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Periodic Report Summary - EPIRADBIO (Combining epidemiology and radiobiology to assess cancer risks in the breast, lung, thyroid and digestive tract after exposures to ionizing radiation(…))

Project context and objectives:

The main aim of EPIRADBIO is to combine epidemiology and radiobiology to assess cancer risks in the breast, lung, thyroid and digestive tract after exposures to ionizing radiation with cumulated equivalent doses of the order of 100 mSv or below.

Such exposures are of central importance for radiation protection, since they correspond to:
-the dose limit for occupational exposure (100 mSv in five years)
-exposures currently occurring in the work place (generally below 100 mSv over lifetime)
-exposures currently occurring due to medical diagnostics, e.g, by CT scans causing equivalent doses in the order of 10 mSv per examinations

While most of these exposures are from low-LET radiation, also exposures to high-LET radiation occur. Thus, EPIRADBIO will explore cancer risks not only from the important types of low-LET radiation but also from alpha radiation. It is of urgent importance to analyse these cancer risks, because:
-recent epidemiological results challenge the assumption of a dose-dose-rate effectiveness factor (DDREF) made presently for radiation protection
-the non-linear dose response of non-targeted effects and the differences in gene expression in the low-dose and medium/high-dose region question the linear-no-threshold dose-effect model used presently for radiation protection.

Due to lack of statistical power, conventional epidemiology has limitations in studying organ specific health effects from exposures with equivalent doses of the order of 100 mSv and below. Radiobiology can provide insights into basic mechanisms of carcinogenesis after radiation exposures but does not give quantitative results for risks of humans. EPIRADBIO proposes an innovative approach combining epidemiology and radiobiology in order to address the problem: Incorporation of radiobiological results into the evaluation of epidemiological data on the basis of molecular epidemiology and models of carcinogenesis.

In order to supply a new basis for the risk assessment underlying current radiation protection, the key objectives of EPIRADBIO are to:
-perform measurements of telomere lengths, array-based comparative genomic hybridisation and other 'omics' with cancer tissue and blood samples from members of outstanding radioepidemiological cohorts in order to characterize key processes of carcinogenesis in humans exposed to low dose radiation
-analyze radiation responses of stem cells and low dose perturbation of intercellular communication in 2D and 3D models using human, normal breast and lung epithelial cells in order to elucidate further key processes of carcinogenesis and supplement the studies of samples from epidemiological cohorts
-integrate the new radiobiological results in models of carcinogenesis in order to include this knowledge in an evaluation of key epidemiological data
-derive cancer risks including individual risk factors after exposures to ionizing radiation with cumulative equivalent doses in the order of 100 mSv or below for supporting radiation protection.

Project Results:

Sub-project 1 Genomic instability and individual susceptibility

WP 1.1: Individual susceptibility to genomic instability: Epidemiology and radiation biology studies in French haemangioma patients
Authorisations have to be obtained to perform studies on the cohort in the context of the EPIRADBIO project. A biobank was formed, technics required for the analysis of telomere length by Flow Cytometry were established, and the existing protocols for cytogenetic and microscopy analysis were adapted to the needs of EPIRADBIO and the study of the French haemangioma cohort, respectively. Protocols for cytogenetic analysis and fluorescent microscopy are already well established and will be used 1) to confirm results obtained by Flow Cytometry (mean telomere length) and 2) for the analysis of chromosomal aberrations and telomere heterogeneity. To validate data of mean telomere length obtained using both microscopy analysis and Flow Cytometry, the Southern blot technic is introduced in the laboratory.

WP 1.2: Genomic instability in tumoral tissues of radiation-related breast and lung cancer
Serial frozen sections were prepared from breast cancer samples from exposed and non-exposed female workers at Mayak. In the majority of cases, macrodissection after visual control was performed on the slides in order to enrich the tumor cell population. From all currently suitable samples, DNA was extracted using QIAmp Mini DNA kit with RNAse treatment. Quality control was performed by using the BIOMED-2 multiplex control primer set. At least 400 bp fragments were amplifiable from all samples. From the same tissue samples and additional control samples of normal breast tissue, RNA and miRNA was extracted after lysis using the MagnaLyser. miRNAs were extracted using the High Pure miRNA Isolation Kit with DNase treatment. Currently, array CGH and miRNA expression profiling by next generation sequencing (Illumina platform) is carried out.

For lung cancer samples from MAYAK workers, techniques adapted for FFPE material with reduced DNA quality have been established.

