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Supplementary Figure 1 - Search strategy for identification of studies on salivary biomarkers of exposure, susceptibility and effect relevant for ionizing radiation research.

Salivary biomarkers have important potential to facilitate breakthroughs in epidemiologic studies, management of emergency situations, and detection and surveillance of diseases by medical staff. During the last decade, an increasing number of studies on salivary biomarkers have been published as a consequence of the impressive development of new high-throughput technologies. Here, we present a review of salivary biomarkers potentially useful in ionizing radiation (IR) research, particularly in molecular epidemiologic studies. Although several salivary biomarkers of cancer and other IR-associated diseases have been identified, few salivary biomarkers of exposure and no biomarker of susceptibility or effects specific to IR have been reported so far. Further studies are therefore needed to fully assess the potential of saliva as a source of biomarkers in the radiation research field. Although the use of saliva samples is not without drawbacks, it could represent an ideal noninvasive alternative to blood, particularly in children and in the context of large molecular epidemiology studies on the effects of low doses of IR, where, given the expected limited magnitude of effects, an extensive number of samples is required to reach statistical significance.See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.”Cancer Epidemiol Biomarkers Prev; 23(12); 2673–80. ©2014 AACR.

Supplementary Figure Legend from Usefulness of Saliva Samples for Biomarker Studies in Radiation Research

International audience; 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.

Acute myeloid leukemia (AML) is an aggressive cancer that progresses rapidly with a poor prognosis. Cytogenetic analysis provides the most accurate determination of diagnosis and prognosis however, about 42-48% of AML patients have a cytogenetically normal karyotype. Genetic analysis can provide further information and the identification of new mutations could result in improved risk stratification, prognosis and better understanding of the mechanisms of AML leukaemogenesis. In this study, we analyzed genetic alterations in 16 human AML cases by Haloplex sequencing with confirmation of two previously unreported mutations in the genes DNMT3A and RUNX1 by Sanger sequencing or pyrosequencing. The two novel mutations consist of two frameshift mutations identified in two different AML patients and reported as deleterious by bioinformatic analysis. These mutations confirm the exclusion and co-occurrence of specific gene mutation patterns in AML and may provide further information for patient diagnosis and prognosis.

The NATO HFM 291 research task group (RTG) on "Ionizing Radiation Bioeffects and Countermeasures" represents a group of scientists from military and civilian academic and scientific institutions primarily working in the field of radiobiology. Among other tasks, the RTG intends to extend their work on risk estimation and communication to bridge the gap in appropriate judgment of health risks given a certain radiation exposure. The group has no explicit psychological background but an expertise in radiobiology and risk assessment. The group believes that, as one of the essential first steps in risk communication, it is required to put radiation risk into perspective. Radiation risk requires a weight in comparison to already-known risks. What we envision is to Compare Radiation exposure-associated health Risks (CRRis App) with daily life health risks caused by other common exposures such as cigarette smoking, driving a car, etc. Within this paper, we provide (1) an overview of health risks after radiation exposure, (2) an explanation of the task and concept of an envisioned CRRis App, (3) an overview of existing software tools related to this issue, (4) a summary of inputs and discussions with experts in the field of radiation protection and risk communication during the ConRad conference, and finally, (5) identification of the next steps in the development of the App.

The aim of this review is to investigate the literature pertaining to the potential risks of low-dose ionizing radiation to Lynch syndrome patients by use of computed tomography (CT), either diagnostic CT colonography (CTC), standard staging CT or CT surveillance. Furthermore, this review explores the potential risks of using radiotherapy for treatment of rectal cancer in these patients. No data or longitudinal observational studies of the impact of radiation exposure on humans with Lynch syndrome were identified. Limited experimental studies utilizing cell lines and primary cells exposed to both low and high radiation doses have been carried out to help determine radio-sensitivity associated with DNA mismatch repair gene deficiency, the defining feature of Lynch syndrome. On balance, these studies suggest that mismatch repair deficient cells may be relatively radio-resistant (particularly for low dose rate exposures) with higher mutation rates, albeit no firm conclusions can be drawn. Mouse model studies, though, showed an increased risk of developing colorectal tumors in mismatch repair deficient mice exposed to radiation doses around 2 Gy. With appropriate ethical approval, further studies investigating radiation risks associated with CT imaging and radiotherapy relevant doses using cells/tissues derived from confirmed Lynch patients or genetically modified animal models are urgently required for future clinical guidance.

