Skip to main content

Sustaining access to Tissues and data frOm Radiobiological Experiments

Final Report Summary - STORE (Sustaining access to Tissues and data from Radiobiological Experiments)

Executive Summary:

The sharing of data and biomaterials from publicly-funded experimental radiation science adds enormous value to the original investment. Sharing will yield substantial scientific added value through re-analysis and new investigations. This does not only account for new technologies in data analysis. Moreover, the rapid progress in radiation biology allows analysis of old material with new techniques. The emergence of high throughput ’omics’ technologies has led to an explosion in the rate and volume of data generation and data sharing through the scientific literature alone has become impossible. It is well recognised in the community that potential benefits will accrue from a widespread culture of sharing research data and bioresources by making data available immediately after publication, and resources, within a defined period. Additionally, the use of legacy data is particularly important for radiation biology, because a large number of studies conducted between the 1950s and ‘90s are unrepeatable due to ethical and financial restrictions. Still, such legacy data can be usefully reanalysed in the light of new paradigms.
Thus, it is not only important to rescue endangered primary data; even more crucial is to archive it at the time it is generated. This lesson has been learned by the radiobiology community, who took on that challenge 25 years ago when developing the International Radiobiological Archives, with was further developed to become the internet-based data repository ERA.
For the purpose of rescuing endangered data from past experiments and saving data from modern studies, the STORE infrastructure has been developed. STORE allows the storage and retrieval of data from past, current and future radiobiological studies. Securely accessible software allows full data control to its originator: information can be stored without a public access; it can be made available to selected co-workers only; it can be released to the whole scientific community. STORE can also act as a directory to collections of tissue samples, FFPE blocks, and slides etc. or to other relevant data bases, e.g. the one of the German Uranium Miners Cohort Study. STORE offers the possibility of hosting whole histopathology slide scans as a virtual archive. It has to be emphasised that STORE is suitable for both information from radiobiological experiments and for studies amongst human populations. As an example, STORE hosts the data of the German Thorotrast study – a cohort study amongst Thorotrast patients –, as well as the data of the respective animal experiments and a pointer to biomaterial from these experiments.
Knowledge management does not only imply making data and biomaterial available. Information will only turn into knowledge if it is linked together in biologically meaningful ways; however, having the information is the necessary first step. To allow the extraction of all possible information from a platform such as STORE, experience from other similar platforms has been made available. STORE is capable to include annotations to the information, give references to scientific publications or incorporate grey literature such as internal laboratory reports. This has been done successfully in the former EU FP6-funded project ERA-PRO, within which the electronic form of ERA was created (http://era.bfs.de).
In the STORE project, knowledge management also included the development of Standard Operating Procedures (SOP) for evaluating the quality of radiobiological archive tissue and defining test systems describing the usefulness of such material. The resulting data have been quantified and the procedures performing best in terms of the call rates of data points and experimental noise, compared to those from the fresh-frozen or freshly-made formalin-fixed paraffin-embedded (FFPE) control, have been validated. The SOPs have been made available on the STORE web page (http://www.rbstore.eu). Last but not least, new methods applicable for archival material (e.g. XFM) were successfully tested during the course of the project.


Project Context and Objectives:
The value of an online accessible and easy-to-use radiobiological resource (data warehouse) lies in retaining the primary data from experiments to allow reanalysis, reinterpretation and re-evaluation of results from, for example, carcinogenicity studies, in the light of new knowledge in radiation biology. Consequently, there is an imperative need to keep these data available for the research community. More than that, it will establish the basis for long-term use and for exchange between scientists from different countries and from various fields. It will as well help to minimise animal experiments as far as possible by making information available on those experiments that have already been conducted.
With the on-going globalisation of science it has become essential to develop common infrastructures such as data bases. Finding and integrating data in such data bases relies on standardisation and harmonisation of data structure semantics and metadata. This is an issue being energetically addressed through initiatives such as Science Commons, together with those of many ‘omics‘ and model organism communities. The European Commission in its Horizon 2020 programme is strongly interested in bringing scientists from different fields together, which will be made easier if good metadata description for each data set is made available. The STORE project aimed at developing an initial infrastructure for the radiation biology community in this area. Thus, STORE helps to stimulate adoption and dissemination of standards, and development of those particularly needed for radiation science.


