• Integration activities
• Jointly executed activities
• Activities to spread excellence
• Management activities
  

The integration activities are split into five work packages:

• WP 5.1 Training and education (lead by AP Dei Tos, Treviso Hospital) is directed at exchanging information within (WP 5.1a) and outside (WP5.1b) the network in the form of courses and scientific meetings. We will plan annual network meetings (for the full duration of the project), which are preferred to coincide with the meeting of the EMSOS (The European Musculo Skeletal Oncology Society). A closed meeting to discuss ongoing and planned activities within EuroBoNeT is planned together with open training sessions, open for everyone, to disseminate data and consensus generated within the network. Specialized training will be organized by several institutes and mostly already ongoing training (WP5.1b). Members EuroBoNeT will inform each other on these courses. Exchange of PhD students and Post-docs will take place to those participants that are have the best proven expertise and possibilities on the required technologies. Further education is followed in a web-based manor.
  
  
• WP5.2 Technology platforms are set up for:
  
  
• Development of novel innovative technology to study bone tumour biology
• Dissemination of technological tools and knowledge within and outside the network
• Connect the different research lines and work packages via horizontal technology platforms
• Standardization of protocols and reagents to compare data from different institutions through a technology platform leader (TPL) for each platform
• Integrated analysis of data using standard statistical and bioinformatics methods

The TP are subdivided in 5 themes: Morphology,DNA analysis, RNA analysis, Protein analysis, and Functional analysis. Technology platform leaders are assigned for each technology platform. New technology platforms will be implemented when necessary and available. If a particular technology is needed but not provided within the network new participants who can provide this expertise (especially SME'S) will be invited to join the network or financial means will be made available by EuroBoNeT to obtain expertise within one or more institutes.

Technology platforms EuroBoNeT

• WP5.3 BioBank. An important issue when dealing with rare tumours is to set up a BioBank to gather information on patient material i.e. available tissue (fresh frozen and fixed), DNA, RNA, protein, serum, and clinical data of the patients. This will facilitate exchange of materials between institutes. At the moment of submitting we are uncertain about the organisation of the BioBank, because discussions are and will be propagated with the leading institutes cooperating in the previously EC granted TUBAFROST consortium, which has set standards for biobanking in Europe (www.tubafrost.org) 1. This already documented knowledge will ensure rapid effectiveness of this work package. The managing institute of TUBAFROST (Erasmus MC, Rotterdam) is part of our NoE and will provide us with their expertise regarding virtual BioBanks, organise and hire personnel required for the establishment and maintenance to the virtual BioBank. With the creation of a BioBank several problems are solved. With the EuroBoNeT put into use the number of samples investigated per study will be enlarged. This will increase the statistical power of the experiments, and in addition we will be able to exclude population bias. At present this exchange of material happens only occasionally1, but with more close collaborations the number of multi-participant studies will increase. The BioBank will contain information on which partner has which tumour available for research by other partners. The samples will remain in the ownership of the original partner and will only be sent to other partner (on loan) when requested. The virtual BioBank will contain information of the type of sample and some clinical data. Sample types could comprise of for example frozen or paraffin tumour sample, serum, cell lines, tumour DNA/RNA and corresponding normal DNA. We hope to collect information of at least 100 tumour samples in the virtual BioBank. Of the cell lines we hope to collect some 25, but this depends on what is available by the partners. We expect that in the first 18 months the bulk of the tumours will be registered from the archival material present with the partners, and in the following years have this number increased in small steps (due to the rarity of these tumours) by registration of newer samples.
  
  
• WP 5.4 The Web committee is directed at developin

• Web based database of ongoing research activities inside the network. Work package leaders are responsible for reporting on the progress of their work package. Research line leaders are responsible for maintaining the database and will guard the contribution of all partners to the work packages.
• Literature alert system to inform all partners on published data or presentations on scientific meetings that may be important for our programme.
• A forum for network related research queries, diagnostic issues.A Webmaster will be hired (by Treviso Hospital) to set up the website. This will contain both open and closed sections.The open section will contain subjects such as general information on the project, web-based discussion forums, newsletters, Standard Operating Protocols and information on courses/conferences. In the closed section the virtual BioBank, information and forms required for reporting to the European committee, etc.

• WP 5.5 Translational Research For dissemination of the data generated within EuroBoNeT we plan to perform efforts for translating results into applicable diagnostic tests. Often it takes a long period of time to implement findings of basic research into the clinic. By implementation of a translational research platform within a consortium that generates the data it may be expected that this time lag be diminished. Within EuroBoNeT validation of diagnostic/prognostic tests can be performed, because a number of participants (no 1, 2, 3, 4, 5, 7, 8, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22 and 23) are involved in clinical practice of Bone tumours. Validation of the possible markers will be performed on archival material, of samples with sufficient follow-up data, following the ethical guidelines.
  
  

Research line 1: Cartilaginous tumours
Chondrosarcomas are the second most frequent bone sarcomas after osteosarcomas. A number (peripheral chondrosarcomas) arise secondarily within a preexisting benign osteochondroma, a tumour that develops at young age during active skeletal growth. The remainder is centrally located of which a subset arises within the context of enchondromatosis, a disabling disease in which patients have multiple enchondromas with a unilateral predominance. These patients are relatively young and have an increased risk (20­30%) of malignant transformation towards central chondrosarcoma. If we can understand the molecular background of malignant transformation of enchondroma and osteochondroma this may lead to identification of new diagnostic and prognostic tools as well as molecular targets for therapy, since so far surgery is the only option. The 2002 WHO definition states that "the term chondrosarcoma is used to describe a heterogeneous group of lesions with diverse morphologic features and clinical behaviour"2. This phrase points exactly to the problem in chondrosarcoma research in the past. The investigations have been hampered by the inclusion of heterogeneous groups involving all different subtypes, making the identification of new diagnostic and prognostic tools as well as molecular targets for therapy extremely difficult.
Integrating activities for RL 1:

• Consensus on sub classification

Especially for chondrosarcoma it is important to carefully characterize and subdivide cases based on clinical, radiological and pathological criteria. Criteria will be based on the 2002 WHO classification3 and will be discussed and agreed upon during the first EuroBoNeT meeting in order for each contributor having the same well defined and homogeneous groups (Central vs. peripheral, Grade I, II and III, sporadic vs. enchondromatosis (central) or EXT (multiple osteochondromas) related tumours) allowing comparison of results (WP1.1). Of the different subtypes we will try to collect at least 10 specimens for research although this may not be feasible for the very rare cases, such as for instance Enchondromatosis samples.

• EXT mutation analysis

For all osteochondromas in patients with two or more lesions, as well as for all peripheral chondrosarcomas arising secondarily in these lesions in order to be included in the project EXT mutation analysis will be performed in a standardized way (see TP 2.2 mutation analysis). This is the only way we can guarantee adequate correlation of results to mutational status (> 400 samples, WP1.3).

• Use and share of technical platforms

By using the same technology platforms according to standard operating procedures (SOPs) it will become possible to combine results obtained on different well characterized smaller series of different contributors in order to increase statistical power. Common bio-mathematical platforms will be achieved in order to enable a direct comparison between gene expression and mutation profiles obtained on the different subgroups in the involved laboratories (WP1.1, WP1.2, WP1.3, WP1.4: DNA/RNA isolation protocols, platform for expression arrays, genomic arrays Real Time PCR, etc).

• Establishment of a BioBank

Since the classification into several subgroups is essential for cartilage tumour research this hampers reaching significant numbers in the separate subgroups for most research groups. This problem has hampered chondrosarcoma research in the past, but can be overcome by sharing and exchanging tumour material (RNA, DNA, tissue, cell lines). This will be accomplished using the web-based virtual database Bio-Bank in order to guarantee the availability of sufficient numbers within the different subgroups


Research line 2: Osteogenic tumours and related sarcomas
Osteogenic tumours are the most predominant tumours among malignant tumours of the skeletal system. Nowadays almost 60% of all patients are long-term survivors with a high quality of life. However, a substantial number of patients still die of the disease. Despite this significant increase of survival it became now obvious over the almost last 20 years that with the former conventional, mainly clinical approach a significant increase of treatment results could not be elaborated by any of the various, multinational study trials anymore. It became clear that clinical approaches would be only of limited help since new therapies mainly depend on an increased understanding of biological mechanisms in this disease. The application for a Network of Excellence for bone tumours takes therefore place in a situation of an international consensus about the urgent need of a tight coordination between clinical efforts and intensified basic biological research activities. In this regard, the Network is a 1:1 complementation and backbone of ongoing clinical studies and is fully supported by these.

• Unified patho-morphological evaluation of newly diagnosed malignant, osteogenic bone tumours The development of new therapies requires common diagnostic procedures and nomenclature in order to characterize the spectrum of osteogenic bone diseases. An expert panel will be defined including reference pathologists from all ongoing clinical studies represented in EURAMOS (WP2.3).
• Establishment of an interdisciplinary working group The working group will contain clinicians from the EURAMOS study and members of the network to achieve consensus about the most urgent questions and transfer of knowledge into a clinical practice. This will include the definition of new research lines, as well as the formulation and introduction of new therapies, including legal and ethical aspects.
• Establishment of a virtual tumour bank The chemotherapeutic and surgical treatment of osteogenic tumours in common takes place in specialized clinical centres. Out of this situation it is obvious that the presence of technical platforms and technical expert knowledge does not necessarily parallel the access to tumour material. It is therefore essential for all members of the working group to guarantee the availability of sufficient numbers and amounts of newly diagnosed osteosarcoma cases.
• Use and share of technical platforms In the era of global gene expression analysis and whole genome mutation analysis, data accumulate rapidly and its interpretation is hampered by the use of different technical platforms. The group found consensus about the use of the Affymetrix platform in the investigation of global gene expression. It will be the aim to develop common bio mathematical platforms in order to enable a direct comparison between gene expression and mutation profiles obtained in the involved laboratories

Research line 3: Osteoclastogenesis and Giant cell tumours of bone
The programme of investigations in RL 3 to study osteoclastogenesis and giant cell tumours of bone brings together a group of clinicians and scientists who together have a wide range of expertise in the study of bone neoplasms and bone cell biology. The range of cell biology and molecular studies outlined in RL3 should provide a comprehensive analysis of the pathogenic factors that are likely to contribute to the development and progression of giant cell tumour of bone. These studies could not be carried out by a single centre and are designed to be complementary with information being provided from different investigation modalities. It is anticipated that not only will there be considerable exchange of information and tissue for individual studies carried out by members of the network but also some standardisation of research techniques and methods to study osteoclast biology and the patho-biology of giant cell tumours of bone. The osteoclastogenesis studies (WP3.2) will include analysis of the cellular components of giant cell tumour of bone (i.e. osteoclast-like giant cells) and mononuclear cells; the role of these cells in osteolysis (i.e. the growth of the tumour in bone) and tumour invasion, and investigation of the mechanisms whereby osteoclasts accumulate in giant cell tumours and cause considerable bone destruction. The effect of inhibitors of bone resorption on the formation and function of osteoclast-like giant cells in giant cell tumour of bone will also be investigated and a wide range of molecular investigations programmed in order to provide a scientific basis for the development of therapeutic strategies to treat giant cell tumour of bone. These studies should also provide important information on the behaviour of giant cell tumour of bone. The gene expression profile studies should permit study of a wide range of genetic markers by different groups; analysis of this combined data should facilitate identification of the molecular mechanisms involved in giant cell tumour of bone pathogenesis and aggressive behaviour/metastasis. Telomere analysis should provide information on the nature of cells in giant cell tumour of bone at different stages of aggressiveness (WP3.1). Molecular and osteoclast biology investigations will also be studied regarding osteoclast formation and the development of sarcomas (including giant cell tumour of bone) in Paget's disease (WP3.3).

• Unified pathological criteria One anticipated outcome of the establishment of this network facility to carry out the various studies planned will be standardisation of the criteria for assessing giant cell tumour of bone and its degree of aggressiveness. In addition, the new information derived from the studies outlined should lead to the development of new molecular markers to indicate the likely behaviour of giant cell tumours; they should also permit the development of diagnostic markers to distinguish giant cell tumour from other giant cell lesions of bone (WP3.1).
• Use of the virtual BioBank Most of the applicants have access to banks of frozen tissue of giant cell tumours of bone and related giant cell-lesions of bone. This is important as giant cell tumour of bone and other giant cell lesions of bone are relatively uncommon tumours and it is often difficult to obtain adequate numbers of these tumours for study purposes in a single centre. In addition, giant cell tumour of bone can present as a localised bone tumour, a locally aggressive bone tumour or, in a minority of cases, with metastases principally in the lungs. Examples of all these types of giant cell tumour of bone will be made available to the network for the studies planned.

Research line 4: Ewing's sarcoma family of tumours
The Ewing’s sarcoma family of tumours (ESFT) includes:

• Ewing’s sarcoma;
• primitive neuro-ectodermal tumour;
• Askin’s tumour;
• paravertebral small-cell tumour;
• atypical Ewing’s sarcoma.

ESFT represents a peculiar entity in oncology. In spite of its absolute rarity (about 300-400 cases per year in Europe), ESFT is one of the most frequent solid neoplasms in paediatric age. Due to this fact, its impact on the health system is particularly important. ESFT primarily affects white and Hispanic young people and is extremely rare in individuals of African or Asian origin. The reason for this striking ethnic distribution is not known, although interethnic differences exist for certain alleles of one of the genes consistently disrupted in these neoplasms. Tumours can develop in almost any bone and soft tissue. Approximately 25% of patients have detectable metastatic disease to lung, bone and bone marrow at diagnosis, but nearly all patients have micro-metastases, as evidenced by a less than 10% cure rate with local therapy alone. The adoption of multimodal treatments with very aggressive chemotherapeutic regimens, have significantly improved the chance of survival of ESFT non-metastatic patients, shifting the 5-year survival rates to around 60%. This improvement was obtained by the standardisation of treatment modalities through the design of multicentric trials, which have been adopted by several National and International groups in Europe including most of the Network partners. This collaboration will take advantage from the development of this research line. Despite these important clinical results, which are usually difficult to obtain in rare diseases, several problems related to histogenesis, prognosis and treatment response are still open.

• Histogenesis

The histogenesis of ESFT is still uncertain and the normal counterpart of ESFT cells is still unknown. These neoplasms share the presence of specific chromosomal translocations, which produce an EWS/ETS gene rearrangement [in more than 95% of cases, the gene fusion is EWS/FLI-1, due to the t(11;22) (q24;q12) or EWS/ERG due to the t(21;22) (q22;q12)], as well as the expression, at extremely high levels, of an antigen determined by the MIC2 gene (also known as CD99), and are currently defined along a limited gradient of neural differentiation. However, the neuro-ectodermal origin and whether or not neuro-ectodermal differentiation of ESFT has a prognostic importance is still a matter of dispute. Other histogenetic possibilities cannot be excluded because ESFT can also exhibit some epithelial and mesenchymal characteristics and can arise in organs not directly related to the neural crest (i.e., the kidney).

• Prognosis

The lack of prognostic factors obliges the use of non differentiated treatments for all patients, leading to over-treatment of those patients who could benefit of less toxic therapies. The reduction of delayed side effects is particularly important in this disease considering the young age of the patient and their long life expectancy. Several biological studies have been recently carried out on the identification of the peculiar features of ESFT, offering promising molecular targets to be potentially used for therapy. The biological basis for the prognostic differences among the various fusion genes is unknown, but a recent report has shown a significant association with levels of expression of insulin-like growth factor (IGF-IR) and the proliferation rate. Moreover, secondary cytogenetic and molecular alterations of ESFT (gains of chromosome 8 and 12, loss of chromosome 16, deletions at chromosome 1p36, and p53 mutations) have recently been associated with a worse prognosis, further indicating a possible association between genetic variants and clinical features.

• Therapy

In the current state of ESFT treatment there is a survival “plateau” (around 60% for patients with localized disease and 25% for high-risk groups) due to the lack of new drugs and toxicity that impedes more intense use of existing drugs. The identification of new targets for innovative therapeutic strategies are, therefore, strongly needed for this tumour (WP4.2, WP4.3). Progresses are generally hampered by the rarity of the disease (in Europe about 400 cases/year) implying a limited number of cases for effective research. Moreover, because ESFT is an orphan disease, no private company will develop new therapeutic tool and charge on itself the costs to conduct pre-clinical investigation.
Solutions and areas for further development
This research line, by identifying the clinical relevance of a number of markers, may allow the differentiation of patients in terms of risk to recur. This will enable more aggressive treatments where these are justified, and avoid toxicity in cases where such treatments may be known to be unnecessary, with particularly significant consequences for the quality of life of the patients. Moreover, successful treatment of therapy-resistant patients requires new strategies. Indeed, there is desperate need for new therapeutic approaches in ESFT. Practically, most patients relapsing after front-line chemotherapy, that have used virtually all active drugs currently available, are suitable for inclusion in phase II studies on new targeted therapies. Thanks to the presence of different laboratories with an extremely specialised and differentiated experience on the subject, a thorough study of the pre-clinical effectiveness of new targeted therapeutic strategies will be performed with the aim of the identification of the Achilles’ heel in this disease and the consequent development of a tailored biological therapy to be used in association with conventional chemotherapy. With the same aim, the feasibility of (tumour-antigen-specific) T cell-mediated immunotherapy will be studied in a preclinical setting. With the same aim, the feasibility of (tumour-antigen-specific) T cell-mediated immunotherapy will be studied in a preclinical setting (WP4.2). By providing an organisational framework for collaboration the project will also allow multi-centre collection and analysis of cases as well as suitable collaborative research to allow genetic studies for the screening of high-risk patients and patients responding differently to chemotherapy (WP4.3).
Specific Scientific & Technological Objectives
Due to the development of the Horizontal Technology Platforms, this line is aimed to standardize researches on histogenesis, prognosis and therapy in Ewing family of tumours. The main objective of this network is to evaluate the prognostic and therapeutic relevance for each of these factors and any correlations among them, to provide the rationale for the design of new options for therapy to patients escaping conventional treatments (WP4.3). Accordingly, the prognostic impact for each of these factors, by the retrospective and prospective analysis of a large number of tissue samples collected in the different centres, and the pre-clinical effectiveness of new strategies specifically targeting some of these molecules will be defined. Only the joint effort of large Institutions can provide enough experience and enough cases. The centres involved in this research line have been already in contact and have actively collaborated inside the European Musculo-Skeletal Oncology Society (EMSOS). The existence of a STREP Project (PROTHETS) focused on this topic, will increase the possibilities to enlarge the number of researchers and centres who could contribute to the knowledge in this field. The centres involved in this research line are: Bologna, Düsseldorf, Helsinki, Leiden, Oxford, Salamanca, and Valencia. PROTHETS is limited to Ewing’s sarcoma, whilst EUROBONET involves all bone tumours. Similarities can be found only in WP3 of the Research Line 4, however studies in PROTHETS are very preclinical investigations and limited to very selected targets (CD99, IGFIR, erb-B2, NOV, EWS-FLY1). In EUROBONET, most of the researchers involved in Ewing are not involved in PROTHETS and the studies will focus on larger molecular aspects, including pharmacogenomics and drug response. In addition, the results of PROTHETS will be used as baseline for preclinical and clinical applications within EUROBONET. This aim is stressed by the fact that the network coordinator of PROTHETS is also the responsible of the Research Line Ewing family of tumours in EUROBONET