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.

The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.

DNA methylation alteration is crucial for the initiation and development of Acute Myeloid Leukemia. However, only a few epigenetic biomarkers of AML have been discovered so far. DNA methylation of CpG rich gene promoters has the highest impact on gene expression level, so biomarkers should be sought in these genomic regions. Principal Component Analysis of methylation level reveals that male and female AML patients differ in CpG rich-gene promoters. Statistical comparison between males and females conducted for each CpG site confirms statistical relevance of differences only in CpG-rich promoter regions. P-value integration results in detection of AML-specific hypermethylated genomic regions and shows that almost 10% of CpG-rich promoters are differentially methylated between males and females. Functional analysis of genes with differentially methylated promoters in AML results in 322 enrichments for women and 1893 for men. Genes are mainly related to homeobox, cell development and morphogenesis. Survival analysis indentifies gender-specific potential epigenomic prognostic markers. Dissimilarity of the survival-significant gene sets between males and females is 75%.

In vitro experiments show that the cells possibly responsible for radiation-induced acute myeloid leukemia (rAML) exhibit low-dose hyper-radiosensitivity (HRS). In these cells, HRS is responsible for excess cell killing at low doses. Besides the endpoint of cell killing, HRS has also been shown to stimulate the low-dose formation of chromosomal aberrations such as deletions. Although HRS has been investigated extensively, little is known about the possible effect of HRS on low-dose cancer risk. In CBA mice, rAML can largely be explained in terms of a radiation-induced Sfpi1 deletion and a point mutation in the remaining Sfpi1 gene copy. The aim of this paper is to present and quantify possible mechanisms through which HRS may influence low-dose rAML incidence in CBA mice. To accomplish this, a mechanistic rAML CBA mouse model was developed to study HRS-dependent AML onset after low-dose photon irradiation. The rAML incidence was computed under the assumptions that target cells: (1) do not exhibit HRS; (2) HRS only stimulates cell killing; or (3) HRS stimulates cell killing and the formation of the Sfpi1 deletion. In absence of HRS (control), the rAML dose-response curve can be approximated with a linear-quadratic function of the absorbed dose. Compared to the control, the assumption that HRS stimulates cell killing lowered the rAML incidence, whereas increased incidence was observed at low doses if HRS additionally stimulates the induction of the Sfpi1 deletion. In conclusion, cellular HRS affects the number of surviving pre-leukemic cells with an Sfpi1 deletion which, depending on the HRS assumption, directly translates to a lower/higher probability of developing rAML. Low-dose HRS may affect cancer risk in general by altering the probability that certain mutations occur/persist.

Humans have learned to harness the power of radiation for therapeutic ends, with 50% of all patients diagnosed with cancer undergoing radiotherapy as part of their treatment [...]

Therapy-related and more specifically radiotherapy-associated acute myeloid leukaemia (AML) is a well-recognized potential complication of cytotoxic therapy for the treatment of a primary cancer. The CBA mouse model is used to study radiation leukaemogenesis mechanisms with Sfpi1/PU.1 deletion and point mutation already identified as driving events during AML development. To identify new pathways, we analysed 123 mouse radiation-induced AML (rAML) samples for the presence of mutations identified previously in human AML and found three genes to be mutated; Sfpi1 R235 (68%), Flt3-ITD (4%) and Kras G12 (3%), of which G12R was previously unreported. Importantly, a significant decrease in Sfpi1 gene expression is found almost exclusively in rAML samples without an Sfpi1 R235 mutation and is specifically associated with up-regulation of mir-1983 and mir-582-5p. Moreover, this down-regulation of Sfpi1 mRNA is negatively correlated with DNA methylation levels at specific CpG sites upstream of the Sfpi1 transcriptional start site. The down regulation of Sfpi1/PU.1 has also been reported in human AML cases revealing one common pathway of myeloid disruption between mouse and human AML where dysregulation of Sfpi1/PU.1 is a necessary step in AML development., This work identifies new leukaemia pathways involving genetic and epigenetic changes after irradiation exposure.

