In vitro cellular and proteome assays identify Wnt pathway and CDKN2A-regulated senescence affected in mesenchymal stem cells from mice after a chronic LD gamma irradiation in utero.

Reliable data on the effects of chronic prenatal exposure to low dose (LD) of ionizing radiation in humans are missing. There are concerns about adverse long-term effects that may persist throughout postnatal life of the offspring. Due to their slow cell cycle kinetics and life-long residence time in the organism, mesenchymal stem cells (MSCs) are more susceptible to low level genotoxic stress caused by extrinsic multiple LD events. The aim of this study was to investigate the effect of chronic, prenatal LD gamma irradiation to the biology of MSCs later in life. C3H mice were exposed in utero to chronic prenatal irradiation of 10 mGy/day over a period of 3 weeks. Two years later, MSCs were isolated from the bone marrow and analyzed in vitro for their radiosensitivity, for cellular senescence and for DNA double-strand break recognition after a second acute gamma-irradiation. In addition to these cellular assays, changes in protein expression were measured using HPLC-MS/MS and dysregulated molecular signaling pathways identified using bioinformatics. We observed radiation-induced proteomic changes in MSCs from the offspring of in utero irradiated mice (leading to ~ 9.4% of all detected proteins being either up- or downregulated) as compared to non-irradiated controls. The proteomic changes map to regulation pathways involved in the extracellular matrix, the response to oxidative stress, and the Wnt signaling pathway. In addition, chronic prenatal LD irradiation lead to an increased rate of in vitro radiation-induced senescence later in life and to an increased number of residual DNA double-strand breaks after 4 Gy irradiation, indicating a remarkable interaction of in vivo radiation in combination with a second acute dose of in vitro radiation. This study provides the first insight into a molecular mechanism of persistent MSC damage response by ionizing radiation exposure during prenatal time and will help to predict therapeutic safety and efficacy with respect to a clinical application of stem cells.

Identifying a Diagnostic Window for the Use of Gene Expression Profiling to Predict Acute Radiation Syndrome.

In the event of a mass casualty radiological or nuclear scenario, it is important to distinguish between the unexposed (worried well), low-dose exposed individuals and those developing the hematological acute radiation syndrome (HARS) within the first three days postirradiation. In previous baboon studies, we identified altered gene expression changes after irradiation, which were predictive for the later developing HARS severity. Similar changes in the expression of four of these genes were observed using an in vitro human whole blood model. However, these studies have provided only limited information on the time frame of the changes after exposure in relationship to the development of HARS. In this study we analyzed the time-dependent changes in mRNA expression after in vitro irradiation of whole blood. Changes in the expression of informative mRNAs (FDXR, DBB2, POU2AF1 and WNT3) were determined in the blood of eight healthy donors (6 males, 2 females) after irradiation at 0 (control), 0.5, 2 and 4 Gy using qRT-PCR. FDXR expression was significantly upregulated (P < 0.001) 4 h after ≥0.5 Gy irradiation, with an 18-40-fold peak attained 4-12 h postirradiation which remained elevated (4-9-fold) at 72 h. DDB2 expression was upregulated after 4 h (fold change, 5-8, P < 0.001 at ≥ 0.5 Gy) and remained upregulated (3-4-fold) until 72 h (P < 0.001). The earliest time points showing a significant downregulation of POU2AF1 and WNT3 were 4 h (fold change = 0.4, P = 0.001, at 4 Gy) and 8 h (fold change = 0.3-0.5, P < 0.001, 2-4 Gy), respectively. These results indicate that the diagnostic window for detecting HARS-predictive changes in gene expression may be opened as early as 2 h for most (75%) and at 4 h postirradiation for all individuals examined. Depending on the RNA species studied this may continue for at least three days postirradiation.

Low-dose radiation therapy for COVID-19 pneumopathy: what is the evidence?

In the current dismal situation of the COVID-19 pandemic, effective management of patients with pneumonia and acute respiratory distress syndrome is of vital importance. Due to the current lack of effective pharmacological concepts, this situation has caused interest in (re)considering historical reports on the treatment of patients with low-dose radiation therapy for pneumonia. Although these historical reports are of low-level evidence per se, hampering recommendations for decision-making in the clinical setting, they indicate effectiveness in the dose range between 0.3 and 1 Gy, similar to more recent dose concepts in the treatment of acute and chronic inflammatory/degenerative benign diseases with, e.g., a single dose per fraction of 0.5 Gy. This concise review aims to critically review the evidence for low-dose radiation treatment of COVID-19 pneumopathy and discuss whether it is worth investigating in the present clinical situation.

Education and training to support radiation protection research in Europe: the DoReMi experience.

A review is presented to the program of education and training setup within the DoReMi Network of Excellence. DoReMi was funded by Euratom under the EU 7th Framework Programme to coordinate the EU research into risks from low-dose ionizing radiation. It was seen to be necessary to form a network of expert institutions in order to tackle the scientific questions with the resources available. From the start, importance was given to the need to stimulate and support education and training to build up the capability of the research community. DoReMi dedicated a workpackage to education and training that put in place a number of activities that have been successful in attracting new students into the area and introducing research scientists to new topic areas and technologies. The program of education and training in DoReMi provided a significant contribution to the low-dose radiation research community and has been further developed and extended in the following Euratom-funded project OPERRA and the European Joint Programme CONCERT.

Cancer immunology and radiobiology: Oliver Scott's struggle for the perfect tumour model in translational research.

Oliver Scott is best known for his research into the role of tumour hypoxia in radiation oncology. Yet no less important were Oliver's activities in the development of concepts and methods for performing translational research on the effect of ionising radiation on tumour in experimental animals, stressing the importance of using strictly inbred animals for transplantation of tumours which had arisen in exactly the identical mouse strain. Otherwise residual immunity would lead to uncontrollable bias in the results of cure experiments, invalidating conclusions. These pioneering views are no less valid in today's cancer research.

Successful Teaching of Radiobiology Students in the Medical Management of Acute Radiation Effects From Real Case Histories Using Clinical Signs and Symptoms and Taking Advantage of Recently Developed Software Tools.

In 2015, the Bundeswehr Institute of Radiobiology organized a North Atlantic Treaty Organization exercise to examine the significance of clinical signs and symptoms for the prediction of late-occurring acute radiation syndrome. Cases were generated using either the Medical Treatment Protocols for Radiation Accident Victims (METREPOL, n = 167) system or using real-case descriptions extracted from a database system for evaluation and archiving of radiation accidents based on case histories (SEARCH, n = 24). The cases ranged from unexposed [response category 0 (RC 0, n = 89)] to mild (RC 1, n = 45), moderate (RC 2, n = 19), severe (RC 3, n = 20), and lethal (RC 4, n = 18) acute radiation syndrome. During the previous exercise, expert teams successfully predicted hematological acute radiation syndrome severity, determined whether hospitalization was required, and gave treatment recommendations, taking advantage of different software tools developed by the North Atlantic Treaty Organization teams. The authors provided the same data set to radiobiology students who were introduced to the medical management of acute effects after radiation exposure and the software tools during a class lasting 15 h. Corresponding to the previous results, difficulties in the discrimination between RC 0/RC 1 and RC 3/RC 4, as well as a systematic underestimation of RC 1 and RC 2, were observed. Nevertheless, after merging reported response categories into clinically relevant groups (RC 0-1, RC 2-3, and RC 3-4), it was found that the majority of cases (95.2% ± 2.2 standard deviations) were correctly identified and that 94.7% (±2.6 standard deviations) developing acute radiation syndrome and z96.4% (±1.6 standard deviations) requiring hospitalization were identified correctly. Two out of three student teams also provided a dose estimate. These results are comparable to the best-performing team of the 2015 North Atlantic Treaty Organization exercise (response category: 92.5%; acute radiation syndrome: 95.8%; hospitalization: 96.3%).

Special radiobiological features of second cancer risk after particle radiotherapy.

In absolute terms: second cancer risks from radiotherapy of first cancers in adults are small compared to the benefits from radiotherapy but this is not so for radiotherapy of childhood cancers. Moreover, the radiation dose dependence of cancer induction differs between organs and tissues. The organ-specific dose dependence of second cancer risks may indicate the existence of different radiobiological mechanisms. As an inevitable consequence of the age dependence of organ sensitivity to second cancer induction, the organ/tissue weighting factors which have been proposed by ICRP for calculating effective dose (the dose unit Sv) and for risk estimation in the general population should not be used in medical radiation exposures. In adult cancer radiotherapy, the most common unwanted effect is local tumour recurrence whereas both, severe late normal tissue damage and radiation-induced second cancers are rare, around 1% of locally controlled cancer patients. In childhood cancers, local failures are rare (<10% in some cancers) yet second cancers are more common than uncontrolled primaries. The main reason for considering particle radiotherapy for childhood cancers is the possibility to exploit their physical characteristics to reduce the radiation exposure to organs and tissues close to and distant from the primary cancer which is to be targeted. However, the relative biological effectiveness of the radiation doses within the proton beam is not a constant and the relative biological effectiveness of the neutrons is not known as far as the mechanisms of late normal tissue damage and second cancer risk are concerned. In view of the highly charged discussions of the potential risks of treatment-induced seecond cancers from the neutron contamination of exposure doses in out-of-PTV critical organs a comprehensive European project called ANDANTE was performed which integrated the disciplines of radiation physics, molecular biology, systems biology modelling and epidemiology in order to investigate the RBE of induction of cancer from exposure to neutrons compared to photons. Since out-of-field "effective" neutron doses from proton therapy are smaller than the photon stray doses whichever reasonable RBE is chosen for comparison, and since the absolute risk of radiation-induced second cancer rates are in the order of 1% in the cohorts of adult patients who have been treated in the past with methods which caused relatively high out-of-field doses to large body volumes, it is highly unlikely that such patients treated in future with highly conformal particle therapy are at a higher radiation-induced second cancer risk than those patients treated with photons and described before. Still, the potential risks of second cancers from scattered proton radiotherapy for childhood cancers may cause concern. Yet, the overall risk of undesired consequences of radiation exposure of children which are more complex and manifold than in adult patients (including developmental, neurocognitive, hormonal and growth impairment effects) are likely to be very much reduced by the better focussing of the radiation dose in the target offered by particle radioherapy. This benefit may far outweigh the still hypothetical second cancer risk from particle radiotherapy in pediatric radiotherapy.

Master of Science (MSc) Program in Radiation Biology: An Interdepartmental Course Bridging the Gap between Radiation-Related Preclinical and Clinical Disciplines to Prepare Next-Generation Medical Scientists.

Radiation biology is a highly interdisciplinary field at the interface of biology, physics, and medicine. It is characterized by rapid advances in biological and technical knowledge. The potential for using these advances to optimize medical care, radiation protection, and related fields can be exploited only with complementary activities to support the education of young academics. A small number of academic institutions have committed resources into radiation-related courses and curricula; however, few offer a comprehensive interdepartmental research and training program. At the Technical University of Munich (TUM), a full Master of Science (MSc) course in radiation biology has been established. This article describes the TUM MSc radiation biology program, discusses the scope of the field, the teaching goals, and the interdisciplinary curriculum. Detailed information on the full MSc program can be found continuously updated at www.radonc.med.tum.de/masterradiationbiology.

Tangential Field Radiotherapy for Breast Cancer-The Dose to the Heart and Heart Subvolumes: What Structures Must Be Contoured in Future Clinical Trials?

BACKGROUND AND PURPOSE: The aim of the present study was to evaluate if it is feasible for experienced radiation oncologists to visually sort out patients with a large dose to the heart. This would facilitate large retrospective data evaluations. And in case of an insufficient visual assessment, to define which structures should be contoured and which structures can be skipped as their dose can be derived from other easily contoured structures for future clinical trials. MATERIAL AND METHODS: Planning CTs of left-sided breast cancer patients treated with 3D-conformal radiotherapy by tangential fields were visually divided into two groups: with an estimated high dose (HiD) and with an estimated low dose (LoD) to the heart. For 46 patients (22 HiD and 24 LoD), the heart, the left ventricle, the left anterior descending artery (LAD), the right coronary artery, and the ramus circumflexus were contoured. A helper structure (HS) around the LAD was generated in order to consider if contouring uncertainties of the LAD could be acceptable. We analyzed the mean dose (Dmean), the maximum dose, the V10, V20, V30, V40, and the length of the LAD that received 20 and 40 Gy. RESULTS: The two groups had a significant different Dmean of the heart (p < 0.001). The average Dmean to the heart was 4.0 ± 1.3 Gy (HiD) and 2.3 ± 0.8 Gy (LoD). The average Dmean to the LAD was 26.2 ± 7.4 Gy (HiD) and 13.0 ± 7.5Gy (LoD) with a very strong positive correlation between Dmean LAD and Dmean HS in both groups. The Dmean heart is not a good surrogate parameter for the dose to the LAD since it might underestimate clinically significant doses in 1/3 of the patients in LoD group. CONCLUSION: A visual assessment of the dose to the heart could be reliable if performed by experienced radiation oncologists. However, the Dmean heart is not always a good surrogate parameter for the dose to the LAD or for the Dmean to the left ventricle. Thus, if specific late toxicities are evaluated, we strongly recommend contouring of the specific heart substructures as a heart Dmean is not highly specific.

Late proliferating and inflammatory effects on murine microvascular heart and lung endothelial cells after irradiation.

BACKGROUND AND PURPOSE: Radiotherapy of thoracic tumors increases the risk to develop cardiac diseases at later time-points. We compared time kinetics of radiation-induced changes of surface markers related to proliferation, progenitor cell development and inflammation in lung and heart microvascular endothelial cells (ECs). MATERIAL AND METHODS: Mice received local thorax irradiation with a single dose of 0, 2 or 8 Gy. Following magnetic bead separation and biotin-streptavidin competition, cell surface markers of isolated ECs from the lung and heart were analyzed 5, 10, 15 and 20 weeks after irradiation by flow cytometry. RESULTS: Irradiation with 8 Gy resulted in a temporary and differential up-regulation of proliferation markers (HCAM, Integrin ß-3, Endoglin, VE-cadherin, VEGFR-2) on ECs. Mucosialin a progenitor marker increased in lung ECs 15-20 weeks and inflammatory markers (PECAM-1, ICAM-1, ICAM-2, VCAM-1) started to increase 10 weeks after thorax irradiation with 8 Gy. Interestingly, ICAM-1 and VCAM-1 remained up-regulated 20 weeks after irradiation in heart and lung ECs. CONCLUSIONS: The persistently elevated expression density of ICAM-1 and VCAM-1 on ECs may suggest that an irradiation at 8 Gy induces late inflammatory responses in heart and lung ECs.

Adhesion molecule expression and function of primary endothelial cells in benign and malignant tissues correlates with proliferation.

BACKGROUND: Comparative analysis of the cellular biology of the microvasculature in different tissues requires the availability of viable primary endothelial cells (ECs). This study describes a novel method to isolate primary ECs from healthy organs, repair blastemas and tumors as examples of non-proliferating and proliferating benign and malignant tissues and their functional characterization. METHODOLOGY/PRINCIPAL FINDINGS: Single cell suspensions from hearts, lungs, repair blastemas and tumors were incubated consecutively with an anti-CD31 antibody and magnetic micro-beads, coupled to a derivative of biotin and streptavidin, respectively. Following magnetic bead separation, CD31-positive ECs were released by biotin-streptavidin competition. In the absence of micro-beads, ECs became adherent to plastic surfaces. ECs from proliferating repair blastemas and tumors were larger and exhibited higher expression densities of CD31, CD105 and CD102 compared to those from non-proliferating normal tissues such as heart and lung. The expression density of CD34 was particularly high in tumor-derived ECs, and that of CD54 and CD144 in ECs of repair blastemas. Functionally, ECs of non-proliferating and proliferating tissues differed in their capacity to form tubes in matrigel and to align under flow conditions. CONCLUSIONS/SIGNIFICANCE: This method provides a powerful tool to generate high yields of viable, primary ECs of different origins. The results suggest that an altered expression of adhesion molecules on ECs in proliferating tissues contribute to loss of EC function that might cause a chaotic tumor vasculature.

Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes?

In this pilot study we compared for the first time the radiation sensitivity of mouse lens epithelial cells (LECs) and mouse lymphocytes. We freshly prepared LECs and lymphocytes and irradiated them with γ-rays ((137)Cs; doses ranging from 0.25 to 2 Gy). DNA damage and repair were evaluated by alkaline comet assay and γH2AX foci assay. Using the comet assay, we observed a dose-dependent increase in DNA damage in both cell types. The faster formation of single- and double-strand breaks in LECs of C57BL/6 mice at doses below 1 Gy needs to be confirmed in other mouse strains. Immunofluorescence for γH2AX foci showed a higher degree of lesions in LECs from C57BL/6J mice compared to those of JF1 mice and to lymphocytes of both strains. Correspondingly, repair of DNA damage proceeded faster in LECs of C57BL/6J mice compared to LECs of JF1 mice and lymphocytes of both strains. It is obvious that the lymphocytes of both strains repaired DNA lesions more slowly than the corresponding LECs. In conclusion, our results demonstrate that LECs of C57Bl/6 mice show a steeper dose-response than lymphocytes in both types of experiments. It shows that both test systems are able to be used also at doses below 0.25 Gy. The observed difference in DNA repair between the LECs from C57BL/6J mice compared to the LECs from JF1 mice and to the lymphocytes of both strains warrants further experiments to identify the underlying molecular mechanisms.

Biological mechanisms of normal tissue damage: importance for the design of NTCP models.

The normal tissue complication probability (NTCP) models that are currently being proposed for estimation of risk of harm following radiotherapy are mainly based on simplified empirical models, consisting of dose distribution parameters, possibly combined with clinical or other treatment-related factors. These are fitted to data from retrospective or prospective clinical studies. Although these models sometimes provide useful guidance for clinical practice, their predictive power on individuals seems to be limited. This paper examines the radiobiological mechanisms underlying the most important complications induced by radiotherapy, with the aim of identifying the essential parameters and functional relationships needed for effective predictive NTCP models. The clinical features of the complications are identified and reduced as much as possible into component parts. In a second step, experimental and clinical data are considered in order to identify the gross anatomical structures involved, and which dose distributions lead to these complications. Finally, the pathogenic pathways and cellular and more specific anatomical parameters that have to be considered in this pathway are determined. This analysis is carried out for some of the most critical organs and sites in radiotherapy, i.e. spinal cord, lung, rectum, oropharynx and heart. Signs and symptoms of severe late normal tissue complications present a very variable picture in the different organs at risk. Only in rare instances is the entire organ the critical target which elicits the particular complication. Moreover, the biological mechanisms that are involved in the pathogenesis differ between the different complications, even in the same organ. Different mechanisms are likely to be related to different shapes of dose effect relationships and different relationships between dose per fraction, dose rate, and overall treatment time and effects. There is good reason to conclude that each type of late complication after radiotherapy depends on its own specific mechanism which is triggered by the radiation exposure of particular structures or sub-volumes of (or related to) the respective organ at risk. Hence each complication will need the development of an NTCP model designed to accommodate this structure.

Late radiation-induced heart disease after radiotherapy. Clinical importance, radiobiological mechanisms and strategies of prevention.

The clinical importance of radiation-induced heart disease, in particular in post-operative radiotherapy of breast cancer patients, has been recognised only recently. There is general agreement, that a co-ordinated research effort would be needed to explore all the potential strategies of how to reduce the late risk of radiation-induced heart disease in radiotherapy. This approach would be based, on one hand, on a comprehensive understanding of the radiobiological mechanisms of radiation-induced heart disease after radiotherapy which would require large-scale long-term animal experiments with high precision local heart irradiation. On the other hand - in close co-operation with mechanistic in vivo research studies - clinical studies in patients need to determine the influence of dose distribution in the heart on the risk of radiation-induced heart disease. The aim of these clinical studies would be to identify the critical structures within the organ which need to be spared and their radiation sensitivity as well as a potential volume and dose effect. The results of the mechanistic studies might also provide concepts of how to modify the gradual progression of radiation damage in the heart by drugs or biological molecules. The results of the studies in patients would need to also incorporate detailed dosimetric and imaging studies in order to develop early indicators of impending radiation-induced heart disease which would be a pre-condition to develop sound criteria for treatment plan optimisation.

Second malignancies in high­dose areas of previous tumor radiotherapy.

PURPOSE: To characterize second tumors that developed in or near the high-dose areas of a previous radiotherapy, regarding their frequency, entities, latency, and dose dependence. PATIENTS AND METHODS: 9,995/15,449 tumor patients of the Radiation Oncology Department in Ulm, Germany, treated between 1981 and 2003, survived at least 1 year after radiotherapy. By long-term follow-up and review of treatment documentation, 100 of them were identified who developed an independent second cancer in or near the irradiated first tumor site. RESULTS: Major primary malignancies were breast cancer (27%), lymphoma (24%), and pelvic gynecologic tumors (17%). Main second tumors were carcinomas of the upper (18%) and lower (12%) gastrointestinal tract, head and neck tumors (10%), lymphoma (10%), breast cancer (9%), sarcoma (9%), and lung cancer (8%). Overall median second tumor latency was 7.4 years (1-42 years). For colorectal cancer it was 3.5 and for leukemia 4.3 years, but for sarcoma 11.7 and for breast cancer 17.1 years. The relatively frequent second tumors of the upper gastrointestinal tract were associated with median radiation doses of 24 Gy. By contrast, second colorectal cancer and sarcoma developed after median doses of 50 Gy. CONCLUSION: The 5- and 15-year probability to develop a histopathologically independent second tumor in or near the irradiated first tumor site, i.e., after intermediate or high radiation doses, was 0.5% and 2.2%, respectively. To identify potentially radiogenic second malignancies, a follow-up far beyond 5 years is mandatory. The incidence and potential dose-response relationship intermediate will be analyzed by a case-case and a case-control study of the Ulm data.

Radiation-induced cardiovascular diseases: is the epidemiologic evidence compatible with the radiobiologic data?

The Life Span Study of Japanese atomic bomb survivors demonstrates that radiation exposure significantly increased the risk of developing ischemic heart disease, in particular myocardial infarction. Similarly, epidemiologic investigations in very large populations of patients who had received postoperative radiotherapy for breast cancer or for peptic ulcer demonstrate that radiation exposure of the heart with an average equivalent single dose of approximately 2 Gy significantly increased the risk of developing ischemic heart disease more than 10 years after irradiation. These epidemiologic findings are compatible with radiobiologic data on the pathogenesis of radiation-induced heart disease in experimental animals. The critical target structure appears to be the endothelial lining of blood vessels, in particular arteries, leading to early functional alterations such as pro-inflammatory responses and other changes, which are slowly progressive. Research should concentrate on the interaction of these radiation-induced endothelial changes with the early stages of age-related atherosclerosis to develop criteria for optimizing treatment plans in radiotherapy and also potential interventional strategies.