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08:30-09:15 Session Keynote Speaker
Monte Carlo simulation of radio-induced DNA damage

ABSTRACT. Increasing knowledge of the mechanisms of cell damage formation induced by ionising radiation has been a fundamental research area in radiation protection for more than 30 years. Indeed, this knowledge can then be used to improve the assessment of the subsequent risk at different types of exposure (internal, external, different particles, energies) and at different dose levels. Microdosimetry first and nanodosimetry second, are two formalisms that, by investigating the energy deposition generated by irradiation at the cellular and intracellular scale, contribute to this research. Although both approaches can be used experimentally, the great advances in computational capabilities in recent years have made simulation, and more particularly Monte Carlo (MC) simulation, the method of choice for this type of research. Thus, using the experimental data and the knowledge acquired on the different stages of the interaction between radiation and biological matter, this method can be used to calculate the damage at the level of a cell population, particularly the damage produced to the DNA which will greatly condition the fate of the cells. In this presentation, the objective is to give an overview of the work done at IRSN concerning the simulation of DNA damage. Based on the use of the Geant4-DNA code1, 2, our damage simulation chain3 pays particular attention to the geometrical models of DNA as we have shown that taking into account a realistic representation of the hetero and euchromatin domains influences the number and complexity of damages4,5. In addition, an increasingly complex simulation of the chemical step is needed to improve the results and to be able to address specific issues such as the effects of flash radiotherapy or the use of metallic nanoparticles in radiotherapy. To this end, the latest developments of the Geant4-DNA collaboration implemented in our calculation chain will be presented. Finally, current developments concerning the extension of the simulation chain to take into account variability at the level of an irradiated cell population (extension in scale)6 and DNA damage repair mechanisms (extension in time) will also be discussed.

09:15-09:45 Session Invited Speaker
Radiation Chemistry based modeling investigations of the FLASH mechanism
PRESENTER: Emanuele Scifoni

ABSTRACT. The mysterious differential effectiveness of ultra-high dose rate (UHDR) irradiations, returning a protective effect on normal tissues for same antitumor efficacy as compared to conventional dose rates, the so-called FLASH effect, observed in numerous preclinical experiments, triggered in the last 3-4 years an exponentially growing number of biophysical modeling works attempting to investigate and explain it from the mechanistic point of view. Since it was appearing that such a phenomenon should imply several physical, chemical and biological stages of the radiation action, different spatio-temporal scales were considered and analyzed in these modeling approaches. An overview of these investigations will be concisely reported, with a focus on the ongoing joint efforts of GSI and TIFPA in this context, especially in the attempt of combining different scales. In particular, radiation chemical based approaches, employing TRAXCHEM [1-2], the GSI radiation chemical track structure code and its specific extensions, allowing to go from the physical stage to the homogeneous chemical stage will be mentioned and a novel dedicated extension of the Generalized Stochastic Microdosimetric model (GSM2)[3-4] for UHDR regime, aiming at combining the DNA damage and repair kinetics with the chemical stages on several levels. Impact of LET [5] and dose delivery features will be discussed as well.

09:45-10:15 Session Oral Presentation
Characterizing the performance of a proton tomography system for x-ray CT cross-calibration
PRESENTER: Elena Fogazzi

ABSTRACT. Purpose: INFN research projects have recently studied the feasibility of proton Computed Tomography (pCT) as a possible clinical method to cross-calibrate x-rays CT (xCT), aiming at improving the dose computation accuracy in proton therapy treatment plansa. Herein, we report on the performance of our pCT apparatus.

Methods: The pCT system was tested with both a Sedentex-CT Image Quality and an Electron-Density (ED) phantoms. A filtered backprojection algorithmb, taking into account the protons’ most likely path, was applied to reconstruct the phantoms’ Relative Stopping Power (RSP) 3D maps. The edge test device inside the Sedentex-CT phantom allowed investigating the spatial resolution of the apparatus. The pCT RSP values of the ED phantom inserts were compared with multilayer ionization chamber measurements to estimate the system accuracya.

Results: Proton tomographies of the Sedentex-CT phantom were analyzed, resulting in a spatial resolution of about 0.67 lp/mm (Fig.1). Direct measurements of RSP values of the ED phantom inserts showed a mean absolute percentage error of 0.74%. Finally, statistical analysis suggested a minimum obtainable noise magnitude of about 0.005 for RSP.

Conclusions: The obtained performances allow designing a first clinical application for our pCT system. To this purpose, the RSP maps of a set of stabilized, heterogeneous, biological phantoms have been acquired and will be compared voxel-by-voxel with the corresponding xCT images, obtaining a cross-calibrated xCT calibration curvec. Once validated, this procedure might be offered to proton therapy centers not equipped with a pCT system, representing a new and direct calibration method to be adopted in treatment planning (Fig.2).