In the past decade, radiation therapy (RT) entered the era of personalized medicine, following the striking improvements in radiation delivery and treatment planning optimization, and in the understanding of the cancer response, including the immunological response. The next challenge is to identify the optimal radiation regimen(s) to induce a clinically relevant anti-tumor immunity response. Organs at risks and the tumor microenvironment (e.g. endothelial cells, macrophages and fibroblasts) often limit the radiation regimen effects due to adverse toxicities. Here, we reviewed how RT can modulate the immune response involved in the tumor control and side effects associated with inflammatory processes. Moreover, we discussed the versatile roles of tumor microenvironment components during RT, how the innate immune sensing of RT-induced genotoxicity, through the cGAS-STING pathway, might link the anti-tumor immune response, radiation-induced necrosis and radiation-induced fibrosis, and how a better understanding of the switch between favorable and deleterious events might help to define innovative approaches to increase RT benefits in patients with cancer.

Background This communication reports the identification of a new panel of transcriptional changes in inflammation-associated genes observed in response to ionising radiation received by radiotherapy patients. Methods Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumours. Nanostring nCounter analysis of transcriptional changes was carried out in samples prior and 24 h post-delivery of the 1st radiotherapy fraction, just prior to the 5th or 6th fraction, and just before the last fraction. Results Statistical analysis with BRB-ArrayTools, GLM MANOVA and nSolver, revealed a radiation responsive panel of genes which varied by patient group (type of cancer) and with time since exposure (as an analogue for dose received), which may be useful as a biomarker of radiation response. Conclusion Further validation in a wider group of patients is ongoing, together with work towards a full understanding of patient specific responses in support of personalised approaches to radiation medicine.

For triage purposes following a nuclear accident or a terrorist event, gene expression biomarkers in blood have been demonstrated to be good bioindicators of ionizing radiation (IR) exposure and can be used to assess the dose received by exposed individuals. Many IR-sensitive genes are regulated by the DNA damage response pathway, and modulators of this pathway could potentially affect their expression level and therefore alter accurate dose estimations. In the present study, we addressed the potential influence of temperature, sample transport conditions and the blood cell fraction analyzed on the transcriptional response of the following radiation-responsive genes: FDXR, CCNG1, MDM2, PHPT1, APOBEC3H, DDB2, SESN1, P21, PUMA, and GADD45.Whole blood from healthy donors was exposed to a 2 Gy X-ray dose with a dose rate of 0.5 Gy/min (output 13 mA, 250 kV peak, 0.2 mA) and incubated for 24 h at either 37, 22, or 4 °C. For mimicking the effect of transport conditions at different temperatures, samples incubated at 37 °C for 24 h were kept at 37, 22 or 4 °C for another 24 h. Comparisons of biomarker responses to IR between white blood cells (WBCs), peripheral blood mononuclear cells (PBMCs) and whole blood were carried out after a 2 Gy X-ray exposure and incubation at 37 °C for 24 hours.Hypothermic conditions (22 or 4 °C) following irradiation drastically inhibited transcriptional responses to IR exposure. However, sample shipment at different temperatures did not affect gene expression level except for SESN1. The transcriptional response to IR of specific genes depended on the cell fraction used, apart from FDXR, CCNG1, and SESN1.In conclusion, temperature during the incubation period and cell fraction but not the storing conditions during transport can influence the transcriptional response of specific genes. However, FDXR and CCNG1 showed a consistent response under all the different conditions tested demonstrating their reliability as individual biological dosimetry biomarkers.