Overall objective

The provision of a data warehouse is expected to raise awareness of the existence of these resources, many of which are, anecdotally, underused due to their availability and unknown existence. The generation of benchmarks for sample preservation and usability by preparation of SOPs will also help to disseminate formal standards by which the usefulness of archival material can be assessed.

The main objectives of STORE were to:

1. develop a “Data warehouse” database and portal to allow ready accessibility to both data and material from biological samples via the web.
2. develop benchmarking procedures on how to best quality assess, store and optimally utilise radiobiological samples, such as tissues embedded in paraffin blocks, so that persistence and accessibility of these samples can be guaranteed for the future. This was done by development of Standard Operating Procedures (SOPs) on validating new biological analytical methods on archive samples.
3. assess the scientific and financial viability of a central data and bioresource repository and evaluating models for cost recovery based on STORE experience.

To realise the goals of this project the work was divided into four work packages. The establishment of a public data repository as well as investigating international regulations on transfer of data was realised by work package 1 (WP1). The second work package (WP2) developed operational parameters for a radiobiological archive and examine issues of scientific and financial sustainability. In Work Package 3 (WP3) SOPs for evaluating the materials were developed. The WP4 was related to management tasks. Each of the Work Packages was sub-divided into tasks.


Project Results:
WP 1 Data Warehouse

The development of novel and hugely powerful high-throughput technologies, together with the globalisation of scientific research, present the radiation research community with unprecedented challenges for the management, archiving and distribution of data and bioresources. The traditional paradigm of publishing scientific data and results in the literature is no longer effective as new technologies produce large volumes of diverse types of data and biological materials. Critical to the maintenance, distribution, and archiving of these data and materials are stable publicly funded databases and repositories. In response to this need the European Commission has funded the STORE project whose aim is to provide an infrastructure for the sharing of primary data from radiobiological research and to investigate the modes by which biomaterials such as tissues, histological specimens and slides might be safely archived and efficiently shared. The major aim of the project was to provide a central database infrastructure to facilitate the deposition and sharing of pre- and post-publication data derived from radiation biology experiments, human exposures and biomaterials and reagents used in this domain. In order to achieve this, a database infrastructure into which such depositions could be made has been developed.


The development of data standards, database architecture, user interfaces and security were the major activities. We have developed a database platform, exploiting semantic technologies such as ontologies and formal annotation, into which primary data such as raw data, spread sheets, system files (e.g. based on SAS® or SPSS®), images, mass spectroscopy data, FACS, proteomics and transcriptomics data might be deposited, either pre- or post-publication. The aim was to facilitate and encourage sharing of the raw data on which publications are based on with the rest of the community. The database allows the storage and retrieval of data from past, current and future radiobiological and –epidemiological studies. This database allows users to upload and store their data without losing control of it. Secure access software allows the originator to decide whether to release data either to the whole scientific community, to selected co-workers or to keep them locked, i.e. data are only accessible to the originator himself. STORE can also act as a directory to physical collections from radiobiological experiments (tissue samples, FFPE blocks, slides etc.). In that case STORE offers to upload a link to where researches can find more information.
Additionally, STORE offers the possibility of hosting entire histopathology slide scans as a virtual archive. This will allow fast and interactive inspection on the web (virtual pathology).


The STORE database is available through http://fp7store.eu or directly on http://www.rbstore.eu. STORE already includes information from human cohort studies, from animal experiments and links to other data bases.

The main STORE system is structured in the following way:

• The main objects are projects, for example a project funded by the EC. Project details include the project name, project number and work package details. Each project may have a number of datasets. These could be the equivalent of studies or simply a collection of related files. Each dataset can have a number of files or links to files on other databases.
• STORE users can be project members, contributors to datasets and curators of files, but only the owner/ creator of each object can make changes or delete items. The creator has the power of approval or disapproval of objects.
• Files can be downloaded and downloads can be restricted to approved users only.
• Visitors can download files, comment on files and create shopping baskets to collect files for later download and analysis.
• Uploaded files are saved on the main STORE server as plain files. Links to other databases require the server details and a unique ID which provide a direct URL to these files on other servers. This is also how files are currently connected to the NDP servers.
• The NDP servers don't currently accept direct uploads because of the size of the files (up to 4GB for each image). Users’ images are copied directly onto the server and they are indexed and added to the system automatically. Once the files are indexed their unique ID can then be uploaded into the main STORE database. We are currently working on an upload interface for zoomable images.

The following figure shows the structure of STORE database schema. On the left hand side are the tables which hold project and work package details. Each project can have a number of datasets (the second row of tables) and each dataset can have a number of files. All file details are stored in the rest of the tables, except for the top left table which holds user details.

The SQL code can be downloaded as part of the source code package (see above), the file is called store.sql.


All of the software developed in STORE is available as open source in line with accepted ethical practice.

STORE worked extensively on adopting and implementing existing semantic standards for data description where they exist and have created small ontologies and CVs where needed; the result is that the data in the STORE database may be integrated seamlessly with any other database using currently accepted semantic and data-type standards. The overall approach is an example of the implementation of the recommendations of the committee for the W3 semantic web for health care.

The database may be accessed through the STORE portal: http://www.rbstore.eu.


WP 2 Sustainability and Accessibility of the Biological Archives

Baseline requirements for sustainable archives have been put down, based on different scenarios.
It is obvious that archives or archiving platforms are only viable if continuous funding, even at a relatively low level, is secured. With the end of the STORE project funding there is a very powerful and growing concern about guaranteeing the viability of the infrastructure. Significant resources for managing, sustaining and making data accessible are required. There is a need for both financial and scientific sustainability which will depend on the sharing imperative being embraced by the radiation science community and acceptance of responsibility for maintaining the infrastructure by all of the stakeholders across national and disciplinary boundaries. A first step towards sustainability is taken by making STORE a part of the large DoReMi network of excellence.
The Commission is strongly encouraged to enable the establishment of a long-term physical repository for biological material. But this cannot be done within STORE given its resources. The establishment of a central tissue archive is not practical or sustainable without substantial investment and on-going infrastructure funding. It is for this reason that STORE will seek to raise awareness of the existence of relevant tissue archives for PIs requiring both tissue and data. It shall facilitate the contact between tissue archives and prospective applicants. The database provides SOPs for quality control as well as a small guide to assist in the shipment of archival biological and any radioactive materials in compliance with existing regulations. The manual is to be used as a reference ONLY. Accordingly, it includes a respective disclaimer.

One special Task of WP 2 was the evaluation of data and biomaterial held in the archives of the Southern Urals Biophysics Institute (SUBI). During periods of activities of SUBI experimental laboratories (1955-1996), their predecessors (1949-1954) and successors (since 1997), large-scale research in the area of radiobiology and radiotoxicology was carried out. In these experiments, more than 120,000 animals of different species were used.
The main topics were:

• Radionuclides metabolism in organism at different ways of administration and internal dosimetry;
• Determined and indirect effects and mechanisms of their development;
• Late effects of neoplastic and non-neoplastic nature and mechanisms of their development;
• Experimental justification of radiation safety standards and medical protection system on the basis of available knowledge.

At the beginning of the project, an unorganised archive of biological material from different animal species is one of important SUBI resources for acquiring of new knowledge based on state-of-the-art technologies. The biological material is a result of the above mentioned experiments and is stored in different forms (paraffin, formalin, histological slides). An initial estimation said that the maximum volume of the biomaterial could be up to 500,000 samples obtained from 23,515 animals.
For the evaluation of the SUBI archives, six experiments were selected during a site visit by members of STORE and the Advisory Board. It could be shown that for these six experiments, paraffin embedded tissues from these animals had been preserved and are available through SUBI. The tissues were successfully linked to the relevant data for each individual animal. This information was abstracted from lab log-books.

In order to make this archive accessible an electronically database was developed. This makes it easy to organise the SUBI radiobiology archive and search through the results. It is written as a web application that runs on a local server for and will be made public on the web via linking to the STORE database.
Further, a standardized approval procedure was developed to allow trans-border shipment of the biological material stored in SUBI by requests from international research organizations within the framework of pilot and long term scientific projects. In that context, scientists from SUBI were trained on re-embedding the biological material before analysing it or before shipment to partners.

With respect to scanning slides, it could be shown that the slides held at SUBI are good enough for scanning with an automated systems like “Olympus dotSlide®”, but not with a Hamamatsu® high resolution scanner because of their thickness. Anyway, it has to be noted that only a fraction of the slides from the past experiments is still in a shape where scanning will give meaningful results.


In summary it can be noted that SUBI, though it suffered from the changes that took place after the dissolution of the USSR, is a significant resource that should be further exploited.


WP3: Standard Operating Procedures (SOPs)

The rapid expansion in technology-driven analytical methods offers great rewards to those who intend to revisit the past. As mentioned, STORE wants to give technical assistance in exploiting archival material for radiobiological research. Accordingly, another key part of the project was to produce standard operating procedures (SOPs) that would allow the radiation biology community to extract molecular information from archived material, e.g. formalin-fixed paraffin embedded samples (see following figure).


Generally, there is a need to validate methods before their introduction into routine use. To confirm that the SOPs employed for specific tests were suitable for its intended use a method validation was carried out under controlled conditions for determination of their robustness and reproducibility by the beneficiary IBBL, Luxembourg. Each SOP will be loaded into the STORE database. Re-validations will be performed whenever the conditions change for which a specific method has been validated or whenever this method is changed and the change is outside the original scope of the method.

The following SOPs were developed and successfully validated:

• SOP for extraction and quantitative analysis of proteins from FFPE tissue
• SOP for DNA extraction and testing
• SOP to determine the quality of DNA and RNA from tissue of unknown pre-treatment and/or storage conditions
• SOP for genomic DNA isolation from FFPE tissue blocks
• SOP for Multiplex-PCR check of genomic DNA isolated from FFPE tissue for its usability in array CGH analysis
• SOP for preparation of FFPE sections prior to processing to nucleic acid
• Preparing FFPE pellets from cell lines
• Quantitative reverse transcription real-time PCR with mRNA from fresh-frozen or formalin-fixed paraffin embedded tissue
• HER2:HER proximity ligation assay on formalin fixed paraffin embedded tissue using the DUOLINK system

These efforts led to a number of publications in scientific journals.
Using these validated standard operating procedures can minimise differences of the variability of results generated by different laboratories analysing the same archived radiobiological material. An overall benefit of valid SOPs for archived material is minimizing work effort, along with improved comparability and credibility of the ensuing results. STORE recommends that these SOPs are followed in order to ensure optimum value of the biological material.
Users are cautioned that deviations in the method described may impact the results (and the validity of the method). Current copies of the SOPs will be readily accessible in electronic format on STORE’s web-site after the process of internal validation is completed. STORE also investigates which methods and procedures developed for fresh or fresh frozen cell cultures or tissues work best on archived radiobiological material.

The figure above shows a summary of the validation performance: A – for processing methods, B – for characterisation methods.


In conclusion, the STORE processing methods were shown to be fit-for-purpose, reproducible and robust for DNA, RNA and protein extractions from FFPE materials. The STORE characterisation methods were shown to be fit-for-purpose and robust for Quality Control of the extracted DNA, RNA and proteins.


Potential Impact:
The project was a feasibility study on developing an online repository for data from radiobiological studies and experiments, on establishing a central archive for biological samples from those experiments and on developing SOPs for using those samples with modern biological techniques.

The expected final result was an online tool for the following purposes:

• storing of data from past, present and future radiobiological studies, both human studies and animal experiments;
• pointing to data and biological material available at various laboratories and/or data bases, both on radiobiological experiments and human studies;
• creating SOPs on how to best use biological material from past experiments for modern biological assays.

The project was successful in various aspects:

The radiobiological resource is accessible online and easy to use for the radiobiological community. More than that, it establishes the basis for long-term use and for exchange between scientists from different countries and from various fields. It helps as well to minimise animal experiments as far as possible by making information available on those experiments that have already been conducted.

STORE has developed a database platform that will allow the storage and retrieval of (raw) data from past, current and future radiobiological studies or, if necessary, provide ‘pointers’ referring to the data in public databases. Secure access software allows data to be released either to the whole scientific community, to selected co-workers or to be secured, i.e. data are only accessible to the originator, to allow users to retain control. STORE data base is open to individual investigators and to funding agencies as a potential central repository for data sharing. This is greatly helping to raise awareness among the radiobiology community of the existence of these resources, many of which may be underused because their availability is unknown. STORE also acts as a directory to physical collections from radiobiological experiments (tissue samples, FFPE blocks, slides etc.). For slides, STORE offers the possibility of hosting whole histopathology slide scans as a virtual archive. This will allow fast and interactive inspection on the web.

Benchmarks are set within the project for sample usability, and Standard Operating Procedures (SOPs) are set for adopting new technologies in the preparation of material from tissue samples. STORE provides the necessary SOPs for the evaluation of archived tissue usability.


It is also worth to mention that:
 It is important to save data and biological materials from publicly funded projects. Every year, one or more local archives are lost, as curators retire or storage space is taken away for other purposes. It is essential that local endeavours are transformed into national and international efforts to provide safe storage for endangered materials and data. STORE provides a one-stop portal to maximise the use of biological materials and data, and promotes collaboration across the wider radiobiological community by facilitating data exchange and sharing materials. The European Commission itself in its Horizon 2020 programme is strongly interested in bringing scientists from different fields together, which will be made easier when there is good metadata description for each data set.

 The establishment of a central archive is only possible if the necessary financial support is being guaranteed by national or transnational funding agencies. Significant resources for managing, sustaining and making data accessible are required. There is a need for both financial and scientific sustainability which will depend on the sharing imperative being accepted by the radiation science community. Additionally, an acceptance of responsibility for maintaining the infrastructure by all of the stakeholders across national and disciplinary boundaries is essential. A first step towards this goal is taken by making STORE a part of the large DoReMi network of excellence. The project will be further developed in the frame of DoReMi’s work package “Infrastructures”.


Thus, STORE has the potential of becoming a useful and powerful tool within the radiation research community.


List of Websites:

Project public website:

http://fp7store.eu and http://www.rbstore.eu


Coordinator contact details:

Dr. Bernd Grosche
Bundesamt für Strahlenschutz
Ingolstädter Landstraße 1
85764 Neuherberg
Germany

Phone: +4930183332260
Fax: +4930183332205
E-mail: bgrosche@bfs.de


List of Beneficiaries

Beneficiary Number Beneficiary name Beneficiary short name Country
1 (coordinator) Federal Office for Radiation Protection BfS D
2 Helmholtz Centre Munich HMGU D
3 University of Cambridge UCam-PDN UK
4 Southern Urals Biophysics Institute SUBI RF
5 The University of Edinburgh-Edinburgh Cancer Research Centre UEDIN UK
7 Imperial College London Imperial UK
8 Rijksinstituut voor Volksgezondheid en Milieu (National Institute for Public Health and the Environment) RIVM NL
9 Integrated Biobank of Luxembourg IBBL Lu