Gamma radiation for Cultural Heritage preservation at Calliope Facility (ENEA Casaccia R.C., Rome, Italy)
PRESENTER: Alessia Cemmi

ABSTRACT. Ionizing radiation treatments for preservation of Cultural Heritage artefacts attacked by bio-deteriogen agents (insects, microfungi, moulds) present several advantages over classical procedures, due to their biocide effects1. Nevertheless, in various countries, some resistance to ionizing radiation processing is still shown by Cultural Heritage operator communities. The reason for this mistrust is often due to the incorrect knowledge of the physical-chemical modifications (side-effects) induced by ionizing radiation on the irradiated materials. For these reasons, recently further efforts and experimental tests were performed with the aim of providing reliable results, obtained by different experimental techniques widely accepted and recognized, to describe the side-effects occurrence. Moreover, a very important issue is related to the irradiation parameters optimization for the minimization of the radiation-induced modifications on the artefacts, guaranteeing their safeguard and giving reliable and standardized procedures. At the Calliope 60Co gamma irradiation facility (ENEA Casaccia R. C., Rome, Italy) several studies were performed on cellulose-based substrates by means of different experimental techniques (infrared spectroscopy, Electron Spin Resonance spectroscopy, colourimetric analyses, polymerization degree evaluation) 2, 3. In this research, the optimal irradiation conditions (in terms of irradiation dose and dose rate, environmental atmosphere) were also investigated. A fundamental part of research activities has covered the study of radiation-induced modification over time, performing accelerated and artificial ageing tests, and verifying the synergy between radiation and temperature.

1Uses of Ionizing Radiation for Tangible Cultural Heritage Conservation, IAEA Radiation Technology Series 9 (2017). 2 S. Baccaro, A. Cemmi, Nukleonika 62 (2017) pp.261-267, DOI: 10.1515/nuka-2017-0038. 3A. Cemmi, I. Di Sarcina, G. Ferrara, ENEA Technical Report RT/2020/1/ENEA.

10:15-10:45Coffee Break
10:45-11:15 Session Oral Presentation
Dosimetric characterization of a new resin-coated silicon carbide detector

ABSTRACT. The research in the development of new detectors for relative dosimetry has been very active in the last two decades. The physical characteristics of Silicon Carbide (SiC), such as wide bandgap, ultra-low leakage current, high electron saturation velocity, almost near tissue-equivalence and high radiation resistance, have attracted the attention of the interested scientific community [1-9]. Moreover, SiC based devices present dose rate independent response and linearity with energy in a wide dynamic range [10, 11]. These features make SiC also suitable for dosimetric applications with both conventional and high intensity beams. In this work a new generation of SiC device based on p-n junction technology was investigated for dosimetric applications. The detector was manufactured in the context of a collaboration between INFN (Istituto Nazionale di Fisica Nucleare, Italy), IMM-CNR (Microelectronic and Microsystems Institute) and STM (ST-Microelectronics, Catania). The adopted detector, built by using new technological processes, presents a detection area of 1 cm2 and is embedded in epoxy resin in order to make it waterproof. The study aimed at evaluating the potential use of the SiC detector as a relative dosimeter, in accordance with the dosimetric protocols in force (IAEA TRS-398) [12]. The detector response was tested in water with x-ray and electron beam. The released absolute dose during each experimental session was evaluated using a standard ionization chamber. In this work the performance and the preliminary characterization of this new detector will be presented and discussed.

BNCT treatment verification by on-line prompt gamma measurement using CdZnTe (CZT) detectors
PRESENTER: Nicoletta Protti

ABSTRACT. Boron Neutron Capture Therapy (BNCT) is a hadrontherapy based on the reaction 10B(n,α)7Li induced by low energy neutrons. Thanks to the short ranges in tissues of the high LET secondaries, the lethal damages affect only tumour DNA sparing the nearby healthy cells. To properly exploit its selectivity, real time knowledge of B10 concentrations and the local thermal neutron flux distribution must be known. Today the quantities are measured independently and through indirect methods. This leads to a lack of precision in dose estimation [1]. The B10 concentration and the thermal flux are coupled by the reaction rate of B10 neutron captures. The intensity of 478 keV γ ray emitted after the B10 reaction is a direct measurement of this rate and thus it equals to an on-line monitoring of the B10 dose giving a real time treatment verification [2]. The energy is pretty close to that of PET, nonetheless the single γ emission requires an equipment closer to SPECT scanners. In addition, the huge (n+γ) background characterising BNCT represents an important difference between the still missing detector and those of Nuclear Medicine [3]. Recently, the Physics Department of Pavia University has acquired four modules of the “DoseCapture” system developed by due2lab s.r.l. for BNCT-SPECT [4]. Each module is based on an array of 4 Frisch Grid CdZnTe (CZT) detectors coupled to a squared hole Pb collimator. To reduce neutron activation of the Cd113 naturally occurring in the alloy, Li6 enriched TLDs surround five out of six sides of the array. A fast digital electronics complete the system. The modules have been tested using the highly thermalised collimated neutron beam of the Prompt Gamma Neutron Activation Analysis facility housed in the research nuclear reactor of the LENA laboratory of Pavia University. The presentation will report the results of this first measuring campaign which positively supports the development of a B10 dose verification system using CZT γ detectors and tomographic imaging. References: [1] I.Murata et al., Progress in Nuclear Science and Technology, Vol. 1, p.267-270 (2011) [2] T.Kobayashi et al., Medical Physics 27, 2124 (2000): doi: 10.1118/1.1288243 [3] S.Altieri et al., Il Nuovo Cimento 41 C (2018) 208 [4]

11:15-12:15 Session AWARD: Bacq & Alexander - Carine Michiels
How cell biology and radiation physics met to boost radiotherapy

ABSTRACT. In close collaboration with Prof S. Lucas a,d A-C. Heuskin, since 15 years, studies are performed to increase the efficacy of radiotherapy using hadrons instead of photons. Indeed, the use of high energy charged particles like protons or carbon ions presents clear benefits thanks to their depth dose profile and high energy transfer. Currently, research topics include the study of the mechanisms responsible of high LET hypersensitivity and the use of proton irradiation in combination with DNA repair inhibitors to induce synthetic lethality. We also developed a unique nanohybrid compound, composed of gold nanoparticles coupled to targeting antibodies, being able to act as radiosensitizing agents for radiation therapy. We demonstrated for the first time that the enhancing effects of these nanoparticles is not due to “physical” effects but to the fact that the gold nanoparticles induced an inhibition of the antioxidant defenses within the targeted cells. Thioredoxin reductase is the main target of this effect. In addition, the responses of different types of cancer cells as well as cell of tumor microenvironment (TME) after exposure to different radiation modalities as well as to chemotherapy and targeted therapies are being investigated More recently, the group started to assess the impact of the different types of radiation on the immunogenicity and immunomodulatory capacity of cancer cells and other cells of the TME. For instance, it was determined that unlike X-rays, proton irradiations with high linear energy transfer (LET) partially reprogramed macrophages and promoted a pro-inflammatory phenotype. This discovery provides insight into mechanisms that can regulate the eradication of cancer cells by the immune system. Another project has been investigating the regulation of the expression of PD-L1 by RT. The results showed that X-ray irradiation as well as charged particle irradiation increased the gene expression and cell surface abundance of PD-L1 in multiple cancer cell lines. Using chemical inhibitors and siRNA, it was demonstrated that this mechanism involves the activation of the DNA damage response pathway and relies on the transcriptional activity of IRF1, activated upon irradiation. Furthermore, charged particle radiation increases PD-L1 abundance more strongly than X-ray and this effect seems to be potentiated by an increasing LET at doses that are equally lethal to cancer cells). These results provide further evidence that different radiation modalities at equivalent doses induce different immune-modulatory pathways. All together, these projects aim at enhancing the efficacy of radiotherapy by new developing radio-sensitization mechanisms coupled to evidence of the underlying mechanisms.

13:15-14:30Lunch Break
13:15-14:30 Session POSTER SESSION 1: (SR, RP&C and HE&RP&RC)
Radiology during pregnancy: risks, radiation protection in medical practice, and communication with the patient
PRESENTER: Harmen Bijwaard

ABSTRACT. Purpose: Radiation protection practices differ between hospitals when it comes to radiology during pregnancy. To remedy this best practices were identified by a literature review, interviews with radiographers, and interviews and a survey among pregnant women.

Materials and Method: The literature review was used as input for both the interviews and the survey. The interviews were conducted with 52 radiographers in focus group sessions. Three main topics were selected: (1) dose reduction, (2) confirmation of pregnancy, and (3) risk communication. In addition, 150 recently pregnant women were involved: 10 were interviewed and 140 filled in a questionnaire.

Results: The literature review showed: (1) deterministic effects will in radiological practice never occur, because of the threshold dose of around 100 mGy; (2) stochastic effects have not been observed in recent studies, but may occur when the fetus is inside the X-ray beam. However, even for high dose procedures, the risk is very low. The outcomes of the focus groups were: (1) no consensus about shielding when the fetus is outside the X-ray beam, (2) better justification by requesting physicians, (3) a need for a multi-lingual, informative website and (4) a need for a list of dose-reducing measures. (5) Both the requesting physician and the radiographer should inquire after pregnancy (because of the time between request and actual procedure). The interviews with pregnant women showed that radiation risk information fell short. The survey indicated that 68% would like to have information about radiation risks beforehand. Most women (56%) chose a pictogram matrix as their preferred way to visualize the risk.

Conclusions: The scientific literature shows that risks of radiology during pregnancy are minimal, but anxiety among patients can be reduced by proper communication about radiation risks and uniform evidence-based procedures in all hospitals.

Stromal Vascular Fraction mitigates radiation induced gastro-intestinal syndrome
PRESENTER: Lydia Bensemmane

ABSTRACT. Accidental or intentional radiation exposures have serious health consequences for exposed individuals and can affect a large number of people. Large volume irradiation at high irradiation doses induces multiple tissue lesions. The gastro-intestinal tract is particularly sensitive to irradiation and lethality. At dose more than 10 Gy results in diarrhea, dehydration, sepsis with mortality within 10 days post-exposure. Radiation-induced gastrointestinal syndrome (GIS) results from direct cytocidal effects on intestinal stem cells and crypt stroma impairing epithelial regeneration. Given the logistical hurdle and the urgency for treatment in large numbers of casualties, there is a tremendous need for effective therapeutic measures, even if implemented several days after radiation exposure. The stromal vascular fraction (SVF) derived from adipose tissue is an easily accessible source of cells with angiogenic, anti-inflammatory, immunomodulatory, and regenerative properties. We examined whether SVF restores the irradiated intestinal cells niche and mitigates the GIS. Mice exposed to abdominal radiation (18Gy) received an intravenous injection of SVF (2*106 cells) on the day of irradiation. When injected before 24hours post-irradiation, the SVF limited the weight loss and inhibited the intestinal permeability and mortality after abdominal irradiation[1]. Histological analyses of intestine showed that SVF stimulated the regeneration of the epithelium by promoting the restoration of the cell population in the intestinal stem cell compartment. The intestinal “organoid” model that mimics the vivo response confirmed that SVF treatment stimulated the intestinal stem cell compartment. SVF has an anti-inflammatory effect by repressing pro-inflammatory cytokines, increasing the presence of anti-inflammatory monocyte subtypes CD11b+Ly6clowCX3CR1high in the ileum. The SVF by inducing regeneration of intestine could be a promising therapeutic approach for the treatment of GIS.

[1] Bensemmane, C Squiban, C Demarquay, N Mathieu, M Benderitter, B Le Guen, F Milliat, C Linard, Stem Cell Res Ther, 2021,12:309.

Radon progeny adsorption on facial masks
PRESENTER: Andreas Maier

ABSTRACT. The radioactive noble gas radon (222Rn) is responsible for around half of annual radiation exposure from natural sources. Radon will decay into several short-living progeny (218Po, 214Pb, 214Bi and 214Po) which can be inhaled and will deposit their energy in the lung. Therefore, progenies are responsible for more than 95% of the total effective dose and together with radon are classified as carcinogenic for lung cancer. Filtration of the progenies will reduce the dose to the lung and is typically done by HEPA-filter or electrostatic air cleaners. Due to the Covid19 pandemic, wearing of face masks as a safety measure is recommended by WHO. In our study we investigate the filtration properties of FFP2 mask and surgical mask (II R) for radon progeny. Therefore, FFP2- and II R-mask were attached to a measurement device (EQF 3220, Sarad GmbH), which can distinguish between radon progeny in dependence of their size distribution, ranging from unattached (< 5 nm) and clustered (20-100 nm) to attached (> 100 nm) progeny. In parallel, it is measuring the radon activity-concentration during experiments. This setup is placed inside a radon chamber1. Usual measurement times were up to 5 hours, allowing radon to achieve radioactive equilibrium with its short living progeny after approximately 3 hours. Therefore, only data 180 minutes after start of radon exposure were analyzed. The measured progenies were normalized to the radon activity concentration. By comparing background measurements without filter and experiments with FFP2- and II R masks, the percentage of retained radon progenies was determined. For unattached progeny the percentage of retained radon is almost the same for FFP2 (98.8 ± 0.6%) and II R mask (98.4 ± 0.7 %). For clustered progeny, there are minor differences (FFP2: 85.2 ± 18.1 %; II R: 79.5 ± 22.1 %). Therefore, we show that both masks are effective in filtering radon progeny and thus are capable of reducing the total effective dose to the lung.

1Maier A., van Beek P., Hellmund J., Durante M., Schardt D., Kraft G., Fournier C. Experimental setup for radon exposure and first diffusion studies using gamma spectroscopy., Nucl. Instr. and Meth. in Phys. Res. B, 362 :187-193 (2015).

Radon solubility in human blood and components
PRESENTER: Annika Hinrichs

ABSTRACT. Radon as a natural occurring radioactive noble gas contributes significantly to annual radiation dose. Despite being carcinogenic, it is also used to treat inflammatory diseases like rheumatoid arthritis. Detailed knowledge of the radon distribution within the blood is needed to evaluate partition coefficients. However, data for human blood is scarce and therefore we tested human whole blood, plasma and erythrocytes. In order to understand the underlying mechanisms determining radon solubility bovine protein solutions of hemoglobin and albumin were tested. Additionally oleic acid and pentanol were used to test additivity of solubility as assumed by ICRP1. There it is stated that solubility in tissue can be calculated on the basis of its composition, for instance solubility in breast tissue is calculated as a combination of fatty and muscle tissue. Samples were exposed to a defined radon activity concentration under controlled conditions for one hour. After sealing the samples radon tight, the radioactive equilibrium between radon and its γ-emitting progeny (214Pb, 214Bi) is achieved after a few hours. Extrapolating the activity of γ-emitting nuclei measured with γ-spectroscopy, the initial radon concentration was determined. Subsequently, using the samples mass and radon activity concentration during exposure the solubility is calculated. Comparing the human samples, plasma showed highest solubility followed by erythrocytes and whole blood. Solubility in protein solutions was in the same range and only significantly decreased when hemoglobin was heat denatured before exposure. Measurements of oleic acid and pentanol seem to contradict the additivity of solubility assumed by ICRP. Based on this, a re-evaluation of solubility data should be envisaged. Our measurements indicate that a there are more complex mechanisms on the microscopic scale determining solubility than pure tissue composition.

1ICRP, 2017. Occupational Intakes of Radionuclides: Part 3. ICRP Publication 137. Ann. ICRP 46(3/4)

Interphase Fluorescence In Situ Hybridization (FISH) for interphase chromosomal aberration-based biological dosimetry

ABSTRACT. Metaphase spreads stained with Giemsa or painted with chromosome specific probes (FISH) have been in use since long for retrospective dose assessment (biological dosimetry). However, in cases of accidental exposure to ionizing radiation the culturing of lymphocytes to obtain metaphase chromosomes and analysis of chromosomal aberrations is time-consuming. Similarly, analyzing chromosomal damage in G0/G1 cells or non-dividing cells by premature chromosome condensation (PCC) is laborious. Following large scale radiological emergencies, the time required for analysis is more important than precision of dose estimate. Painting of whole chromosomes using chromosome-specific probes in interphase nuclei by the FISH technique will eliminate the time required for cell culture and allow a fast dose estimate, provided that a meaningful dose response can be obtained by scoring color changes between chromosomal domains visible in interphase nuclei. In order to test the applicability of interphase FISH for quick biological dosimetry, whole blood from a healthy donor was irradiated with 8 Gy of gamma radiation. Irradiated whole blood was kept for 1 h at 37°C to allow DNA repair and thereafter processed for FISH with probes specific for chromosome 1 and 2. Damaged chromosomal fragments, distinguished as having an extra color domain, were observed in interphase nuclei of lymphocytes irradiated with 8 Gy. These fragments were efficiently detected and quantified by the FISH techniques utilizing both confocal microscopy and fluorescence microscopy. Furthermore, our investigation showed a clear dose response curve for exposure to 0, 1, 2, 4 and 8 Gy of gamma radiation. These results demonstrate interphase FISH as an interesting test for biodosimetry and for studying cytogenetic effects of radiation in non-dividing cells.

Shielding of the femur reduces the severity of the gastrointestinal subsyndrome of ARS and increases survival in mice
PRESENTER: Ingunn Hanson

ABSTRACT. Acute radiation syndrome (ARS) appears in individuals that receive a large, acute radiation dose to a significant amount of the body. The condition is classified according to the affected organ system into the hematopoietic, gastrointestinal and neurovascular subsyndromes. Depending on the dose received, the mortality can reach 100% within days after exposure. Today, there is no preventative or curative treatments available for ARS, and treatment options generally consists of supportive care including blood transfusion and bone marrow transplant, administered according to the symptoms presented.

In this study, C57BL/6j mice received a 225 kV x-ray dose of 8.5 Gy at a dose rate of 59 Gy/h. For 50% of the mice, a lead shield was placed in front of the left hind leg to shield the femur and mimic bone marrow transplant, as demonstrated by Van Bekkum et al.1 The rest received 8.5 Gy as a total body dose. We monitored pain scores according to a system developed by Nunamaker et al.2, and weighed the mice twice per day in the critical period.

All unshielded mice reached humane end points within 10 days. Symptoms included high pain scores, depilation, diarrhea and weight loss. Necroscopies revealed empty and partially hemorrhaged stomachs and intestines, suggesting that death was caused by the gastrointestinal subsyndrome. This was confirmed by histological examination of the small intestine.

The mice that were partially shielded all survived until termination of the study (110 days). In the first 10 days, symptoms in this group included moderate pain scores, depilation and weight loss. After 10 days, the pain scores stabilized at a low level and all mice eventually gained weight above the baseline level.

Our results show that the subsyndromes of ARS are not easily separable, and that early treatment of the hematopoietic subsyndrome has potential to alleviate the gastrointestinal subsyndrome to a point that significantly increases the chance of survival in the affected individuals.

Low doses of chronic 137Cs contamination impairs olfactory capacity of mice with sexes differences

ABSTRACT. Resuspension of Cesium 137 (137Cs) present around the contaminated Chernobyl Exclusion Zone is possible by diverse external elements. Wildfire occurring in April 2020 [1] or military moving in February 2022 induce respectively a release of 137Cs and possibly a stirring up dust. People living around this zone can therefore be exposed to low doses of 137Cs by inhalation and contaminated food. Little is known about effects of 137Cs inhalation, although, a study found a link between moderate doses of ionizing radiation and olfaction [2]. This study has shown that people exposed to external radiation presented impaired olfactory function. The aim of our study is to understand if low doses of 137Cs contamination could also impairs this olfactory function. ApoE-/- male and female mice received water ad libitum supplemented or not with 20, 100 or 500kBq/L of 137Cs for 6 months. We expect that 137Cs present in the litter is inhaled by mice. In parallel, to evaluate their olfactory capacity, some mice received predator odor to test the induction of stress. The weight of mice was monitored, behavior test to explore olfactory activity and stress are realized before euthanasia and olfactory epithelium and bulb were collected. Our results show that male mice exposed to 137Cs spend less time exploring unknown odors than controls mice. They also spend less time doing olfactory exploration in general. On the other hand, this effect seems absent in female but they failed to stress in response to predator odors assuming an impairment of olfactory system. Moreover, only non exposed animals to 137Cs lost weight du to stress induced by predator odor. Immunostaining of olfactory epithelium is ongoing to explore potential alterations which could explain the loss of olfactory function and the difference between males and females. Thereby, we found that low doses of chronic 137Cs contamination could impair olfaction of mice in different levels between males and females.

[1] Baró, R.; Maurer, C.; Brioude, J.; Arnold, D.; Hirtl, M. The Environmental Effects of the April 2020 Wildfires and the Cs-137 Re-Suspension in the Chernobyl Exclusion Zone: A Multi-Hazard Threat. Atmosphere 2021.

[2] Tonacci A, Baldus G, Corda D, Piccaluga E, Andreassi M, Cremonesi A, Guagliumi G, Picano E. Olfactory non-cancer effects of exposure to ionizing radiation in staff working in the cardiac catheterization laboratory. Int J Cardiol. 2014 .

Effects of low and moderate doses of ionizing radiation on a aortic aneurysmal model of Angiotensin II infusion ApoE-/- mice
PRESENTER: Teni Ebrahimian

ABSTRACT. Exposure of populations to ionizing radiation show a correlation between this exposure and the occurrence of cardiovascular disease (CVD)1,2,3. While systemic review of published data did not find a clear link of low doses radiation to circulatory diseases4,5. In vivo experimental studies suggested that chronic low dose rate ionizing radiation induced a protective effect on atherosclerosis in rodent studies with a decrease in inflammation in atheromatous plaques and plaques size6,7. The first cause of CVD morbidity and mortality is atherosclerosis and is followed by aortic aneurysm. In our study we evaluated the impact from low to moderate dose of ionizing radiation in aortic abdominal anevrysm. We used a model of male ApoE-/- mice at the age of 10-12 weeks. Those mice were implanted with subcutaneous osmotic pump (model 2004 Alzet) filled with a solution of angiotensin II (Angio II) to perfuse at 1000 ng/kg/min during 7 or 28 days8. Mice received X-ray total body irradiation with the same dose rate at different doses (50, 500, 1000, 5000 mGy and control) 3 days before or after the Angio II infusion. We observed in mice irradiated before aneurysm induction, a significant decrease in the incidence of pathology with the highest doses (1000 mGy and 5000 mGy) and a significant decrease in the systolic aortic diameter with 500, 1000 and 5000 mGy. Moreover, at 1000 mGy, we shown a decrease in the actin contained in the smooth muscle cells suggesting vascular wall remodeling. Whereas in mice irradiated after Angio II infusion, we didn’t observed any tendency in pathological incidence or the dilatation mesurement. Our results suggest a dose and microenvironment dependant effect on a mice model of aortic anevrysm.

Low doses of chronic 137Cs contamination effects on atherosclerosis
PRESENTER: Teni Ebrahimian

ABSTRACT. Epidemiological studies have shown a relationship between high doses of ionizing radiation (IR) and cardiovascular diseases, however studies are uncertain for low doses [1]. Populations living in radiation-contaminated territories, such as Chernobyl and Fukushima, are chronically exposed to external and internal γ radiation due to the 137Cs released in the environment. Thus, a central question in radiation protection research is to understand if low-doses exposure to IR play a role in progression of cardiovascular diseases. Previous animal studies have shown some protective effects of chronic low dose γ exposure on vascular system [2]. These data were obtained in male mice with chronic contamination to 137Cs until 100kBq/L in drinking water for 6 months. However, chronic higher doses in male and female mice have never been studied. In our study, we evaluated a potential threshold effect on cardiovascular system up to 100kBq/L in male and female mice. ApoE-/- male and female mice receiving water ad libitum supplemented or not with 20, 100 or 500kBq/L of 137Cs for 6 months before euthanasia and collection of plasma, aorta and heart. We measured differences in absorbed doses between males and females mice which is confirmed by a higher activity per gram of skeletal muscle in male for the 500kBq/L groups. Histological, immunohistochemical analyses of aortic plaque phenotype didn’t show any differences in plaque size and lipid content in male mice contaminated with different concentrations of 137Cs. However, the identification of immune cells recruited near the plaque could change with the increasing dose from 100 to 500kBq/L of 137Cs and data for female are still expected. Thereby, this work could help to identify the potential existence of a dose threshold, below that which harmful effects are not exhibited, and beneficial effects are potentially observed. Furthermore, these findings permit to explore differences between males and females.

[1] Gillies M & al., Mortality from Circulatory Diseases and other Non-Cancer Outcomes among Nuclear Workers in France, the United Kingdom and the United States (INWORKS). Radiat Res. 2017

[2] Le Gallic C & al., Chronic Internal Exposure to Low Dose 137Cs Induces Positive Impact on the Stability of Atherosclerotic Plaques by Reducing Inflammation in ApoE-/- Mice. PLoS One. 2015

Radon exposures delay the development of psoriatic skin lesions in a mouse model
PRESENTER: Sylvie Lerchl

ABSTRACT. The chronic inflammatory skin disease psoriasis is on the indication list for radon therapy. Therapeutic success is reported but the mechanisms leading to observed clinical benefits are unknown. The pathology of psoriasis is caused by an autoimmune reaction inducing hyperproliferation of keratinocytes and infiltration of immune cells into the skin. The proinflammatory cytokine IL-17A is described as a key player in this mechanism. To investigate the effects of radon exposures on a psoriatic phenotype, we used transgenic DC-IL-17Aind/ind-mice that constitutively express IL-17A and therefore gradually develop psoriatic skin lesions (plaques); breeded in the group of Prof. Clausen, Gutenberg University Mainz 1. Before plaque induction, mice received single (~539 kBq/m³, 1 h) or multiple (10x ~39 kBq/m³, 1 h) radon treatments as in patient therapy 2. The occurrence of skin lesions was observed and scored from 0–6 (0=normal skin, 4=plaque induction, 6=spread lesion) for ~20 weeks after treatments. In a second setup, mice were sacrificed 3 days or 2 weeks after multiple radon exposures to collect tissue samples for molecular analyses. After radon exposures, the development of psoriatic skin lesions was delayed for several weeks as compared to sham exposed mice, with more pronounced effects in multiple than in single exposed animals. Molecular analyses indicated transient immunosuppressive effects of radon in skin and lymph nodes. In particular, following multiple radon treatments, NGS data showed a downregulated gene expression of factors related to initiation phase of psoriasis in skin. Furthermore, distinct subtypes of dendritic cells were depleted in lymph nodes. Instead, the amounts of anti-inflammatory components like regulatory T cells were increased in these organs. This study provides the first in vivo evidence for alleviative effects of radon on a chronic psoriatic disease model which so far has only been reported by patients where placebo-effects cannot be excluded.

Capturing the complex biology of low-dose effects using multimodal data integration: analysis of pros and cons using a mouse model of colon cancer as a case example
PRESENTER: Dmitry Klokov

ABSTRACT. Biological mechanisms of low-dose radiation (LDR) health effects are very complex. Analytical methods using systems biology approaches to analyze complex multimodal and/or multiomics data have shown great potential for revealing networks of causally related biological changes spanning molecular, cellular, tissue and organism levels. However, these methods are facing multiple challenges pertinent to the structure and nature of the data. Examples are: 1) “fat data” or “high dimensionality” of data – a combination of thousands to millions of variables are obtained from a single sample (e.g. RNA-seq or DNA-seq) with a small number of replicates/animals per group; 2) two types of heterogeneity of data, one being biological heterogeneity (e.g. omics vs. physiological data) and the second being data scale heterogeneity (e.g. millions of variables in omics vs. few physiological variables); and lastly, 3) the origin of the data when different data modalities are coming not from the same individual animal/subject, which is a common feature of animal radiobiological experiments. We present here our systems biology approach to tackle some of these common challenges using data collected in a recently completed study wherein the effects of LDR on intestinal tumorigenesis were examined in the APCmin/+ mouse model of human colon cancer. The data comprised of RNA-seq transcriptomes, DNA-seq methylomes, blood cellularity and biochemistry, blood cytokine profiles, and tumor formation. In step one, each data modality was analyzed separately, followed by step two where challenges 1) and 2) above were addressed using the RGCCA/SGCCA1 multiblock algorithms. Finally, integrative analysis was carried out to extract biologically relevant information, such as biological pathways. Our results suggest that the approach used can help decipher biologically relevant information for complex pathological conditions from a highly heterogenous data sets encompassing various levels of biological organization. One of the major limitations of the used algorithms is, however, the inability to address the type 3) challenge above, which informs planning of future LDR animal studies. Not only can this approach be applied to animal studies of the effects of LDR on other health conditions, e.g. on atherosclerosis, but also it can facilitate the development of adverse outcome pathways by identifying causally related key biological events.