This communication reports the identification of a new panel of transcriptional changes in inflammation-associated genes observed in response to ionising radiation received by radiotherapy patients. Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumours. Nanostring nCounter analysis of transcriptional changes was carried out in samples prior and 24 hours post-delivery of the 1st radiotherapy fraction, just prior to the 5th or 6th fraction, and just before the last fraction. Statistical analysis with BRB Array Tools, GLM MANOVA and nSolver, revealed a radiation responsive panel of genes which varied by patient group (type of cancer) and with time since exposure (as an analogue for dose received), which may be useful as a biomarker of radiation response. Further validation in a wider group of patients is ongoing, together with work towards a full understanding of patient specific responses in support of personalised approaches to radiation medicine.

Large-scale radiation emergency scenarios involving protracted low dose rate radiation exposure (e.g. a hidden radioactive source in a train) necessitate the development of high throughput methods for providing rapid individual dose estimates. During the RENEB (Running the European Network of Biodosimetry) 2019 exercise, four EDTA-blood samples were exposed to an Iridium-192 source (1.36 TBq, Tech-Ops 880 Sentinal) at varying distances and geometries. This resulted in protracted doses ranging between 0.2 and 2.4 Gy using dose rates of 1.5–40 mGy/min and exposure times of 1 or 2.5 h. Blood samples were exposed in thermo bottles that maintained temperatures between 39 and 27.7 °C. After exposure, EDTA-blood samples were transferred into PAXGene tubes to preserve RNA. RNA was isolated in one laboratory and aliquots of four blinded RNA were sent to another five teams for dose estimation based on gene expression changes. Using an X-ray machine, samples for two calibration curves (first: constant dose rate of 8.3 mGy/min and 0.5–8 h varying exposure times; second: varying dose rates of 0.5–8.3 mGy/min and 4 h exposure time) were generated for distribution. Assays were run in each laboratory according to locally established protocols using either a microarray platform (one team) or quantitative real-time PCR (qRT-PCR, five teams). The qRT-PCR measurements were highly reproducible with coefficient of variation below 15% in ≥ 75% of measurements resulting in reported dose estimates ranging between 0 and 0.5 Gy in all samples and in all laboratories. Up to twofold reductions in RNA copy numbers per degree Celsius relative to 37 °C were observed. However, when irradiating independent samples equivalent to the blinded samples but increasing the combined exposure and incubation time to 4 h at 37 °C, expected gene expression changes corresponding to the absorbed doses were observed. Clearly, time and an optimal temperature of 37 °C must be allowed for the biological response to manifest as gene expression changes prior to running the gene expression assay. In conclusion, dose reconstructions based on gene expression measurements are highly reproducible across different techniques, protocols and laboratories. Even a radiation dose of 0.25 Gy protracted over 4 h (1 mGy/min) can be identified. These results demonstrate the importance of the incubation conditions and time span between radiation exposure and measurements of gene expression changes when using this method in a field exercise or real emergency situation.

Background In this exploratory study, the impact of local irradiation on systemic changes in stress and immune parameters was investigated in eight patients treated with intensity-modulated radiation therapy (IMRT) or stereotactic ablative body radiotherapy (SABR) for prostate adenocarcinoma to gain deeper insights into how radiotherapy (RT) modulates the immune system. Patients and methods RT-qPCR, flow cytometry, metabolomics, and antibody arrays were used to monitor a panel of stress- and immune-related parameters before RT, after the first fraction (SABR) or the first week of treatment (IMRT), after the last fraction, and 3 weeks later in the blood of IMRT (N = 4) or SABR (N = 4) patients. Effect size analysis was used for comparison of results at different timepoints. Results Several parameters were found to be differentially modulated in IMRT and SABR patients: the expression of TGFB1, IL1B, and CCL3 genes; the expression of HLA-DR on circulating monocytes; the abundance and ratio of phosphatidylcholine and lysophosphatidylcholine metabolites in plasma. More immune modulators in plasma were modulated during IMRT than SABR, with only two common proteins, namely GDF-15 and Tim‑3. Conclusion Locally delivered RT induces systemic modulation of the immune system in prostate adenocarcinoma patients. IMRT and SABR appear to specifically affect distinct immune components.

Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected, moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers.