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The Chernobyl Tissue Bank – Coordinating International Research on Radiation Induced Thyroid Cancer

Final Report Summary - CTB (The Chernobyl tissue bank - coordinating international research on radiation-induced thyroid cancer)

Executive summary:

The Chernobyl accident on 26 April 1986, occurred when an experiment at the nuclear power plant when disastrously wrong. The resultant explosion and fire in the graphite core led to the release of more than 1019 Becquerel of radioisotopes, including significant quantities of radioisotopes of iodine (I). The major effect of the Chernobyl accident so far demonstrated has been the very large rise in incidence of thyroid carcinoma in children living in the regions exposed to the highest levels of fall-out. Thyroid cancers are very rare in the unexposed population of this age and there is now little doubt that the increase observed thus far is due to exposure to radioisotopes of I, but the effects of exposure to low dose radiocaesium are as yet unclear.

The objective of the Chernobyl Tissue Bank (CTB) is to provide a resource for research into the health consequences of the Chernobyl accident and to promote collaborative, rather than competitive, research on a limited biological resource. It comprises a substantial collection of the thyroid tumours that have arisen in areas of Ukraine and Russia which were exposed to significant fallout from the damaged Chernobyl reactor. The collection also contains a number of samples from cases of thyroid tumours that have arisen in children living in the same areas, but who were born after the radioactive I had decayed and have not therefore been exposed to radiation. This group forms a very valuable control cohort for the cases exposed to radiation, but also provides an opportunity to study early onset thyroid disease.

Three sponsors have supported the project during the funding period now reported: the European Commission (EC), the National Cancer Institute (NCI) of the United States of America (USA) and the SMHF of Japan.

The project aims to:

(a) collect and curate biological specimens with appropriate consent from patients operated for thyroid cancer or cellular follicular adenoma who were born on or after the 26 April 1967 (i.e. aged 19 or under at the time of the Chernobyl accident);
(b) provide quality assured materials (both in terms of pathology and molecular biology) to the wider research community;
(c) provide a database to the wider research community that links dosimetry data with information on age at clinical presentation and at the accident, residency and pathology of tumour;
(d) promote the CTB to encourage scientists to use the biomaterial and data resources;
(e) provide a web-accessible data warehouse containing research data from projects that have used materials from the CTB with a clearly stated access policy that respects the rights of the providers of both published and unpublished research data;
(f) inform and communicate research findings relevant to exposure of populations to low level radiation in the environment.

Anonymised data about the patients and the samples is stored in a comprehensive database, which has been extensively redeveloped during this funding period. A separate database houses research results from scientists who have used CTB materials. Applicants wishing to use the biomaterials and / or research data stored in the CTB are directed through a simple on-line application process via the CTB portal.

In order to maximise the amount of information that can be obtained from small pieces of tumour and to conserve this valuable material for future generations of scientists, researchers are provided with aliquots of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) extracted from well documented pathological specimens.

The project has achieved all its objectives and will continue to collect and distribute biological material and data. Valuable experience and expertise from the Chernobyl studies have helped to inform the response to the public health issues arising following the incident at the Fukushima nuclear power plant in Japan in 2011.

Project context and objectives:

The accident at Chernobyl resulted in a significant release of radioiodine into the environment. Within 3 - 4 years of the accident an increasing incidence of thyroid cancers was reported in children living in the areas most exposed to the radioactive fallout. The thyroid is the only organ in the body to concentrate and bind I; exposure to the thyroid from 131-I is 1 000 - 2 000 times the average body dose. 131-I has a short physical half-life, which results in quick elimination from the environment. Patients who were born more than 9 months after the accident were therefore not exposed to radioiodine either in utero or as young children.

Whilst having serious consequences for the population, particularly those under 19 years old living in the immediate vicinity of the power station at the time of the accident, and those involved in the early phases of the clean-up of the accident and therefore exposed to high levels of radiation from the fallout, the Chernobyl accident has provided a unique opportunity to collect samples of a human cancer, with a low natural incidence, for which both the etiology and the time of exposure to the etiological agent is known. Thyroid cancer is normally rare in children; it is therefore likely that the majority of the thyroid cancers that have arisen in this population are a direct consequence of exposure to radioiodine in fallout. Many of the children in the exposed areas of Belarus, Ukraine and Russia received thyroid doses in excess of 1Gy.The BEIR VII model of the risk of radiation induced thyroid cancer based on studies of groups of children exposed to external sources of radiation predicts a lifetime excess relative risk (ERR) of around 10 per Gy for exposure in early childhood, falling to an ERR of about 2 per Gy at age 20 at exposure. There is a marked increase in background risk of thyroid cancer with attained age and the shape of this increase varies with gender. In females in the United Kingdom (UK) the incidence rises to a plateau of around 6 cases per 100,000 at an attained age of 40 years, and in males there is a steady rise in incidence with age. This pattern is similar in other countries.

The current risk models suggest that there is no attenuation of the ERR with attained age. This suggests that there will be an inevitable relative increase in the number of cancers in the exposed population as the affected cohort ages. Although the increase in incidence is large, differentiated thyroid cancer diagnosed in the young has a good prognosis, and a recent review has suggested that although there is a predicted recurrence rate of 30 %, the death rate is predicted to be of the order of 1 %. The data presented here only reflect cancer incidence, but there is evidence from the Ukraine-American cohort study that benign tumours of the thyroid are also increasing in incidence as a result of the radiation exposure although at a lower rate than cancers. Whilst not life-threatening, these lesions require surgery to confirm absence of malignancy and consequently have a significant health impact on the individuals concerned.

The CTB was established in 1998 to collect, store and disseminate biological samples for research into thyroid tumours that have arisen in the children exposed to radiation following the Chernobyl accident. The CTB now includes material and information from all patients with thyroid carcinomas and cellular follicular adenomas from the contaminated oblasts of the Russian Federation (Bryansk, Kaluga, Tula and Oryol) and Ukraine (Kiev, Kiev city, Cherkasse, Chernigov, Rovno, Zhitomyr and Sumy) who were born on or after 26 April 1967 (i.e. aged under 19 at the time of the Chernobyl accident) and operated on or after 1 October 1998 and have consented to their tissue and blood samples being stored in the CTB and disseminated for research. This cohort of patients is unique in that, in the vast majority, the cause of their thyroid tumours will have been exposure to radiation at a young age. Both the dates of exposure and operation are known, which permits calculation of the time taken for the tumour to become clinically apparent (i.e. the latency of the tumour).

The active tissue banks are based at the Institute of Endocrinology and Metabolism of the Ukrainian Academy of Medical Sciences in Kiev, Ukraine (IEM, Kiev) and at the Medical Radiological Research Centre of the Russian Academy of Medical Sciences in Obninsk (MRRC RAMS) and the project is coordinated from imperial College, London. Initially Belarus was also part of the project but has been excluded since 2006 for political reasons.

During this funding period the objectives for the project have been to:

(a) collect and curate biological specimens, with appropriate consent, from patients operated for thyroid cancer or cellular follicular adenoma in Ukraine and Russia, who were born on or after 26 April 1967 (i.e. aged 19 or under at the time of the Chernobyl accident);
(b) provide quality assured materials (both in terms of pathology and molecular biology) for research to the wider research community;
(c) provide a database to the wider research community that links dosimetry data with information on age at clinical presentation and at the accident, residency and pathology of the tumour;
(d) provide a web-accessible data warehouse for future bioinformatic studies on data from research projects funded by the EC and other sponsors;
(e) promote the CTB to encourage scientists to use the biomaterial and data resources;
(f) coordinate further research studies where multiple research groups access different types of biological material from the same patient thereby enabling a 'systems' or 'integrated' biology approach to thyroid cancer.

Patients presenting with thyroid lesions at the clinics in Ukraine and Russia, who meet the entry criteria for their tissues to be included in the CTB, are invited to contribute samples to the project by the attending clinician. Thyroid lesions are generally very small and therefore although, where possible, pieces of tumour and normal thyroid will be frozen, in many cases only fixed, paraffin embedded (FFPE) blocks can be taken. Each patient is given a tissue bank code authorised by the appropriate person responsible for the management of the resource in each country and each sample is identified by a unique code that incorporates the patient's code number. All cases are reviewed by the CTB Pathology Panel, an international panel of the thyroid pathologists, which meets annually to agree a consensus diagnosis before a case is incorporated into the CTB. Information about age, sex, oblast and country of residence, together with the pathological diagnosis and the dose received, are recorded in the database for each specimen stored in the CTB, and are made available to researchers who receive material from the bank. Basic clinical information is also recorded on each patient in the CTB database. This permits correlation with other databases (for example further information on dosimetry, clinical status of disease, radioiodine treatment and response etc.). Each of the banks houses only material and information from its own population.

The CTB seeks to maximise the amount of information that can be obtained from the small tissue samples by providing multiple aliquots of RNA and DNA extracted from the tissues, rather than the tissue blocks themselves, so several researchers can use the extracted material from the same lesion. All the extractions are now carried out at the collecting centres, which have been supplied with the appropriate equipment and reagents during this funding period. Staff training has also been undertaken.

An important feature of the project is that it also collects biosamples from patients resident in the areas of Ukraine and Russia exposed to radioactive fallout, but who were not exposed to radioiodine as they were born more than 9 months after the accident. These cases form an age- and residency-matched cohort of patients who develop spontaneous thyroid neoplasia. This is the ideal cohort for comparison with those who were exposed to radioiodine in 1986. The current number of cases in this valuable cohort is 338, with a further 214 coming from areas other than the exposed oblasts. The number of cases in this cohort is much lower than those exposed to radioiodine - the incidence is approximately the same as the background spontaneous rate from uncontaminated regions - of the order of 1 per million per year.

It is already clear from the results of the HUGO project that it is likely to be the interaction of suites of genes which is responsible for both susceptibility to development of human cancer and the biological mechanism which influences tumour growth. The CTB allows multiple researchers access to the same material, permitting comparison of different techniques for the same molecular marker, as well as analysis of multiple molecular markers. In addition, it provides the opportunity for additional markers (some of which may not yet have been discovered) to be studied at later time points. Researchers have the opportunity to carry out multiple analyses on individual samples from the same piece of tumour and to compare these with analyses on a separate area of tumour. This enables scientists to investigate the heterogeneity of a given tumour - a factor that may prove very important in the future design of therapeutic strategies. Most current molecular biological methodologies require the extracts from frozen material, but the CTB also aims to be able to supply the next generation of scientists, who may be in a position to benefit from a much more detailed analysis carried out on paraffin embedded sections. Samples of peripheral DNA from blood lymphocytes and samples of serum from each patient are also available to permit the study of the interaction of the hormonal / immunological environment with the genetics. Linkage of dosimetry data, which has been completed in the current funding period, provides additional information for researchers looking to stratify cases on the basis of molecular changes in the thyroid tumours occurring following radiation exposure. Researchers wishing to access the resources of the CTB are required to submit an application describing their proposed research. This is reviewed by an external panel of experts before approval for access is given. On completion of the project, researchers are requested to return their data to the CTB. While publication of research results is an essential component of any research project, there is considerable value to the scientific community in being able to share research results in advance of publication. This maximises the value of the read-out from modern molecular technologies, which in many cases are expensive to use and produce very large volumes of data.

During this funding period an extensive review of the information technology (IT) infrastructure was performed with the result that a new structure, the CTB Data Warehouse, was designed and developed. This structure incorporates the CTB Samples database, which is the repository for the information about the patient and the samples that have been taken, and the Research database, which is the repository for research results from studies using CTB materials. The Samples database was extensively redesigned and has now replaced the existing DB2 database. The Research database has been developed de novo.

Since 2001, the CTB has been funded jointly by three sponsors, the EC, NCI of the USA, and the Sasakawa Memorial Health Foundation of Japan. In the funding period now reported, the EC has provided support for 2 salaries at the Coordinating Centre (the project manager and a computer programmer to help with the web-accessible database), salaries for staff in the collection sites at IEM and MRRC and for two other European partners, who have led the work packages (WPs) to obtain the dosimetry data and to develop the data warehouse. Travel funds for meetings and site visits and a contribution to the consumable costs at IEM and MRRC have also been provided. The NCI has provided funds for the management and development of the database as well as equipment costs for IEM and MRRC, and a contribution to computing and consumable costs. The support from the SMHF has provided funding for consumables and travel.

The CTB has already had a considerable impact on tissue banking for research and on the dissection of mechanisms involved in radiation related cancer. A number of similar projects exist to amass biological archives from specific patient groups, however, few of these projects seek, from their outset, to collate research data produced from the samples collected. The CTB archive is different in that it deals with a relatively rare tumour whose aetiology is known, therefore enabling study of the interaction of environment and physiology in the development of a particular cancer. However, it is clear from the research that has already been carried out on material from the CTB that continued collection and distribution of multiple types of biospecimens from the same patient are important in the further dissection of the way in which the environment, genetics and physiology all interact to alter tumour development in exposed populations. The CTB is therefore a paradigm for tissue banking in the 'omic' era.

Those children who were the youngest in the cohort at the time of the accident, are still only 26 years of age. Eventually the increasing background incidence of thyroid tumours as the cohort ages will make it very difficult to differentiate between an increasing sporadic incidence and cases induced by radiation, but at present several important questions remain to be answered: (a) prediction of the size of the outbreak of thyroid cancer to determine the risk per Gray over time, and to study possible factors increasing the risk of development of thyroid cancer; (b) the pathology and molecular biology of the thyroid tumours, to determine whether the type of thyroid cancer changes with time post exposure, to determine whether there are specific tumour markers of radiation exposure, and to increase the understanding of the way in which radiation causes thyroid cancer, particularly in relationship to genetic susceptibility; (c) study of the relationship between age at exposure and risk of developing thyroid carcinoma, and to improve understanding of the relationship between age and sensitivity to exposure to fallout containing radioiodine.

On 26 April 2011 was the 25th anniversary of the Chernobyl accident and 2011 also saw another major incident at a nuclear power plant when the reactors at Fukushima in north east Japan were damaged by an earthquake and the consequent tsunami. Although there were few parallels in the nature of the accidents, with the release of radioactivity at Fukushima being a fraction of the release from Chernobyl and localised to the vicinity of the plant, much of what has been learned from the accident in Soviet Russia helped to inform the response to the Fukushima incident. Following this incident there remains an elevated interest in understanding the effects of exposure to radiation from nuclear power plant accidents and a need to provide the scientific evidence to reassure and educate the public, particularly in Japan, about the negligible risk posed by the levels of exposure from Fukushima.

Although we have not been successful in renewing support from the EC, the CTB will continue with support from the other sponsors and it is hoped that new opportunities to apply for funding will be possible via MELODI.

Details of the project and publications resulting from material supplied by the project are listed on the project website (see http://www.chernobyltissuebank.com online).

Project results:

Overview

The CTB now comprises two banks of biological material and information, consisting of:
(a) samples from tumour, normal tissue and, where possible, metastatic tissue from postoperative specimens;
(b) nucleic acid extracted from these specimens;
(c) vials of serum from patients whose thyroid tissue is held in the bank;
(d) samples of whole blood;
(e) DNA extracted from blood;
(f) a computerised database in which relevant information on the patient (date of birth, date of operation, sex, oblast of residence at the time of the accident and operation) is held, together with location coordinates for each sample of tissue, DNA or RNA extracted from tissue, and blood, serum and DNA extracted from blood;
(g) a record of the calculated dose received by the patient;
(h) research results from approved projects that have used CTB materials, linked to the patient record.

The CTB contains 4 288 cases of thyroid cancer and cellular follicular adenoma from patients who were under 19 at the time of the Chernobyl accident, of which 3 566 (2 267 from Ukraine and 1 299 from the Russian Federation) are available to researchers. Frozen material is available from 2 744 of these cases, and DNA and RNA has already been extracted from approximately 25 %. Collection of blood samples began in late 1999 and samples of serum and whole blood have been collected from approximately 2 000 patients. As noted above, the CTB also contains samples from children born more than 9 months after the accident who have developed spontaneous thyroid neoplasia. There are currently 552 cases in this valuable cohort; the incidence of thyroid cancer being approximately the same as the background spontaneous rate from uncontaminated regions - of the order of 1 per million per year.

The detailed pathological review by the pathology panel categorises the thyroid lesions into 10 diagnostic categories, including categorisation of atypical lesions and those showing borderline malignant changes. The majority of thyroid cancers that have occurred in the affected population post Chernobyl have been papillary carcinoma, and the majority of these have been of one subtype, associated with a particular molecular biology. However, a recent review of the data available from the CTB, in combination with that available from earlier projects, suggests that different pathological types of thyroid cancer may arise in a radiation exposed population with differing latencies. It is now highly likely that there is a series of cohorts of exposed individuals, with differing levels of risk, influenced by their age at exposure and possibly genetic predisposition, who may develop different types of thyroid cancer with differing latencies.

Biospecimens from the CTB have been provided to the major research groups in Europe, the USA and Japan who are involved in the studies of the consequences of the Chernobyl accident. A simple online application process, accessible either directly or via the CTB website, provides applicants with information about the resources held in the CTB and filters allow the researcher to check the availability of the samples/data they require for their study. All applications are reviewed externally. The provision of extracted nucleic acid from thyroid tissue, rather than each researcher being provided with a small piece of tissue, maximises the amount of data that can potentially be obtained from a single operative specimen and enables multiple molecular biological studies to be carried out for each case. This approach has fostered international collaboration and reduces the chance of competition, and even friction, between groups in their requests for this material. Researchers who obtain material from the CTB agree to provide the results of their study on a case by case basis to enable combined analysis to be carried out at a later date. Historically, all movement of samples between the collecting centres and the coordinating centre and transfer of samples to researchers, were completed under a material transfer agreement (MTA). With the completion of the research database, the Steering Committee agreed to a proposal to change the wording of the MTA to encompass projects that involve either release of samples and clinical data, or samples and clinical data plus research data, or clinical and research data only. This revised material and data transfer agreement (MDTA) has been in use since March 2012.

The component hardware of the CTB Data Warehouse (file servers, storage media and communication interfaces) are housed in a secure environment at Imperial College, London. Controlled access to the information held in the Samples database and the Research database is via the web-enabled CTB portal, accessible directly or from the CTB web site. From March 2013 a research data uploader will be available to facilitate researchers returning data to the Data Warehouse. The IT infrastructure is described in detail under WPs 6, 7 and 9 below.

The project is overseen by a number of committees / panels that were established in consultation with the financial sponsors of the project and the Ministers of Health in Belarus, Ukraine and Russia. The work of these committees / panels is coordinated and managed by the coordinating centre. The project is overseen by a steering committee, which comprises representatives from the funding bodies and senior members of staff of the Institutes in Russia and Ukraine, and a scientific advisory board, which includes experts in tissue banking, information technology, and representatives of the scientific community that use the resource. Representatives on the advisory board also provide additional guidance in specialist areas such as dosimetry and information technology. As noted above, the pathology panel is responsible for the pathological review and determination of a consensus diagnosis for all cases, and the external review panel reviews all applications for access to the resources of the CTB.

Ten WPs were described for the project in this funding period. WPs 1 to 5 comprised the core activity of the tissue bank, i.e. the recruitment of new patients; the collection, processing and storage of tissue and blood samples and the presentation of the cases to the pathology panel. The objectives of WP10 related to the overall coordination and management of the project, including the receipt and management of the peer review of applications to access the CTB and shipment of samples to researchers whose projects had been approved. WPs 6, 7 and 9 related to the development and maintenance of the databases and the IT infrastructure. WP8 covered the assessment of available dosimetry information on the CTB cases, retrieval of the relevant data and inclusion in the Samples database. The EC consortium was comprised of five partners.

A comprehensive suite of standard operating procedures (SOPs) covering all aspects of the collection, preparation, storage and extraction of biomaterials, has been developed in consultation with the professional staff in Ukraine and Russia and with the Imperial College Healthcare Tissue Bank and the Wales Cancer Bank, with which the CTB shares a number of common SOPs (Professor Thomas, the CTB director, is the designated individual (DI), responsible to the Human Tissue Authority of the UK for the Imperial College Healthcare Tissue Bank, and Scientific Director of the Wales Cancer Bank). This cooperation ensures that each of the tissue banks benefits from technical developments in any of the banks. Since September 2012, the CTB Coordinating Centre has been co-located with the Imperial College Healthcare Tissue Bank in refurbished laboratory and office space at the Charing Cross Hospital campus of Imperial College in London. The laboratory is purpose-built and equipped for tissue banking.

Ethic approvals have been agreed with the relevant authorities, conforming to the requirements of each country involved and those of the funding organisations. The SOPs relating to the collection and handling of human material have been approved by the institutional review boards (ethics committee equivalent) of each of the collecting centres and ICREC (the Imperial College Research Ethics Committee), which also maintain the oversight of the project on an annual basis.

Results by WP

WP1: Collection of appropriate informed consent (IEM, MRRC RAMS, ICL)

Appropriate informed consent has been received from all patients whose material is stored in the CTB. The EC has provided support for a designated member of staff in each centre who has been responsible for obtaining informed consent. In cases where the patient is a minor, their parent / guardian signs the consent form on their behalf. Each patient is assigned a unique tissue bank number and all information in the database and released to researchers carries this code. The facility to trace this code back to the individual patient is only accessible by the clinicians in the collecting centres.

In most cases a blood sample is taken pre-operatively. Date of birth, sex and residency at the time of the Chernobyl accident are recorded. The signed forms are kept at the centres in Kiev and Obninsk and are audited by the project manager. The consent forms have been updated during this funding period and agreed by the relevant ethics committees in each of the centres and by ICREC. As noted above, these bodies review the project on an annual basis; no breaches of ethics have been reported.

At the start of the last period of funding in May 2008 it was anticipated that there would be a minimum of 150 new cases from Ukraine and 100 from Russia each year.

WP2: Collection and separation of blood (IEM, MRRC RAMS)

For the majority of cases a sample of blood (approximately 17 mls, collected in two 5.5.mls normal S-monovette tubes and two 2.7 mls EDTA containing S-monovettes) will be been taken pre-operatively. Each tube is labelled with a code which links to the patient. Serum is separated according to an agreed protocol from the samples collected in normal S-monovettes (this yields approximately five 1-ml samples). Serum is stored in tubes labelled with the patients identifying number, in a –20-degree-of-Celsius freezer. The coordinates for each tube are logged in the computer database.

Blood contained in the two EDTA containing tubes is stored at -80 degrees of Celsius. Extraction of DNA from one of the tubes is performed according to the agreed protocol and aliquotted appropriately. The remaining tube remains in the freezer for future use. The number of aliquots of serum and 5 microgram aliquots of DNA extracted from the whole blood sample are documented and their position coordinates noted. Thyroid clinics take place at least three times per week in each of the Institutes, and in order that patients are not missed, the person taking the blood sample and obtaining informed consent is required to be present throughout the clinic.

WP3: Documentation of pathological specimens (IEM, MRRC RAMS, ICL)

A protocol for a uniform method of dealing with the tissue samples has been agreed by the pathology panel and is used in IEM and MRRC RAMS. At no time is more tissue taken during surgery than is appropriate for the diagnosis and treatment of the patient; in many cases a total thyroidectomy is the appropriate operation.

All resected thyroid tissue from the study cohort has been documented. It is imperative that fresh material is taken from the operative specimens as quickly as possible, by a qualified pathologist. Diagnostic tissue samples are formalin fixed and routinely processed. The fresh material is immediately snap frozen and a section cut. Where the size of the tumour permits, three or more samples are taken from tumour and from the surrounding normal thyroid. These are numbered to correspond to those taken for diagnosis. To date, the relative positions of these samples within the thyroid specimen are marked on a diagram that has been scanned into the database, but the facility now exists in the Samples database to complete the diagram electronically. It is likely that this change in practice will be slow to introduce and paper records will continue to be maintained.

Each sample is stored at -80 degrees of Celsius in individual containers until required and is given a coordinate to facilitate location at a later date. In 2010-11 an audit of the documentation and storage of the samples was undertaken. There were no problems with retrieval of the documents and the management of the stored samples was good in both centres, although a risk was identified in the lack of sufficient cold storage capacity. Storage capacity was therefore increased in both centres during this funding period not only to provide additional capacity for new tissue samples and extracts, but also to ensure that back-up storage was available in the event of a freezer failure.

Patients who are registered with the CTB and have a diagnosis of thyroid cancer attend the follow-up clinics at the two Institutes 4-6 months after their operation and thereafter on an annual basis for at least 10 years. The patient is usually asked about their general health, and a routine blood test is performed to check their thyroid hormone and thyroglobulin levels. Ultrasound scans of the neck are also often performed to check for any enlarged local lymph nodes, which may represent an early sign of a recurrence of their cancer. Thyroid cancer in the young patient has a very good prognosis compared with other cancers, but a recurrence rate of about 30 % is expected. The majority of these patients will respond well to further radioiodine treatment, but it is predicted that around 1 % will die of their disease many years later. It will be important to use the data generated from the CTB to determine whether outcome data could be predicted from the molecular biology of the primary cancer. This would aid treatment tailoring for thyroid cancer in non-radiation exposed populations, and therefore may have a much wider application in countries where thyroid cancer incidence is increasing, such as the USA. In addition, acquisition of health outcome data on these patients will give significant insight into the effect on co-morbidity for these patients and whether this is due to exposure to radiation or the treatment they receive. The presence of a significant cohort of patients with non-radiation exposed thyroid cancer (i.e. those born after 1 January 1987) treated at the same clinics will significantly increase our chances of dissecting the late effects of radiation from the late effects of thyroid cancer. Studies of radiation exposed individuals (such as the Life Span Studies of the Hiroshima and Nagasaki bomb survivors) have suggested that there are minimal non-cancer effects in the population but, as yet, there is little available evidence on any effects of long-term exposure to Cs-137 in the environment.

WP4: Provision of material for the pathology review panel (IEM, MRRC RAMS, ICL)

Sections stained with haematoxylin and eosin are provided at the pathology panel meeting by the senior pathologists from Ukraine and Russia for each case included in the CTB. Membership of the pathology panel was reviewed at the start of this funding period and new members recruited from 2009. The panel operates to agreed Terms of Reference and has met annually. However, with the increasing workload it became necessary, with the agreement of the steering committee, to extend the meetings to 2.5 days from 2010. Early in the life of the project, funding was secured to provide a photomicroscope in each of the collecting centres. An image of each section was taken and stored in each centre. An objective for the project from 2008 was to carry out an option appraisal for an updated solution for the storage and online retrieval of digitised images. This was completed in 2011 and discussed by the pathology panel and the scientific advisory board. Both committees strongly supported the proposal to ensure that images of the sections submitted to the pathology panel and of the H&E section from each batch of sections cut from both frozen and FFPE blocks of material used for research, were scanned and stored. Provision of new, efficient scanning microscopes in each of the Centres would have been prohibitively expensive at the present time, although an injection of local (non-CTB) funds into the MRRC provided for a scanning microscope to be installed there. A suitable microscope was not available within Imperial College and therefore the decision was therefore taken to purchase an AxioScan microscope to be installed at the Coordinating Centre, funded by Imperial College with contributions from the NCI and SMHF awards. EC funding was not used for the microscope. The capacity of the scanner is such that scanning of retrospective cases in a reasonable time scale will also be practical. A further advantage of this instrument is that it links to PathXL, the remote image storage software, which is already installed at Imperial College. This offers the possibility of members of the pathology panel reviewing slides in their home Institutions, and limiting face to face discussion to only those cases for which a consensus diagnosis proves to be harder to reach. This will also enable the pathology panel, including the pathologists based in Ukraine and Russia, to collaborate more effectively in research studies using material from the CTB.

WP5: Extraction of nucleic acid (RNA and DNA) from tissue samples and DNA from blood (IEM, MRRC RAMS, ICL)

As noted above, multiple aliquots of DNA and RNA are extracted from frozen tissue and made available to researchers, rather than issuing the tissue block itself. This enables multiple research groups to use material from the same cohort of cases.

During this reporting period, the extraction procedures and quality assurance of the samples have been further refined. RNA and DNA are extracted from frozen tissue samples according to the SOP. Extracts are aliquotted into standard amounts (5 microgrammes in 20 microlitres and 3 microgrammes in 50 microlitres respectively).

The extraction of nucleic acids is an essential part of the project and requires considerable time input from skilled staff in order to maximise the availability of material. Staff from IEM and MRRC RAMS have been trained in the execution of the SOPs and now perform all the routine extractions. The protocols have been developed to provide optimal yield of both DNA and RNA from a single sample. It is essential that at each stage there is adequate documentation of yield of nucleic acid and the number of aliquots produced. The coordinating centre, in collaboration with the molecular biologists from IEM and MRRC RAMS, undertook a detailed review of the effects of different processing and storage procedures on key quality measures for molecular studies. These studies showed that RNA quality is easily compromised if the tissue is defrosted in storage, however provided the tissue is maintained in a frozen state, high quality RNA can be extracted when tissue is stored for up to 10 years.

The critical issues that affect the successful extraction of long strands of DNA and RNA were also examined at the coordinating centre.

Numerous quality control steps are now included in the extraction protocol, ensuring that no contamination between samples occurs during extraction. In addition the quality of the RNA is monitored by use of agilent bioanalysers. The graphical output from these machines is entered into the database and is available on each individual sample of RNA requested by researchers. Many samples are of sufficient quality for the various RNA array platforms used in research. However, even samples that fail this stringent QA can be used for other purposes – e.g. single gene assays using quantitative real-time polymerase chain reaction (qRT-PCR) etc. Quality of DNA is assayed by gel electrophoresis and PCR.

The majority of researchers only require 1 aliquot of nucleic acid, usually derived from matched tumour and normal tissues, for their studies. However, some research projects require more nucleic acid per case and are therefore issued with multiple aliquots from one case. For projects that use sections from formalin FFPE samples, multiple sections are usually issued per case to ensure that researchers have serial sections to use for complimentary histological stains or to ensure that sufficient RNA or DNA can be extracted from them. In corrdinated projects that take a systems biology approach, different analytes can be used from the same tumour. A good example of this is the Genrisk-T consortium that has used RNA and DNA extracted from frozen normal and tumour tissue for identification of mRNA expression and DNA copy number alteration, and single nucleotide polymorphism (SNP), and miRNA extracted from FFPE sections from the same cohort of cases. RNA from the same cases is used to type any rearrangements of the ret oncogenes present in the sample and DNA from tumour to type the presence of the common BRAF mutation found in papillary carcinoma. Other projects are comparing germline SNP analysis from DNA extracted from blood with messenger RNA (Mrna) expression in tissue.

WPs 6, 7 and 9 (ICL, SUT)

Historically, each of the collecting centres has maintained an Access database of cases from that country that have been entered into the CTB and this data was transferred into a central database (a DB2 database) held at the coordinating centre. Three WPs involving the development and maintenance of the IT infrastructure were detailed in Annex 1:

- WP6: Maintenance and further development of the CTB database.
- WP7: Real-time updating of data on the central database.
- WP9: Development of data warehouse for research results.

Whilst initially these three activities were being progressed in parallel, it became clear that an integrated approach was not only feasible, but would offer significant benefits. Consequently, in discussion with the scientific advisory board and with the approval of the steering committee, a new schema for the IT infrastructure was developed. A series of component file servers with associated storage capacity, corresponding front-ends / interfaces and management and search modules, are held on secure servers at Imperial College and comprise the CTB Data Warehouse.

The samples data, previously held in the DB2 database, has been audited and transferred into an improved database schema. The updated Samples database not only provides more efficient data entry and access to the data, but is also compatible with many other database systems. This is important for sustainability so that should funding no longer be available for the project, IEM and MRRC RAMS will each have their own data, accessible and manageable locally on their own computers and on platforms known and used by a large number of users and developers. Automated scripts for transfer of data into other database systems are being built and tested.

The web interface allows members of the CTB to record information in the database on clinical and pathological diagnosis, dose, surgical treatment, the number, type and relative location within the operative specimen of biospecimens taken and the detailed pathology of FFPE specimens. The locations where the specimens are stored and the detailed information on extraction of RNA and DNA (quality assurance data, number and volume of aliquots etc.) are also recorded. Individual samples (tissue blocks and aliquots of nucleic acid) are tracked until issued to research projects. Access to the samples database is restricted to a few DIs at the collecting centres and at the coordinating centre. Staff at the collecting centres are able to view the data for the patients from that country only; designated staff at the coordinating centre can view the full database. Users can report from the database using pre-designed reports designed for, and restricted to, individual user groups. Online data entry has been a significant change for the pathologists in both centres, however, the interface was designed to closely mirror the Forms that have been used to record pathology information thus facilitating a smooth transition to a new system. Various widely used technologies - like Ajax, JavaScript and JQuery - have been utilised in order to provide an intuitive and familiar user interface. Additionally, the reports from the database provide a copy of various Forms for off-line filing, as necessary.

The research database

WP9 of Annex 1 defined the objective to establish a data warehouse for research results generated using CTB materials. The option of being able to share the data and avoid unnecessary duplication of experiments was attractive and the scientific advisory board supported the concept of integrating the Research (results) database as a component of the new CTB Data Warehouse alongside the Samples database and the associated access portal. It was decided to use the Genrisk-T project data as a demonstrator project for the data warehouse. This project, which adopted a multidisciplinary approach to define the genetic component influencing the risk of radiation-induced thyroid cancer at low doses of radiation, was chosen as it had generated data of many different types (pathological, 3’Affymetrix array, BAC array CGH, qRT-PCR and Affymetrix SNP) much of which has already been published. This data was derived from a defined cohort within the CTB and used DNA and RNA derived from the same frozen piece of tissue.

A research data uploader will allow researchers to upload data into the research database, but to view only their own data there. Researchers are asked to return their results using the sample code as the identifier. This enables research data from individual samples and individual patients to be collated together.

The CTB portal (see https://cisbic.bioinformatics.ic.ac.uk/ctb/html_ctb_public/ online for further details)

The CTB portal provides on-line access to the resources of the CTB. Accessible either directly or from the CTB web site, the Portal gives access to a simple, but powerful, search facility which allows a researcher to search the databases and check if samples and / or data are available that match the requirements for their study. Researchers who wish to interrogate the databases must first register for access via the CTB portal. Once access has been approved, data is drawn from the samples and the research databases into the Integrative database in response to individual searches.

Biomaterials are defined by: the type of thyroid tumour (from the consensus diagnosis of the pathology panel), the origin of the sample - blood, tumour tissue or normal tissue, the type of sample - FFPE section, extracted DNA or RNA and key patient information such as age at exposure, residency etc. An additional filter, the dose received by the patient, will be added to the portal in the 1st quarter 2013. The results of the search show the number of cases that match the criteria and the number of samples that are available. The portal went live on the 25th anniversary of the Chernobyl accident. Researchers who require access to the linked research data may add this to their search.

The PI is then guided through the on-line application process to request samples, samples plus data or data only. Management tools embedded within the portal, and accessible only by the secretariat, facilitate the processing of applications and tracking progress through the review and approvals process. A number of checks were introduced into the process to assist the applicant and to ensure that all the required information is submitted. Once the applicant has submitted their application online, the secretariat checks that the application is complete. The status of the application is altered as it progresses through each stage of the process and an e-mail is automatically generated and sent to the PI acknowledging each change of status: (submitted for review, more information required etc.). The software automatically compiles the various sections of the application into a .pdf file, which the secretariat then forwards to the external review panel.

The integrative database produces a comprehensive list of all the cases identified that match the search criteria entered by the applicant. This listing is available only to the secretariat and is a significant step in the automation of the process of selecting appropriate samples for a project. The process can never be totally automated and expert oversight of the pathological information will always be required, however, the initial screening provides a significant improvement.

The CTB portal also provides the access to the research database for researchers who have used samples from the CTB to upload their raw data.

The system was developed with assistance from the Bioinformatics Support Services (BSS) at Imperial College and this collaboration is continuing for on-going user testing and fine tuning of the system.

In the current phase of development, raw data will be stored and the 'originator' of the data will be advised of requests to access the data. Analysed data will not be stored, but, in time, it is hoped that a number of bioinformatic tools can be made available to users of the data warehouse.

Security and integrity of the data in the CTB Data Warehouse is of paramount importance. With regards to the samples database and corresponding front-end, the access to data sets is appropriately restricted according to a country (e.g. Ukraine, Russia) and a role (e.g. pathologist, lab technician, administrator) to which a user belongs. Integration of the Samples database with the rest of the system and transfer of data between different elements of the overall CTB Warehouse are submitted to the same strict security requirements and are designed to minimise the risks of data loss or theft. Regular, time and place restricted updates of the Integrative database from the Samples and Research databases provide the up-to-date link between the research data and the key clinical-data elements required by a researcher.

WP8: Inclusion of dose estimates and I status in the CTB data base

A comprehensive report was prepared by HMGU during the first year of this reporting period detailing the range of dosimetry measurements and estimations made in the contaminated areas since the Chernobyl accident. Imperial College issued an invitation to tender for a sub-contract to extract dose information, where this was available, or to calculate the dose received in the absence of a recorded dose, for all cases in the CTB. The MRRC RAMS and the Ukrainian Institute for Radiation Protection (UIRP) successfully tendered for the work to match the cases in the CTB to dosimetry records in their respective countries and to add this data to the CTB database. The work required close collaboration with the pathologists in the collecting centres to match CTB cases with dose records. A dosimetry working group was convened, which established common criteria for assessment of dose in Ukraine and Russia and set a number of new milestones and deliverables for the dosimetry WP. A project initiation document (PID) was prepared and regularly reviewed by the Working Group.

Individuals for whom the dose to the thyroid was measured and/or estimated fell into four groups:

- Group 1: Individual thyroid dose measured and questionnaire completed detailing relevant parameters such as raion of residence, diet (locally produced milk and vegetables consumption), evacuation from the contaminated area) at the time of and immediately after the accident.
- Group 2: Measurement-based individual thyroid doses, but no questionnaire data.
- Group 3: Average age-gender settlement specific thyroid doses adjusted to personal history (questionnaires completed but no direct measurements).
- Group 4: Average age-gender settlement specific thyroid dose (no questionnaires).

It was agreed that for most biological applications a single figure estimation / measurement of dose and the arithmetic mean was the most that would be required by researchers. The dosimetry working group agreed that a slightly different grouping of patients and different methodologies for calculating dose could be applied in Ukraine and Russia in line with their normal procedures. These details have been documented by each institute in an operating manual and this information will be made available to researchers who require this level of detail.

Only a small number of cases in the CTB have direct thyroid dose measurements (8 % of the cohort in Ukraine and none in Russia), and in the majority of cases (74 %) dose has been calculated on the patient's residence at the time of the accident. The cases that have direct thyroid measurements are those enrolled in the Ukraine-American cohort, an epidemiological study supported by the NCI.

Dose has now been calculated for all cases reviewed by the pathology panel up to and including its meeting in April 2011 and work is continuing to complete the cases reviewed in April 2012. The collaboration with the Ukrainian Institute of Radiation Protection and the MRRC RAMS is continuing so that dose will be calculated for new cases recruited for the CTB.

WP9: Development of the data warehouse is reported above with WPs 6 and 7.

Potential impact:

The CTB has already had a considerable impact on tissue banking for research and on the dissection of mechanisms involved in radiation related cancer. However, it is clear from the research that has already been carried out on material from the CTB that continued collection and distribution of multiple types of biospecimens from the same patient are important in the further dissection of the way in which the environment, genetics and physiology all interact to alter tumour development in exposed populations. The acceptance of a straight line dose response (linear, non-threshold: LNT) to radiation in terms of cancer development has recently been questioned. It is now more widely accepted that different populations, whether separated on a genetic or age-related basis show widely different susceptibilities to induction of cancer. What is now becoming clear is that different types of cancer, even within the same tissue, may have differing latencies. Prolonged detailed study of a carefully described population such as those exposed to radiation in childhood from the Chernobyl accident or those exposed to radiation from Hiroshima and Nagasaki will be the key to gaining a better understanding of lifetime risk following exposure to radiation. As evidenced by the public response to the incident at Fukushima last year, the general public is clearly concerned about the consequences of a nuclear accident. The continued study of the population covered by the CTB project will be important to determine the potential risk to members of the general public from nuclear power generation. Many governments recognise that nuclear power is almost inevitable in order to reduce reliance on carbon, and the resultant problems for the environment, and the public understanding of the health consequences of the Chernobyl accident will play a key role in the politics of nuclear power for some time to come.

Dissemination of Information

Professor Thomas' involvement as a researcher both in the thyroid cancer and low dose radiation fields means that she is a regular speaker at international conferences and meets a wide range of researchers. This breadth of interactions is invaluable not only in stimulating interest among scientists to use the resources of the CTB, but also in providing scientific leadership for the CTB and proposal of new ideas to the scientific advisory board.

The CTB received considerable national and international publicity in 2011 both in relation to publicity surrounding the 25th anniversary of the Chernobyl accident and as a result of the Fukushima accident. Professor Thomas and others from the project were interviewed by both broadcast and print media and the BBC has produced a number of programmes to mark the 25th Anniversary of the Chernobyl accident. Key presentations and programmes are listed below. Professor Thomas also edited a Special Edition of Clinical Oncology (http://www.chernobyltissuebank.com/clinical_oncology.html) which reviewed the health consequences of radiation exposure. This publicity led to a surge in interest in the work of the CTB. The table below gives a selection of the media interviews and presentations given by Professor Thomas at the time of the 25th anniversary of the Chernobyl accident and following the incident at Fukushima.

Following the successful model of the 2011 Symposium, the remaining sponsors of the CTB, the NCI and SMHF, have proposed that two further symposia will be held; one of these will be held as part of an international radiation research meeting. The aim of the symposia will be to provide information to the scientific community and to the wider public about the research results obtained from the CTB.

In addition to the symposia, short publicity pamphlets, which have been successfully piloted during the current funding period, will be provided as hand-outs at major research meetings such as AACR and the Federation of European Cancer Societies and the International Congress of Radiation Research, amongst others.

Professor Thomas and other members of the CTB will continue to promote the tissue bank and the research achievements using the materials and data at scientific meetings.

List of websites: http://www.chernobyltissuebank.com

Dr AR Galpine
Project Manager CTB
Department of Surgery and Cancer
Imperial College London
Room 11L05
Charing Cross Hospital
Fulham Palace Road
London W6 8RF
Tel +44-020-33117342
email: a.galpine@imperial.ac.uk
ctb-figures-and-tables.pdf