WP1.3: Genomic instability in thyroid cancer
Taken together, literature suggests a significant change of miRNA regulation in thyroid cancer and in response to radiation. Patients who developed thyroid cancer in the wake of the Chernobyl incident received high doses of radiation. This raised our interest to evaluate the miRNA profile of a cohort of these patients particularly with regard to the question whether a radiation specific miRNA signature can be identified. Using an approach of combining miRNA Microarray technique with qRTPCR, we found a signature of three miRNAs which show a reduced expression in exposed versus unexposed thyroid cancer tissue. As the miRNAs are expressed at a very low level in both tissues, the effect is only marginal. We have moved on to validate miRNAs of interest from analysis carried out by Munich and by VUMC using qRT-PCR. These results are currently being evaluated and the results will be presented in June. We have experience of this type of approach in other tumour types (breast and hepatobiliary cancers) which is unrelated to radiation but to patient prognosis and have found significant associations. It therefore seems likely that in the case of radiation induced thyroid cancer, the finding that there is no significant association with radiation exposure is a genuine finding. Therefore we are now focusing on changes in methylated DNA and association with radiation exposure, as we had said in the original technical annex.

The results show that there is no strong association between radiation exposure and up- or down-regulation of individual miRNAs. There is, however, a weak association for 3 miRNAs with radiation exposure that could be tested further using an extended dataset to improve statistical power. A preliminary model has been formulated for the integrative analysis of miRNA and mRNA gene expression.

Sub-project 2 Radiation induced perturbation and activation of intercellular communication involved in carcinogenesis

WP2.1: Low-dose induction of proliferation, differentiation and genome instability in stem cells
Progress has been made in setting up the procedures for collecting and isolating cells from primary breast tissue obtained from reduction mammoplasty's at the University of Rostock. Isolated cells from tissue are allowed to form mammospheres in defined medium. Initial ethical approval has also been obtained for accessing primary breast tissue in Belfast with the first samples to become available later this year. Dose-response curves have been obtained by both QUB and UROS for DNA damage induction and cell survival in irradiated MCF10A and compared. HME1 cells have also been characterised in Belfast for both direct damage and bystander responses in the dose range from 0.01 to 2 Gy. 3D cultures of MCF10A have been established. Clonogenic survival and cell cycle progression have been analysed from dissociated cells irradiated in 3D mammospheres showing radioresistance. 3D acini structures have also been generated and are currently being characterised.

WP2.2 : Molecular mechanisms of radiation-induced carcinogenesis - role of tissue-environment and stress responses
EMT (epithelial mesenchymal transition: EMT is a process where epithelial cells lose their polarity and acquire mesenchymal phenotype. EMT occurs during normal tissue morphogenesis and also in carcinogenesis and fibrosis) induction after TGFβ treatment and alpha particle irradiation was studied by analysing the expression of epithelial and mesenchymal differentiation markers in BEAS-2B cells. After irradiation with 0.1 or 1 Gy, vimentin filaments started to re-organize and cells became spindle shaped. Cell samples were also analysed using two-dimensional difference gel electrophoresis (2DE-DIGE) technique with 2-3 biological replicates from each condition. Protein expression profiles were compared to control samples and a few proteins were found to have an altered expression level. Although, EMT was induced by TGFβ in the primary lung epithelial cells NHBE, irradiation alone was not able to elicit the effect. The effect of radiation on EMT was also studied in 3D tissue models in the presence (double treated) and absence of TGFβ. TGF-beta treatment induced clear morphological changes as well as changes in expression pattern of epithelial differentiation markers.

TERRA levels were measured in HeLa cells after irradiation using northern blot hybridization, and real time/reverse transcription-PCR. The initial experiments have been undertaken in the dose range 0.5-2 Gy in order assess the TERRA response at the upper range of low dose of 0.5 Gy relative to that at 2 Gy. In the next period, experiments by UNIPUV are under way to measure TERRA expression and gene amplification induction in various cell lines including normal epithelial lung and breast cells following low doses (100 mGy and below) in acute exposures and low dose rates (between 0.1 and 100 mGy/h) in chronic exposures. The chronic exposures are being carried out in collaboration with Partner SU.

WP2.3: Interactions of transformed and healthy cells - pro- and anti-carcinogenic effects
During this period, the generality of intracellular induction of apoptosis (IIA) through selective removal of precancerous cells via signalling from neighbour normal cells has been established using non-cancerous human lung fibroblast MRC5 cells as an effector cell line and scavengers of signalling molecules. Studies have also been extended to investigate the role of oxygen and diffusion distances of signalling molecules with respect to intercellular signalling of apoptosis to determine the response for a range of physiological conditions. A reduction in apoptosis in the transformed cells is also observed with decreasing oxygen concentration when they are cultured on their own. These changes in conditions contribute to development of mechanistic models. Preliminary data have identified TGF? as the major signalling cytokine participating in intercellular signalling.

In the reporting period, the previously developed mechanistic model has been extended to the second important signalling pathway identified experimentally, the nitric oxide/peroxynitrite pathway. A set of detailed simulations was performed, varying the lifetime and release rates of nitric oxide and investigating the relative importance of the two IIA pathways.

Sub-Project 3 Epidemiology, carcinogenesis and risk

WP 3.1 Breast cancer risk
The follow-up of female Swedish haemangioma patients has been extended up to the end of 2009. Data on breast cancer incidence are collected not only for the patients, but also for their mothers and sisters. The transfer of the Swedish haemangioma dataset to HMGU has been completed. Work has started to analyse these data. Dosimetry work with Monte Carlo calculations is in progress. New absorbed doses to the right and left breast have been calculated using the results from the MC calculations and the determination of distances to the different breast anlage.

The Italian part of the cohort of thyroid cancer survivors had yet been updated, both for new treatments (relapses, metastases, second cancers) and for the new events (second cancers) in 2009. The French part of the cohort has been updated for new treatments and new events, by access to the medical records of all the centres. The improvement of the dosimetry has started. The objective was to perform a standardized dosimetry for 843 thyroid cancer patients for whom an access to the technical records of radiotherapy was not possible. The method is based on the results of individualized dosimetry yet realised for 721 patients.

WP3.2: Lung cancer risk
The transfer of a dataset on cancer in the lung among Mayak workers from SUBI to HGMU has been completed on schedule at month 18. Work has started to develop models that are based on three stages of carcinogenesis, both with and without clonal expansion in the first stage, and effective numerical algorithms for solving the system of partial differential equations have been derived. These models are now applied to the Mayak lung cancer data.

WP 3.3: Thyroid cancer risk
Originally it was foreseen to use results from GENERISK-T as input for models of carcinogenesis. The approach could be strengthened considerably by the availability of tissue from all thyroid cancers that occurred in the UkrAm cohort. HMGU performs molecular measurements with these samples. Results will enter the model development for carcinogenesis. An agreement of EPIRADBIO partnerns with NCI has been signed allowing the transfer of epidemiological data for the UkrAm cohort in order to analyse these data with the developed models of carcinogenesis.

The results of Genrisk-T have been an important input in the further development of the research strategy of EPIRADBIO. As a first step, a focus of the research has been directed on the thyroid cancer samples of the UkrAm cohort. The activities on thyroid cancer among people affected by the Chernobyl have been further strengthened in a second step. Resources have been shifted from the management budget to research to perform molecular measurements for phenomenological healthy thyroid tissues from members of the UkrAm cohort. This will be an additional important input for modelling carcinogenesis in the thyroid.

The descriptive model of Preston et al. (2007) for thyroid cancer in the LSS has been optimized based on the likelihood ratio test. In the model, the ERR depends linearly on dose with an effect modification by attained age and age at exposure. Multi-model inference led to a negligible spreading of uncertainty ranges.

WP 3.4: Radiation risk estimation for radiation protection
The transfer of a dataset on cancer in the digestive tract among Mayak workers from SUBI to HGMU has been completed on schedule at month 18.

Work on evaluation of risk results has commenced by adapting an existing computer code (developed at HPA) that calculates lifetime risks using empirical models to enable calculations with the mechanistic models that will result from WP3.4 and from other parts of the project. The computer code developed at HGMU for evaluating mechanistic models was successfully integrated.

Potential Impact:

The innovative approach of combining epidemiology and radiobiology of EPIRADBIO will contribute to radiation protection by delivering quantitative cancer estimates for exposure scenarios of relevance in our society. The shape of the dose response will be elucidated for exposures to organs with equivalent doses in the order of 100 mSv or below. Tissue sensitivity will be quantified by studying cancer risks in the breast, lung, thyroid and digestive tract. Individual variability in radiation sensitivity will be analysed by studying genomic instability in peripheral lymphocytes of individuals with and without cancer, and by taking account of familial factors (e.g., breast cancer among close relatives or influence of the number of children and age at birth of first child on breast cancer risk) and lifestyle factors (e.g., smoking and alcohol consumption). Cancer risks of different radiation quality types will be assessed by comparing lung cancer risks from plutonium and external radiation of Mayak workers, and breast and thyroid cancer risks from incorporated iodine and external radiation. These studies will also supply information on cancer risks from internal versus external exposure to radiation. By bringing together pathologists, radiobiologists, epidemiologists, statisticians, mathematical modellers and dosimetrists and by including non- radiation research communities, EPIRADBIO applies a multi-disciplinary approach to solve the central problem of radiation protection.

List of Websites:

http://www.epiradbio.eu

Related information

Contact

Jurgen ERTEL, (Head of project management department)
Tel.: +49-8931873022
Fax: +49-8931873364
E-mail
Record Number: 56876 / Last updated on: 2014-07-04
Information source: SESAM
Collaboration sought: N/A