from patients participating in Euro Ewing 2012Table 5).

During period 2, the protocol for the rEECur trial was amended to include consent for the collection of samples from patients taking part in the trial. They are a valuable resource as biospecimens from patients with relapsed ES are scarce. Samples are being collected from patients in the UK, Belgium, France, Hungary, Czech Republic, the Netherlands, Spain, Denmark, Australia, New Zealand and Switzerland and additional funding is being sought for analysis of the samples. The number of samples collected by each country is summarised in Table 6

To enable the EEC to perform the studies outlined in WP3 and WP4, and to audit the number of samples being collected within the EEC, details of tumour biomaterial were required to be uploaded onto a virtual biobank. To facilitate this process, the existing EuroBoNet biobank was updated to include an EEC-specific link within the infrastructure of the EuroBoNet/Tubafrost programme, (http://eurobonet.pathobiology.eu/).

During period 2, issues with the virtual biobank, EuroBoNeT were identified. There was a conflict between the security and the internet provider and then issues with compliance with the GDPR which could only be overcome by more funding than had been allocated. UoB as trial sponsor continued to hold information that was transferred from the EuroBoNeT application to Conticanet (a French database and virtual biobank) on samples within their own database. The final conclusion of EMC was that the budget reserved within EEC for a database biobank application was not sufficient to sustain an existing application over time.

Discussions were held between the CRCTU, WP3 and 4 leaders and staff from Conticanet to determine whether this system would be suitable for EEC samples. Conticanet is a secure database in France that is able to hold clinical and sample data and has long-term funding
https://cordis.europa.eu/project/rcn/78400/factsheet/en. Sample data from previous collections on the EuroBoNeT database were transferred to Conticanet to ensure compliance with the GDPR and Partners at EMC are keeping the EEC informed on progress with this. If a minimal dataset can be agreed on, Conticanet may provide a long-term solution to making biospecimens accessible to researchers outside of the EEC but there are issues around the sharing of clinical information, which to date have prevented this.

In the meantime, UoB as sponsor is responsible for data on the collected samples and have a comprehensive catalogue of samples collected with a process developed for access to these. Any researchers contacting the EEC who wish to access them will be directed towards the CRCTU (UoB) who will enable them to apply through the EEC Sample Sharing Committee (SSC). The SSC was set up in November 2018 to review applications from researchers to use biospecimens collected as part of the Euro Ewing 2012 and rEECur trials. The membership includes CRCTU staff, the Chief Investigators of each trial and EEC Partners.

In March 2019, a call to EEC Partners was announced to invite applications for use of Tissue Microarrays (TMAs) constructed from tumour collected from Euro Ewing 2012 trial participants, a limited and valuable resource. Five applications were received. As several members of the SSC were also applicants, with potential conflicts of interest, the EEC External Advisory Board and the WP3 patient representative reviewed the applications and ranked them according to priority.

4.3.3. Task 3.2: Reference pathology. Central review of cases is mandatory in clinical trials
A reference pathology panel has been established to ensure the availability of high-quality tumour specimens for in-depth molecular characterisation. Tumours are diagnosed using the 2013 WHO classification and immunophenotypic and molecular diagnostic studies are performed where necessary to ensure diagnostic accuracy.

To date, central review has been carried out on samples from 188 trial participants from the UK by the pathologist at the Royal National Orthopaedic Hospital, London. As 242 patients were recruited to the trial, this represents 78% of the total. Immunohistochemistry and molecular data is available for over 90% and 99% respectively, of the patients that have had central review. 99 patient samples (67%) have been centrally reviewed from the total 148 Euro Ewing 2012 trial participants in Spain. All of them have been confirmed with immunohistochemistry and FISH and/ or NGS molecular analysis. Four more other samples are currently being reviewed.

Central review for patients participating through EORTC countries is carried out by Partner 7 (LUMC). 29 cases have been reviewed: one was BCOR-CCNB3, two were CIC rearranged sarcomas, and one was a myoepithelial carcinoma. Of the remainder, molecular confirmation was not possible for five cases. Immunohistochemistry was carried out for all patients. 51 patients were recruited to the trial and so central review was performed on 57%.

4.3.4 Task 3.3: High quality tumour biomaterial
Collection of high-quality fresh tumour biomaterial has continued and is stored at local institutions according to the SOPs generated in Task 3.1. Details of these samples are stored by UoB.

4.3.5 Task 3.4: Tissue microarray
Six tissue microarrays (TMAs) have been constructed by the Royal National Orthopaedic Hospital NHS Trust, London, using tissue from 154 UK patients. In France, samples from 32 patients have been received for a TMA and production will begin shortly. Five TMAs were constructed in Spain using samples from 97 patients with a total of 11 across all Partners providing a unique resource in a such a rare malignancy. These TMAs will be used for the projects reviewed by the EEC Sample Sharing Committee.

4.3.6 Task 3.5: Depth characterization of tumour samples
Depth characterisation of tumour samples has been successful, and Partners have contributed to 16 publications in peer-reviewed journals. Molecular characterisation of tumour samples included genomic (whole genome sequencing, whole exome sequencing, SNP-arrays and target sequencing of recurrent mutations/alterations,), transcriptomic (RNA-Seq) and epigenomic based sequencing methods (whole genome and reduced representation bisulfite sequencing, ChIP-Sequencing).

These approaches allowed Partners to decipher the genomic landscape of ES and highlighted recurrent secondary alterations such as STAG2, TP53 mutations and CDKN2A deletions. Importantly, these alterations were also shown to be of poor prognosis for these patients.

The study of ES-CSC genes was evaluated in genes by Partner 16 (univleeds) and a paper has been submitted to Cancer Discovery. IOR performed exploratory analysis on well-characterized patient samples from primary localized tumours and from metastases focusing on the evaluation of the possible prognostic role of IGF2BP3. A manuscript is in preparation.

4.4 WP4 - Biomarkers of response, toxicity and outcome

4.4.1 Objectives
The aim of WP4 was to examine the miRNA profile in bone marrow aspirates, whole blood and plasma taken at diagnosis to identify the most informative compartment and minimal predictive miRNA signature (in that compartment) of toxicity, response and outcome. A second objective was to quantify the level of EWS-ETS circulating tumour DNA (ctDNA) in plasma at diagnosis, during treatment and on follow up and thirdly to measure the level of EWS-ETS DNA in the cellular fraction of blood to detect circulating tumour cells (CTCs) at diagnosis, and during treatment.

4.4.2 Task 4.1: miRNAs
The identification of circulating biomarkers that reflect tumour biology and patient response will be important tools to increase understanding of ES. miRNAs are short non-protein coding RNAs that post-transcriptionally regulate gene expression. Profiles of miRNAs can classify human cancer types and specific miRNAs are packaged in exosomes to be released into blood. Importantly the miRNAs in blood are stable and can be detected by RTqPCR where they may provide a non-invasive method to predict outcome and monitor evolution of disease in real time. It was found that there was greater heterogeneity of miRNAs detected in plasma (n=66) compared to miRNAs in whole blood (n=60) or bone marrow aspirates (n=50) (Figure 5). This study has demonstrated the feasibility of sample collection for circulating miRNA studies in multiple centres across different countries. Using a penalised logistic regression model/ penalised cox model, to analyse pilot data on the expression/no-expression of miRNAs in blood, plasma and bone marrow samples from 60 patients with Ewings sarcoma four miRNAs that predict febrile neutropenia were identified (univleeds). Univleeds is currently seeking funding and complete toxicity information to validate these initial findings in a larger study cohort.

Sufficient samples that are fit-for-purpose have been collected and analysed to provide pilot information showing that miRNA profiles by sample type identify different patient groups. It has not been possible to evaluate the predictive value of miRNAs in blood samples as the clinical information is at present incomplete and does not include the date that patients were last seen or any of the follow up data fields. However, we have collected and analysed sufficient samples fit-for-purpose to provide pilot information once the clinical information is available. The outputs from these analyses will be used to generate a customised miRNA panel to analyse the additional samples collected once further funding has been secured.

IOR carried out analysis of miR34a on blood samples and a manuscript is in preparation.

4.4.3 Task 4.2: Circulating tumour DNA
We have successfully demonstrated the feasibility of development and application of patient-specific ctEWSR1-ETS assays in an international trial and identified that there is a challenge of obtaining sufficient samples to achieve statistical power. The main limitation has been the low number of tumour samples collected, since this is required to develop each patient-specific EWSR1-ETS assay. The need to develop a patient specific assay will limit the wider clinical adoption of ctDNA studies of this kind. To overcome the need to design a patient-specific assay to detect ctDNA we have developed an NGS pipeline that requires further validation.

We have optimised and validated SOPs for the collection and analysis of blood and bone marrow samples for EWSR1-ETS fusion transcripts and demonstrated that EWSR1-ETS fusion transcripts are detected in blood samples from patients with ES at diagnosis but not after treatment.

Additional studies using the samples collected from the Euro Ewing 2012 trial have been carried out by Partners. Previous studies by Institute Curie have shown that ES with STAG2 alone or associated with TP53 mutations predict a poor overall survival and STAG2 mutations may be associated with resistance to treatment. Therefore, they have designed an additional study aimed at detecting deleterious mutations in plasma at diagnosis. Unlike for the EWSR1-ETS ctDNA study, where the exact translocation breakpoint must be determined from each tumour and patient-specific probes are required, this new approach uses NGS compatible amplified cfDNA libraries for sequencing either the entire genome or specific regions of interest. NGS libraries have been generated so far from 14 cfDNA sample at diagnosis and sequenced using whole genome sequencing (in collaboration with Dr Eleni Tomazou at CCRI, Vienna, Austria) in a pilot experiment and will be used for the detection of deleterious mutations. In addition, these libraries will be used for target sequencing after the capture of STAG2, TP53 and a panel of other recurrently mutated gene in Ewing sarcoma.

EWSR1-ETS fusion mRNA is a surrogate marker of circulating tumour cells (CTCs) and is an ideal tumour specific molecular marker. It was possible to detect these gene fusions in patient samples at a level of one tumour cell in 33,333 normal cells. Studies have been performed in RNA extracted from whole blood samples collected in PAXgeneTM blood RNA tubes at diagnosis (n=50) and post treatment (n=50). EWSR1-ETS fusion RNA was detected in 2/50 (4%) samples; one sample was positive for EWSR1-FLI1 and one for EWSR1-ERG. These fusions were the same as those identified in paired primary tumour at diagnosis. All 50 post treatment blood samples were negative suggesting clearance of CTCs.

The quality and yield of RNA extracted from the frozen EDTA cellular fraction was reduced compared to that extracted from paired blood collected and frozen in PAXgeneTM blood RNA tubes. Therefore, the outcome of the study is the recommendation of the use of PAXgeneTM blood RNA tubes for collection of RNA from patients. This method has the advantage that it is practical for use in centres without access to -80°C freezers.

4.4.4 Task 4.3: Data analysis
Circulating biomarker studies are embedded into the Euro Ewing 2012 trial and eight countries are participating by collecting samples. Sufficient samples (n=170 for prediction of EFS; n=180 for prediction of toxicity) have been collected to achieve the initial study objectives of identifying circulating miRNAs that predict toxicity and outcomes. Partner 16 (univleeds) is currently seeking funding to analyse the samples to complete these studies exploring the potential of miRNAs as circulating biomarkers.

Additional studies, not described in the Description of Work, have also been carried out. Plasma samples have been transferred to Dr Gareth Veal in Newcastle Upon Tyne, UK for companion studies on pharmacokinetics in Ewing sarcoma. Exosomes have successfully been isolated from pre-filtered EDTA plasma samples collected from patients with ES participating Euro Ewing 2012; the identification of exosomes has been confirmed by expression of exosomal proteins (TSG101, CD63) using western blot and flow cytometry, size using the nanosite and ultra-structurally by electron microscopy. The content of ES derived exosomes has been analysed using RNA sequencing and novel candidate RNA biomarkers of risk identified (univleeds).

4.5 WP5 - Dissemination and Patient and Public Involvement (PPI) in the EEC

4.5.1 Objectives
The objectives of WP5 were to establish a patient advocacy group together with training and support and to incorporate Patient and Public Involvement (PPI) activity into all future work of the EEC, including dissemination, oversight of activities and strategic priority setting for next generation research. WP5 also included the aim of ensuring rapid, wide and open communication of all aspects of the project.

4.5.2 Task 5.1: Assembling
The EEC has successfully established, maintained and continued to add to a core group of patient advocates (PAs) with an interest in working with Partners on the EURO EWING Consortium. The PAs have become a well-established group and their increase in confidence and knowledge has benefited the EEC greatly. They have provided input into the EEC at meetings and through email discussions as well as contributing towards the planning of a new trial (Figure 6).

It continues to be the case that there are very few of these specialist groups as the bone cancer patient community is very small, due to the rarity of these diseases. However, working with a small group of highly-engaged patient advocates has proven to be a successful model and the EEC has continued to work in this way while still connecting with patients and carers across Europe through SPAEN

4.5.3 Task 5.2: Training and support
A register of PPI partners was created and a training record was also produced and has been regularly updated. Training and support included the provision of lay summaries before meetings as well as workshops dedicated to increasing PA knowledge and understanding. Information about the EEC and ES, written in lay language is available on the EEC website. This includes information about the two clinical trials, the aims of the EEC project and links to other useful resources.

To help the PAs to understand more about the process of running clinical trials, three PAs visited the Cancer Research Clinical Trials Unit (CRCTU) in Birmingham (UoB). They were introduced to the staff and given an overview of the two EEC trials as well as an introduction to statistical analysis, data capture, laboratory analysis and collection and tracking of biological samples. The feedback from the visit was extremely positive and the PAs felt that it had been very beneficial (Figure 7).Other training opportunities included a face to face session using an online tutorial on cancer genetics from John Hopkins University (https://www.coursera.org/learn/cancer). After each section, there was a discussion about what had been seen and the group helped each other to understand the concepts. PAs also took part in a Q&A session with the CI of the Euro Ewing 2012 trial who was also representing the EE20XX trial planning group. She joined the PAs to answer their questions about the results of the Euro Ewing 2012 trial and to update them on progress with the future trial, EE20XX. This enabled them to have a better understanding in preparation for the EE20XX planning workshop later that day.

4.5.4 Task 5.3: Defining priorities
The objective of this task was to identify PPI activities that would be of value to the EEC and the public and to make sure that resources were focused on those activities that had been identified as priority areas. This was achieved by a process of consultation with patient advocates and EEC Partners during the second period of the project.

The top six priorities identified were:
• Improve availability of tissue samples by: lobbying for opt-out consent system for biological studies rather than the current opt-in; lobbying radiologists/ surgeons/ pathologists to increase the amount of frozen tumour available for research and; lobbying the staff involved in tissue collection to prioritise samples for collaborative international research projects rather than local research or banking
• Providing a community voice to influence how EEC should evolve e.g. define the priorities for the next set of trials, help researchers to define appropriate endpoints e.g. survival v. quality of life and contribute to trial protocol development for the next studies at an early stage
• Encourage advocacy groups to help patients by finding out what patients want to know the answers to after they have been diagnosed
• Influence national health authorities e.g. regarding regulatory decisions or marked file authorisations for new drugs
• Link and collaborate with existing resources in each Partner country as well as existing international collaborative projects
• Researchers and lay members working together to generate information for the public on EEC and ES and the trials e.g. country-specific PIS and summaries for local websites

Over the following periods, the EEC carried out many activities guided by these priorities. These included training on bio-banking, oral and poster presentations by a PA at childhood cancer conferences (Figure 8), establishing links between the EEC and ACCELERATE (https://www.accelerate-platform.org/work-programme/ongoing/why-fair-trials/) PA input into the design of the next ES trial (EE20XX) and PA involvement in the drafting of information for patients on the closure of Randomisation 1 of the Euro Ewing 2012 trial.

4.5.5 Task 5.4: Participation
PA representation was introduced to WP1, 2 and 3. In these roles PAs attended TMG meetings and reviewed applications from Partners to use the TMAs from tumour samples collected from participants in the Euro Ewing 2012 and rEECur trials. They also reviewed applications from EEC Partners for a translational call to encourage ancillary studies.

There continues to be positive feedback from EEC Partners on patient advocacy:
“.....the ’touch with reality’ component is very important for the EEC members who don’t deal directly with patients and families, and I think for the functioning of the whole EEC in reminding us to keep the biological focus on what will change practice and cure more patients.” (clinician)
“.......I would like to add that they are a sense check in terms of trial questions – are the researchers questions of as equal importance as those of the PAs? Also if we all get it right then definitely helps the success for funding.” (clinician)
“Please keep asking simple questions because they are really useful and sometimes researchers can miss the obvious.” (trial coordinator)

4.5.6 Task 5.5: Evaluation
Patient advocates were asked to evaluate PPI in the EEC using questions as prompts e.g. Have you gained anything from being involved in the EEC? Work package leaders were also asked to evaluate PPI and the responses from both groups were summarised and analysed.

Overall, PPI in the EEC was considered a major success and beneficial to the project. It was challenging as ES is a rare cancer, and the majority of patients are children or young adults, a group that is difficult to reach and do not necessarily have the time or enthusiasm for PPI. The lack of established patient advocacy support outside of the UK also impacted on engagement across the community.

4.5.7 Task 5.6: Dissemination and Communication Plan
A dissemination and communication plan was produced that explained how information could be disseminated to both internal and external audiences. As an appendix to the plan, a table was created which listed the researchers involved in each work package to help to ensure that the correct individuals were kept informed.

The EEC communicated with and disseminated information to the wider scientific community and the public through channels including email, the EEC website, social media, publications and conferences. The EEC Twitter account was used to give updates on progress and news and now has 452 followers from the sarcoma community. At the end of the EEC project, a Twitter thread of the EEC in numbers was tweeted to highlight the achievements of the project (Figure 9).
A publication policy was produced which provides guidance on the publication of results and was developed based on the requirements set out in the EEC Grant Agreement and EEC Consortium Agreement.

The EEC coordinated the drafting and submission of a paper to Clinical Sarcoma Research entitled “Report from the 4th European Bone Sarcoma Networking meeting: focus on osteosarcoma”. This paper summarises the meeting held alongside the EEC meeting in June 2017 and highlights the extent of collaboration and networking between EEC partners and the wider ES field https://doi.org/10.1186/s13569-018-0103-0.

A poster was presented at the Connective Tissue Oncology Society (CTOS) conference in Rome in November 2018; How do we achieve the greater collaboration needed to improve outcomes from Ewing sarcoma: The experience of the EURO EWING Consortium (Abstract ID: 3042870) (Figure 10).

A film was made about the EEC with many Partners and PAs being interviewed while at the EEC meeting in September 2019. This was an opportunity to record the perspective of the members of the EEC for the future and to showcase the successes and challenges of the project. The aim was to produce a film that would be a lasting legacy of the project, to provide transparency to the public, to share lessons learned with future investigators and to demonstrate the value of funding similar projects in the future. The filming was carried out by the media department at UCL and a first edit was requested by mid-November 2019.

4.5.8 Task 5.7: Website set up and activation
A EEC website was set up that would provide a secure area for information exchange, contact details and provide news updates to Partners. The website was also an important resource for communicating and disseminating information to patients and the public and was central to the communication and dissemination plan.

4.6 WP6 – Ethics aspects of EEC activities

4.6.1 Objectives

The objectives of WP6 were to ensure that standards of ethics for the EEC programme were commensurate with needs of patients and to ensure the highest quality science, to ensure compliance with the ethical framework of FP7 and to demonstrate compliance with national ethical and regulatory approvals.

4.6.2 Main results

The protocols for Euro Ewing 2012 and rEECur were developed with the input from all of the participating Partners and this was a lengthy and involved process due to the complexities of collaborating across different countries. Regulatory and ethical approval was obtained for the Euro Ewing 2012 trial in the UK, Spain, France, Belgium, Czech Republic, The Netherlands, Denmark, Switzerland, Republic of Ireland and Hungary. Regulatory and ethical approvals were obtained for the rEECur trial in the UK, Belgium, Spain, Norway, Finland, Denmark, Italy, France, Hungary, Czech Republic, Poland, the Netherlands, Sweden, Germany, Switzerland, Australia and New Zealand. The challenges identified in opening a multi-national trial are described in WP1 and WP2 and were presented at the final EEC meeting in September 2019 by Professor Pam Kearns (UoB) (Figure 11 and Figure 12. There were no common issues across participating countries that could be identified. There was a major variation in the time taken to submit documentation to authorities whereas the time taken to gain approvals once submitted was typically around three months and within the guide times set by the regulatory and ethical bodies.

An External Advisory Board was set up with a membership that was independent of the EEC. The members were experts in ES and/ or clinical trials and translational research and had experience of collaborative projects. The EAB met approximately twice a year by teleconference. The EAB were provided with reports on the activity of the EEC and gave feedback to EEC Partners via the Project Coordinator. The EAB gave invaluable assistance throughout the project by giving advice on issues and by providing independent review of applications from EEC Partners for both additional translational research funding and use of biospecimens.










Potential Impact:
The primary goal of the EEC was to improve outcomes from ES to be achieved though collaborative working. The Consortium has successfully conducted two clinical trials, involving about 1,000 randomisations, and by doing so has engaged a wide group of clinicians, scientists and patients who have worked together to achieve the goals set at the start of the Consortium’s work.

The EEC has therefore achieved key European stakeholder engagement around common goals which aim to improve outcomes from this rare cancer. This dedicated clinical network is an essential element to conduct successful and timely clinical research and is anticipated to be sustainable beyond the funding period.

The EEC has successfully conducted practice-changing clinical trials in ES. Treatments used for ES in both a first-line and second-line setting have been investigated and evidence provided that informs selection of treatment for individual patients. This evidence will be important in the care of every future patient with ES in Europe.

New knowledge on the biology of ES has been published which enhances understanding of risk-stratification and personalisation. The collection of clinically-annotated biological materials has been carried out on an unprecedented scale and has created an invaluable resource for investigators.

As a focal point for expertise in this rare cancer, the EEC has already attracted approaches from other investigators outside Europe to increase collaboration and from the pharma industry, keen to work with the EEC in new therapeutic developments. Finally, it is a focal point for patients and families wishing to reach expert care or information.

Overall the impact of the EEC is substantial and broad, far beyond the originating Partner institutions and with a clear identification of the need for sustainment of the collaboration in the interests of continuing work towards the founding aim, of more people in Europe being successfully treated for ES.

List of Websites:
https://arquivo.pt/wayback/20170603174702/http://www.euroewing.eu/