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Purpose: The concept of the adverse outcome pathway (AOP) has recently gained significant attention as to its potential for incorporation of mechanistic biological information into the assessment of adverse health outcomes following ionizing radiation (IR) exposure. This work is an account of the activities of an international expert group formed specifically to develop an AOP for IR-induced leukemia. Group discussions were held during dedicated sessions at the international AOP workshop jointly organized by the MELODI (Multidisciplinary European Low Dose Initiative) and the ALLIANCE (European Radioecology Alliance) associations to consolidate knowledge into a number of biological key events causally linked by key event relationships and connecting a molecular initiating event with the adverse outcome. Further knowledge review to generate a weight of evidence support for the Key Event Relationships (KERs) was undertaken using a systematic review approach., Conclusions: An AOP for IR-induced acute myeloid leukemia was proposed and submitted for review to the OECD-curated AOP-wiki (aopwiki.org). The systematic review identified over 500 studies that link IR, as a stressor, to leukemia, as an adverse outcome. Knowledge gap identification, although requiring a substantial effort via systematic review of literature, appears to be one of the major added values of the AOP concept. Further work, both within this leukemia AOP working group and other similar working groups, is warranted and is anticipated to produce highly demanded products for the radiation protection research community.

Purpose: In a nuclear or radiological event, an early diagnostic or prognostic tool is needed to distinguish unexposed from low- and highly exposed individuals with the latter requiring early and intensive medical care. Radiation-induced gene expression (GE) changes observed within hours and days after irradiation have shown potential to serve as biomarkers for either dose reconstruction (retrospective dosimetry) or the prediction of consecutively occurring acute or chronic health effects. The advantage of GE markers lies in their capability for early (1-3 days after irradiation), high-throughput, and point-of-care (POC) diagnosis required for the prediction of the acute radiation syndrome (ARS)., Conclusions: As a key session of the ConRad conference in 2021, experts from different institutions were invited to provide state-of-the-art information on a range of topics including: (1) Biodosimetry : What are the current efforts to enhance the applicability of this method to perform retrospective biodosimetry? (2) Effect prediction : Can we apply radiation-induced GE changes for prediction of acute health effects as an approach, complementary to and integrating retrospective dose estimation? (3) High-throughput and point-of-care diagnostics : What are the current developments to make the GE approach applicable as a high-throughput as well as a POC diagnostic platform? (4) Low level radiation : What is the lowest dose range where GE can be used for biodosimetry purposes? (5) Methodological considerations : Different aspects of radiation-induced GE related to more detailed analysis of exons, transcripts and next-generation sequencing (NGS) were reported.

Purpose: In case of population exposure by ionizing radiation, a fast and reliable dose assessment of exposed and non-exposed individuals is crucial important. In initial triage, physicians have to take fast decisions whom to treat with adequate medical care. In addition, worries about significant exposure can be taken away from hundreds to thousands non- or low exposed individuals. Studies have shown that the γH2A.X radiation-induced foci assay is a promising test for fast triage decisions. However, in a large-scale scenario most biodosimetry laboratories will quickly reach their capacity limit. The aim of this study was to evaluate the benefit of inexperienced experimenters to speed up the foci assay and manual foci scoring., Materials and Methods: The participants of two training courses performed the radiation-induced foci assay (γH2A.X) under the guidance of experts and scored foci (γH2A.X and 53BP1) on sham-irradiated and irradiated blood samples (0.05-1.5 Gy). The outcome of laboratory experiments and manual foci scoring by 26 operators with basic experience in laboratory work was statistically analyzed in comparison to the results from experts., Results: Inexperienced operators prepared slides with significant dose-effects (0, 0.1 and 1.0 Gy) for semi-automatic microscopic analyses. Manual foci scoring by inexperienced scorer resulted in a dose-effect curve for γH2A.X, 53BP1 and co-localized foci. In addition, inexperienced scorers were able to distinguish low irradiation doses from unirradiated cells. While 53BP1 foci scoring was in accordance to the expert counting, differences between beginners and expert increased for γH2A.X or co-localized foci., Conclusions: In case of a large-scale radiation event, inexperienced staff is useful to support laboratories in slide preparation for semi-automatic foci counting as well as γH2A.X and 53BP1 manual foci scoring for triage-mode biodosimetry. Slides can be clearly classified in the non-, low- or high-exposed category.

Purpose: The workshop on 'Individual Radiosensitivity and Radiosusceptibility' organized by MELODI and CONCERT on Malta in 2018, evaluated the current state of assays to identify sensitive and susceptible subgroups. The authors provide an overview on potential screening assays detecting individuals showing moderate to severe early and late radiation reactions or are at increased risk to develop cancer upon radiation exposure. Conclusion: It is necessary to separate clearly between tissue reactions and stochastic effects such as cancer when comparing the existing literature to validate various test systems. Requirements for the assays are set up. The literature is reviewed for assays that are reliable and robust. Sensitivity and specificity of the assays are regarded and scrutinized for modifying factors. Accuracy of an assay system is required to be more than 90% to balance risks of adverse reactions against risk to fail to cure the cancer. No assay/biomarker is in routine use. Assays that have shown predictive potential for radiosensitivity include SNPs, the RILA assay, and the pATM assay. A tree of risk guideline for radiologists is provided to assist medical treatment decisions. Recommendations for effective research include the setup of common retrospective and prospective cohorts/biobanks to validate current and future tests.

Purpose: Automated detection of dicentric chromosomes from a large number of cells was applied to study age-dependent radiosensitivity after in vitro CT exposure of blood from healthy donors., Materials and Methods: Blood samples from newborns, children (2-5 years) and adults (20-50 years) were exposed in vitro to 0 mGy, 41 mGy and 978 mGy using a CT equipment. In this study, automated scoring based on 13,000-31,000 cells/dose point/age group was performed. Results for control and low dose points were validated by manually counting about 26,000 cells/dose point/age group., Results: For all age groups, the high number of analyzed cells enabled the detection of a significant increase in the frequency of radiation induced dicentric chromosomes in cells exposed to 41 mGy as compared to control cells. Moreover, differences between the age groups could be resolved for the low dose: young donors showed significantly increased risk for induced dicentrics at 41 mGy compared to adults., Conclusions: The results very clearly demonstrate that the automated dicentric scoring method is capable of discerning radiation induced biomarkers in the low dose range (<100 mGy) and thus may open possibilities for large-scale molecular epidemiology studies in radiation protection.

Purpose: Age dependent radiation sensitivity for DNA damage after in vitro blood exposure by computer tomography (CT) was investigated., Materials and Methods: Radiation biomarkers (dicentrics and gammaH2AX) in blood samples of newborns, children under five years and adults after sham exposure (0 mGy), low-dose (41 mGy) and high-dose (978 mGy) in vitro CT exposure were analyzed., Results: Significantly higher levels of dicentric induction were found for the single and combined newborns/children group compared to adults, by a factor of 1.48 (95% CI 1.30-1.68), after exposure to 978 mGy. Although a significant dose response for damage induction and dose-dependent repair was found, the gammaH2AX assay did not show an age-dependent increase in DNA damage in newborns/children compared to adults. This was the case for the gammaH2AX levels after repair time intervals of 30 minutes and 24 hours, after correcting for the underlying background damage. For the low dose of 41 mGy, the power of the dicentric assay was also not sufficient to detect an age-dependent effect in the sample size investigated., Conclusion: A 1.5-fold increased level of dicentric aberrations is detected in newborns and children under five years after 1 Gy radiation exposure.

Purpose: To conduct a feasibility study on the application of the γ-H2AX foci assay as an exposure biomarker in a prospective multicentre paediatric radiology setting., Materials and Methods: A set of in vitro experiments was performed to evaluate technical hurdles related to biological sample collection in a paediatric radiology setting (small blood sample volume), processing and storing of blood samples (effect of storing blood at 4°C), the reliability of foci scoring for low-doses (merge γ-H2AX/53BP1 scoring), as well as the impact of contrast agent administration as potential confounding factor. Given the exploratory nature of this study and the ethical constraints related to paediatric blood sampling, blood samples from adult volunteers were used for these experiments. In order to test the feasibility of pooling the γ-H2AX data when different centres are involved in an international multicentre study, two intercomparison studies in the low-dose range (10-500 mGy) were performed., Results: Determination of the number of X-ray induced γ-H2AX foci is feasible with one 2 ml blood sample pre- and post-computed tomography (CT) scan. Lymphocyte isolation and fixation on slides is necessary within 5 h of blood sampling to guarantee reliable results. The possible enhancement effect of contrast medium on the induction of DNA DSB in a patient study can be ruled out if radiation doses and the contrast agent concentration are within diagnostic ranges. The intercomparison studies using in vitro irradiated blood samples showed that the participating laboratories, executing successfully the γ-H2AX foci assay in lymphocytes, were able to rank blind samples in order of lowest to highest radiation dose based on mean foci/cell counts. The dose response of all intercomparison data shows that a dose point of 10 mGy could be distinguished from the sham-irradiated control (p = 0.006)., Conclusions: The results demonstrate that it is feasible to apply the γ-H2AX foci assay as a cellular biomarker of exposure in a multicentre prospective study in paediatric CT imaging after validating it in an in vivo international pilot study on paediatric patients.