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After large-scale radiation accidents where many individuals are suspected to be exposed to ionizing radiation, biological and physical retrospective dosimetry assays are important tools to aid clinical decision making by categorizing individuals into unexposed/minimally, moderately or highly exposed groups. Quality-controlled inter-laboratory comparisons of simulated accident scenarios are regularly performed in the frame of the European legal association RENEB (Running the European Network of Biological and Physical retrospective Dosimetry) to optimize international networking and emergency readiness in case of large-scale radiation events. In total 33 laboratories from 22 countries around the world participated in the current RENEB inter-laboratory comparison 2021 for the dicentric chromosome assay. Blood was irradiated in vitro with X rays (240 kVp, 13 mA, ∼75 keV, 1 Gy/min) to simulate an acute, homogeneous whole-body exposure. Three blood samples (no. 1: 0 Gy, no. 2: 1.2 Gy, no. 3: 3.5 Gy) were sent to each participant and the task was to culture samples, to prepare slides and to assess radiation doses based on the observed dicentric yields from 50 manually or 150 semi-automatically scored metaphases (triage mode scoring). Approximately two-thirds of the participants applied calibration curves from irradiations with γ rays and about 1/3 from irradiations with X rays with varying energies. The categorization of the samples in clinically relevant groups corresponding to individuals that were unexposed/minimally (0–1 Gy), moderately (1–2 Gy) or highly exposed (>2 Gy) was successfully performed by all participants for sample no. 1 and no. 3 and by ≥74% for sample no. 2. However, while most participants estimated a dose of exactly 0 Gy for the sham-irradiated sample, the precise dose estimates of the samples irradiated with doses >0 Gy were systematically higher than the corresponding reference doses and showed a median deviation of 0.5 Gy (sample no. 2) and 0.95 Gy (sample no. 3) for manual scoring. By converting doses estimated based on γ-ray calibration curves to X-ray doses of a comparable mean photon energy as used in this exercise, the median deviation decreased to 0.27 Gy (sample no. 2) and 0.6 Gy (sample no. 3). The main aim of biological dosimetry in the case of a large-scale event is the categorization of individuals into clinically relevant groups, to aid clinical decision making. This task was successfully performed by all participants for the 0 Gy and 3.5 Gy samples and by 74% (manual scoring) and 80% (semi-automatic scoring) for the 1.2 Gy sample. Due to the accuracy of the dicentric chromosome assay and the high number of participating laboratories, a systematic shift of the dose estimates could be revealed. Differences in radiation quality (X ray vs. γ ray) between the test samples and the applied dose effect curves can partly explain the systematic shift. There might be several additional reasons for the observed bias (e.g., donor effects, transport, experimental conditions or the irradiation setup) and the analysis of these reasons provides great opportunities for future research. The participation of laboratories from countries around the world gave the opportunity to compare the results on an international level.

Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses 2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.

Depending on the ionising radiation exposure scenario, different biological or physical assays or combinations of these assays will be required to successfully recover the scenario and to obtain reliable dose estimates to aid medical decision making for the treatment of potentially exposed individuals. Interlaboratory comparisons (ILCs) are a central tool to validate and improve the performance of methods and laboratories. In most cases, such ILCs are performed under laboratory conditions where the influence of fixed parameters contributing to the uncertainty budget can be minimized. However, in a real-life scenario, the situation is much more complicated and it is therefore crucial to validate methods and laboratory responses under more realistic conditions.

Human C3A cells were exposed to extremely low frequency (50 Hz) magnetic fields (ELF-MF’s) up to 500 µT. They were subjected to the micronucleus assay using a Fluorescence In Situ Hybridization (FISH) technique with an in-house pan-centromere probe. We found no increased frequency in micronucleated cells and no change in the proportion of centromere positive over centromere negative micronuclei compared to the unexposed control cells. These results are in accordance with some, but in contradiction with other previously published investigations underlining that effects of environmental ELF-EMF’s on cellular DNA may be very subtle and that small changes or environmental influences may determine the outcome of a (geno)toxicity study. Interestingly, a low-level (5µT) exposure resulted in less than the background micronucleus frequency., Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics, V. 72 N. 2 (2019)

Purpose: Based on clinical and molecular data, breast cancer is a heterogeneous disease. Breast cancers that have no expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are defined as triple negative breast cancers (TNBCs); luminal cancers have different expressions of ER, PR and/or HER2. TNBCs are frequently linked with advanced disease, poor prognosis and occurrence in young African women, and about 15% of the cases are associated with germline BRCA1/2 mutations. Since radiotherapy is utilized as a principle treatment in the management of TNBC, we aimed to investigate the chromosomal instability and radiosensitivity of lymphocytes in TNBC patients compared to luminal breast cancer patients and healthy controls using the micronucleus (MN) assay. The effect of mutations in breast cancer susceptibility genes on chromosomal radiosensitivity was also evaluated. Methods: Chromosomal radiosensitivity was evaluated in the G0 (83 patients and 90 controls) and S/G2 (34 patients and 17 controls) phase of the cell cycle by exposing blood samples from all patients and controls to 2 and 4 Gy ionizing radiation (IR). Results: In the G0 MN assay, the combined cohort of all breast cancer, TNBC and luminal patients’ exhibit significantly elevated spontaneous MN values compared to controls indicating chromosomal instability. Chromosomal radiosensitivity is also significantly elevated in the combined cohort of all breast cancer patients compared to controls. The TNBC patients, however, do not exhibit enhanced chromosomal radiosensitivity. Similarly, in the S/G2 phase, 76% of TNBC patients do not show enhanced chromosomal radiosensitivity compared to the controls. In both the G0 and S/G2 phase, luminal breast cancer patients demonstrate a shift toward chromosomal radiosensitivity compared to TNBC patients and controls. Conclusions: The observations of the MN assay suggest increased chromosomal instability and chromosomal radiosensitivity in South African breast cancer patients. However, in TNBC patients, the irradiated MN values are not elevated. Our results suggest that the healthy lymphocytes in TNBC patients could handle higher doses of IR.

Purpose: Reliable dose estimation is an important factor in appropriate dosimetric triage categorization of exposed individuals to support radiation emergency response. Materials and Methods: Following work done under the EU FP7 MULTIBIODOSE and RENEB projects, formal methods for defining uncertainties on biological dose estimates are compared using simulated and real data from recent exercises. Results: The results demonstrate that a Bayesian method of uncertainty assessment is the most appropriate, even in the absence of detailed prior information. The relative accuracy and relevance of techniques for calculating uncertainty and combining assay results to produce single dose and uncertainty estimates is further discussed. Conclusions: Finally, it is demonstrated that whatever uncertainty estimation method is employed, ignoring the uncertainty on fast dose assessments can have an important impact on rapid biodosimetric categorization.

International audience; Purpose Reliable dose estimation is an important factor in appropriate dosimetric triage categorization of exposed individuals to support radiation emergency response. Materials and methods Following work done under the EU FP7 MULTIBIODOSE and RENEB projects, formal methods for defining uncertainties on biological dose estimates are compared using simulated and real data from recent exercises. Results The results demonstrate that a Bayesian method of uncertainty assessment is the most appropriate, even in the absence of detailed prior information. The relative accuracy and relevance of techniques for calculating uncertainty and combining assay results to produce single dose and uncertainty estimates is further discussed. Conclusions Finally, it is demonstrated that whatever uncertainty estimation method is employed, ignoring the uncertainty on fast dose assessments can have an important impact on rapid biodosimetric categorization. © 2016 Crown Copyright. Published by Informa UK Limited, trading as Taylor and Francis Group.

Purpose: Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. Materials and methods: Whole blood and separated lymphocyte samples were homogenously irradiated with 60Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. Results: Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. Conclusions: This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories.

International audience; Purpose: To identify and assess, among the participants in the RENEB (Realizing the European Network of Biodosimetry) project, the emergency preparedness, response capabilities and resources that can be deployed in the event of a radiological or nuclear accident/incident affecting a large number of individuals. These capabilities include available biodosimetry techniques, infrastructure, human resources (existing trained staff), financial and organizational resources (including the role of national contact points and their articulation with other stakeholders in emergency response) as well as robust quality control/assurance systems. Materials and methods: A survey was prepared and sent to the RENEB partners in order to acquire information about the existing, operational techniques and infrastructure in the laboratories of the different RENEB countries and to assess the capacity of response in the event of radiological or nuclear accident involving mass casualties. The survey focused on several main areas: laboratory’s general information, country and staff involved in biological and physical dosimetry; retrospective assays used, the number of assays available per laboratory and other information related to biodosimetry and emergency preparedness. Following technical intercomparisons amongst RENEB members, an update of the survey was performed one year later concerning the staff and the available assays. Conclusions: The analysis of RENEB questionnaires allowed a detailed assessment of existing capacity of the RENEB network to respond to nuclear and radiological emergencies. This highlighted the key importance of international cooperation in order to guarantee an effective and timely response in the event of radiological or nuclear accidents involving a considerable number of casualties. The deployment of the scientific and technical capabilities existing within the RENEB network members seems mandatory, to help other countries with less or no capacity for biological or physical dosimetry, or countries overwhelmed in case of a radiological or nuclear accident involving a large number of individuals. © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

Purpose: A European network was initiated in 2012 by 23 partners from 16 European countries with the aim to significantly increase individualized dose reconstruction in case of large-scale radiological emergency scenarios. Results: The network was built on three complementary pillars: (1) an operational basis with seven biological and physical dosimetric assays in ready-to-use mode, (2) a basis for education, training and quality assurance, and (3) a basis for further network development regarding new techniques and members. Techniques for individual dose estimation based on biological samples and/or inert personalized devices as mobile phones or smart phones were optimized to support rapid categorization of many potential victims according to the received dose to the blood or personal devices. Communication and cross-border collaboration were also standardized. To assure long-term sustainability of the network, cooperation with national and international emergency preparedness organizations was initiated and links to radiation protection and research platforms have been developed. A legal framework, based on a Memorandum of Understanding, was established and signed by 27 organizations by the end of 2015. Conclusions: RENEB is a European Network of biological and physical-retrospective dosimetry, with the capacity and capability to perform large-scale rapid individualized dose estimation. Specialized to handle large numbers of samples, RENEB is able to contribute to radiological emergency preparedness and wider large-scale research projects.

Purpose: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G(0)-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. Materials and methods: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy gamma-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. Results: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. Conclusions: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G(0)-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay.

Purpose: In the framework of the ‘Realizing the European Network of Biodosimetry’ (RENEB) project, two intercomparison exercises were conducted to assess the suitability of an optimized version of the cytokinesis-block micronucleus assay, and to evaluate the capacity of a large laboratory network performing biodosimetry for radiation emergency triages. Twelve European institutions participated in the first exercise, and four non-RENEB labs were added in the second one. Materials and methods: Irradiated blood samples were shipped to participating labs, whose task was to culture these samples and provide a blind dose estimate. Micronucleus analysis was performed by automated, semi-automated and manual procedures. Results: The dose estimates provided by network laboratories were in good agreement with true administered doses. The most accurate estimates were reported for low dose points (=< 0.94 Gy). For higher dose points (>= 2.7 Gy) a larger variation in estimates was observed, though in the second exercise the number of acceptable estimates increased satisfactorily. Higher accuracy was achieved with the semi-automated method. Conclusion: The results of the two exercises performed by our network demonstrate that the micronucleus assay is a useful tool for large-scale radiation emergencies, and can be successfully implemented within a large network of laboratories.

Results of the fragment analysis. Illustration of fragment analysis data of an SNP without loss of the mutant allele. (PDF 176 kb)

Purpose: In the framework of the ‘Realizing the European Network of Biodosimetry’ (RENEB) project, two intercomparison exercises were conducted to assess the suitability of an optimized version of the cytokinesis-block micronucleus assay, and to evaluate the capacity of a large laboratory network performing biodosimetry for radiation emergency triages. Twelve European institutions participated in the first exercise, and four non-RENEB labs were added in the second one. Materials and methods: Irradiated blood samples were shipped to participating labs, whose task was to culture these samples and provide a blind dose estimate. Micronucleus analysis was performed by automated, semi-automated and manual procedures. Results: The dose estimates provided by network laboratories were in good agreement with true administered doses. The most accurate estimates were reported for low dose points (≤ 0.94 Gy). For higher dose points (≥ 2.7 Gy) a larger variation in estimates was observed, though in the second exercise the number of acceptable estimates increased satisfactorily. Higher accuracy was achieved with the semi-automated method. Conclusion: The results of the two exercises performed by our network demonstrate that the micronucleus assay is a useful tool for large-scale radiation emergencies, and can be successfully implemented within a large network of laboratories. VII Programa Marco de Investigación y Desarrollo (VIIPM) de la Unión Europea. nº 295513 University Development Cooperation "VLIR Own Initiative Programme" enetre Bélgica and Sudáfrica. ZEIN2011PR387

Primers. The primer sequences (both forward and reverse). (PDF 29 kb)

Results of the fragment analysis. Illustration of fragment analysis data of an SNP with loss of the mutant allele. (PDF 227 kb)

The cytokinesis-block micronucleus assay in peripheral blood lymphocytes is a standardised and validated technique for biodosimetry. Automated scoring of micronuclei allows large scale applications as in population triage in case of radiation accidents or malevolent use of radioactive sources. The dose detection limit (95% confidence) of the micronucleus assay for individual dose assessment is restricted to 0.2 Gy but can be decreased to 0.1 Gy by scoring centromeres in micronuclei using fluorescence in situ hybridization (FISH). In the past the micronucleus assay was applied for a number of large scale biomonitoring studies of nuclear power plant workers and hospital workers. Baseline micronucleus frequencies depend strongly on age and gender. The assay was also already used for biodosimetry of radiation accidents. In a multiple endpoint biodosimetry study for dose assessment of a worker exposed accidentally in 2003 to X-rays, a good agreement was obtained between dose estimates resulting from the micronucleus assay, the scoring of dicentrics and translocations. Automated scoring of micronuclei in combination with centromere signals, allowing systematic biodosimetry of exposed populations, remains a challenge for the future.

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay, In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this StUdy' show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2 Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to I and 2 Gy, respectively, These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method. cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5. and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise. nucleoplasmic bridges were also estimated by the laboratories however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.

Micronucleus(MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis-block micronucleus(CBMN) assay and participation in the HUMN (Human Micronucleusproject) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleated cell (MNC) frequency are evaluated, and a reference range of normal values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted ina database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin B, the percentage of fetal calf serum and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e. normal) subjects was 6,5% and the interquartile range was between 3 and 12%. An increase in MNC frequency with age was evidente in all but two laboratories. The effect of gender, although not so evident in all databases was also present wtih females having a 19% higher level of MNC frequency (95% confidence interval 14-24%).Statistical analysis were performed using random-effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods and scoring criteria explained 75% of the total variance, with the largest contribution attributed to laboratory methods.

All currently known high to intermediate risk “breast cancer genes”, including BRCA1 and BRCA2, are involved in the DNA damage response pathway. Heterozygous germline mutations in these genes predispose to breast and ovarian cancer. In addition, such mutations may also result in enhanced radiosensitivity mediated by chromosomal instability after exposure to ionizing radiation, leading to a higher risk to develop radiation-induced breast cancer. However, results of currently available clinical studies evaluating carcinogenesis and in vitro studies comparing chromosomal radiosensitivity in mutation carriers and non-carriers are inconclusive. Nevertheless, insights into the radiosensitive phenotype of healthy tissues of mutation carriers is of the utmost importance for the safe use of ionizing radiation for diagnostic purposes or radiotherapy treatment. In this thesis, we evaluated in vitro radiosensitivity in carriers of a mutation in DNA damage response genes by means of two different assays. The first assay, the G2 micronucleus assay, is a cytogenetic assay in which MN are analyzed in cells irradiated in the G2 phase of the cell cycle. This assay was developed to evaluate radiosensitivity in cells with a heterozygous BRCA1 or BRCA2 mutation. BRCA1 and BRCA2 have a function in homologous recombination (HR), the main DNA double strand break repair pathway activated in late S and G2 phase of the cell cycle. Furthermore, BRCA1 is also involved in the G2/M cell cycle checkpoint. The G2 micronucleus assay allows evaluation of both functions by means of two distinct endpoints: (1) the radiation-induced micronucleus yield, which reflects DNA double strand break repair capacity and (2) the G2/M checkpoint efficiency ratio, which allows evaluation of the G2 arrest capacity. Before applying the G2 micronucleus assay on BRCA mutation carriers, the assay was validated in a patient with Ataxia Telangiectasia (AT). AT patients are characterized by a manifest increased radiosensitivity. AT patients show biallelic inactivation of ATM, involved in both DNA double strand break repair by means of HR and G2/M checkpoint activation. We demonstrated a severely increased radiosensitivity with both endpoints when applying the G2 micronucleus assay in lymphocytes of this AT patient. In lymphocytes of healthy relatives with a heterozygous ATM mutation the radiosensitivity observed with this assay was intermediate between the AT patient and the control cohort. When applying the G2 micronucleus assay on lymphocytes of healthy BRCA1/2 mutation carriers, we demonstrated significantly enhanced radiation-induced MN yields in both BRCA1 and BRCA2 germline mutation carriers, pointing to an impaired DNA double strand break repair capacity in both groups. Furthermore, an impaired G2 arrest capacity was observed in BRCA1 mutation carriers. In healthy relatives who did not inherit the familial mutation, no enhanced radiosensitivity was observed. Although a significantly enhanced radiosensitivity was demonstrated for the cohort of BRCA1 and BRCA2 mutation carriers compared to the control cohort, individual radiosensitivity evaluation was less straightforward due to overlap in micronucleus yields between both cohorts. Therefore, a scoring system to evaluate individual radiosensitivity was implemented. As both BRCA1 and BRCA2 are involved in HR, we evaluated if the accumulation of RAD51, a key protein involved in this pathway, at the double strand break site can be used to assess HR functionality and radiosensitivity. To this end, a radiation-induced RAD51 foci assay was optimized in a breast epithelial cell line (MCF10A) expressing ±50% reduced BRCA1 and BRCA2 protein levels, obtained by RNA interference. RAD51 foci were analyzed in cells synchronized in S phase by aphidicolin as HR is upregulated during this phase of the cell cycle. We demonstrated significantly reduced RAD51 foci formation, and thus impaired HR capacity, in response to the induction of radiation-induced double strand breaks in the BRCA knockdown cells compared to control cells. As no overlap in RAD51 foci distribution is observed between knockdown and control cells, we think that this assay could better differentiate between normal cells and cells with a heterozygous BRCA1 or BRCA2 mutation than the G2 micronucleus assay. This will be further explored in synchronized lymphocytes of heterozygous germline mutation carriers. In addition to the detection of unequivocal deleterious mutations in BRCA1 and BRCA2, variants of unknown clinical significance (VUS) are detected during diagnostic screening. The associated breast cancer risk is unknown, which creates a challenge for genetic counselling. mRNA analysis to assess variants that might impair proper RNA splicing, a highly regulated process, are widely used. We evaluated the outcome at cDNA level of 21 putative splicing variants in BRCA1 and BRCA2 and demonstrated aberrant splicing for 12 variants, suggesting that these are likely pathogenic. Furthermore, we demonstrated that in silico prediction tools might assist in the evaluation of these putative splicing variants. However, further optimization is warranted to allow reliable application outside the highly conserved consensus splice sites. The results obtained in this thesis may indicate that care should be taken when applying ionizing radiation for diagnostic or therapeutic purposes in individuals with a germline mutation in BRCA1 or BRCA2 as they may be at higher risk of developing radiation-induced breast cancer.

Introduction: Ionising radiation can cause DNA double strand breaks (DSB), that result in chromosomal aberrations if un- or mis-repaired. Individuals with compromised DNA damage repair mechanisms display increased chromosomal radiosensitivity. The G0-micronucleus assay (MN assay) and the γ-H2AX assay are two assays used in radiobiology to study DNA DSB and repair. Breast cancer is the leading cancer amongst South African women, with a lifetime risk of 1 in 34. Since most cancer patients in South Africa present with late-stage disease, chemotherapy and radiotherapy are commonly-used treatments. Several international studies have shown breast cancer patients to be more chromosomally radiosensitive than healthy controls. These studies have not been confirmed on a cancer population living in South Africa. Cervical cancer is the second most common cancer in South Africa; however, it is the leading cancer amongst black women with a lifetime risk of 1/35 compared to 1/82 in white women. Studies show a genetic link to cervical cancer susceptibility and DNA damage repair genes. International studies on radiation-induced DNA damage in lymphocytes of cervical cancer patients remain inconclusive and have never been performed on a South African population. Cervical cancer is caused by infection with the Human Papilloma Virus (HPV). Human Immunodeficiency Virus (HIV), HPV and cervical cancer are epidemiologically linked. Due to the high rate of HIV in South Africa, a significant proportion of cervical cancer patients receiving radiotherapy treatment will be HIV-positive. Studies show an effect of HIV on chromosomal radiosensitivity, however this has not been confirmed on a cancer population. The MN assay on the biopsies and exfoliated cervical cells of cervical cancer patients could be used as a predictive test for response to radiotherapy. The overall aim was to study chromosomal radiosensitivity in South African cervical and breast cancer patients. Materials and methods: Chromosomal radiosensitivity of lymphocytes of cervical and breast cancer patients was examined using the MN assay with the Metafer 4 of Metasystems. Different scoring methods for the Metafer system were compared to each other. The effect of HIV, HPV, ethnicity, clinical parameters and age on micronuclei (MN) values in lymphocytes was investigated. The MN assay was attempted on cells from cervical biopsies and exfoliated cervical cells. The γ-H2AX was performed on the lymphocytes of a group of cervical cancer patients. Results: A new scoring method for the Metafer 4 system that is more reliable in patients with late-stage disease was introduced. Cervical cancer patients had significantly higher MN values with HIV patients having the highest values. HPV, clinical parameters and age had a limited effect on MN values. The MN assay was unsuccessful on biopsies and exfoliated cervical cells of cervical cancer patients. There was no difference in double strand break induction and repair between cervical cancer patients and controls. In breast cancer patients, ethnicity had an effect on MN values, with only white breast cancer patients having significantly higher MN counts. Conclusion: The study showed increased chromosomal radiosensitivity in cervical cancer and white breast cancer patients. Results highlight how such studies are important within the South African context, where factors like HIV, disease stage and ethnicity can have an effect on chromosomal radiosensitivity and where unique genes/polymorphisms may play a role in cancer risk.