Omics molecular biomarkers of localized radiation injury in mice biofluids
PRESENTER: Lucie Ancel

ABSTRACT. A radiological accident, whether from industrial, medical, or malicious origin, may result in localized external exposure to high doses of ionizing radiations. Such exposure leads to the development of a local radiation injury (LRI) whose kinetics and severity depend on the absorbed dose, the duration of exposure and the volume of irradiated tissue. After an asymptomatic phase of variable latency, the LRI manifests as an erythema that may evolve from dry desquamation to deep ulceration and necrosis through unpredictable inflammatory waves. Early diagnosis and prognosis of victims of LRI is therefore crucial for the effectiveness of medical management and the reduction of deleterious effects. To respond to the constraints of a radiological emergency, a fast and non-invasive diagnostic method is needed to facilitate identification and care of victims. This study aims to identify, through an omics approach carried out in biofluids, molecular biomarkers associated with LRI in a preclinical C57BL/6J mouse model of hind limb irradiation using different 10 MV X-ray doses that lead to injuries of different severity grades. We performed broad-spectrum profiling of microRNAs (miRNA) and metabolites in blood and urine, using quantitative real-time polymerase chain reaction and mass spectrometry, respectively. Using a multivariate sparse partial least square discriminant analysis (PLS-DA), we identified panels of miRNAs and metabolites in both biofluids, that can differentiate groups of mice according to the radiation dose or the severity of the injury. Furthermore, an integrative analysis was conducted to establish multi-scale correlations between specific miRNAs/metabolites levels and various biological parameters, i.e. blood cell counts and circulating C-reactive protein levels (inflammation marker), as well as physiological/functional parameters including observational lesion score, cutaneous blood perfusion (laser Doppler), and skin barrier integrity (Tewameter®). The identified biomarker signatures were further confirmed in an independent validation mice cohort. Our results demonstrate relevant plasma and urine molecular signatures associated with LRI in mice and support the use of miRNAs and metabolites in biofluids as suitable molecular biomarkers for the prognosis and diagnosis of LRI.

Design of a preclinical proton minibeam radiotherapy facility
PRESENTER: Jessica Neubauer

ABSTRACT. Spatial fractionated radiotherapy using protons, so-called proton minibeam radiotherapy (pMBRT) was developed for better sparing of normal tissue in the entrance channel of radiation. Preclinical in-vivo experiments conducted with pMBRT in mouse ear models or in rat brains support the prospects. However, the research on radiobiological mechanisms and the search for adequate application parameters delivering the most beneficial minibeam therapy is still in its infancy. Progressing towards clinical usage, pMBRT research should overcome technical and biomedical limitations of the current irradiation test stages and animal models. This work discusses the design of a preclinical pMBRT facility located at an existing 68.5 MeV cyclotron. Two major parts, which have to be designed and constructed are the dose monitor and the range shifter. To be able to monitor high local dose rates a parallel plate ionization chamber was constructed. As electrodes 6 µm thick mylar foils coated with aluminum at a distance of 5 mm were used. Here, we present the final prototype construction and first reference measurements using an x-ray beam. The range shifter needs to be designed such that a beam diameter of ~200µm () is not exceeded. We show beam spreading simulations of different materials, which can be used as range shifter, such as PMMA, carbon and aluminum. Also we present the possibility of using a hybrid range shifter splitted in two parts, one in vacuum and one in air. This on the one hand supports keeping beam size small and on the other hand offers maximum flexibility. Additionally we introduce a possible quality assurance procedure, which has to be conducted to be able to apply the pMBRT in small animals with sufficient small beam size, positioning accuracy as well as lateral and axial dose profiles. For this purpose we show first pilot studies of a cancer model which will be used as a preliminary stage of the animal model.

Model study of 49Ti(p,2pn)47Sc and comparison with first production measurement within the LNL-REMIX experiment

ABSTRACT. Radionuclide 47Sc has gained interest in precision nuclear medicine thanks to its favourable decay properties, suitable for targeted radiotherapy and theranostic applications, also in combination with the β+ emitters 43Sc/44Sc1. However, efficient production routes, appropriate for pre-clinical and clinical investigations, are hard to find and still represent an open issue. Herein we investigate the modeling of 47Sc cyclotron production using proton beams on enriched titanium targets. This theoretical analysis takes also into account the very recent 49Ti(p,2pn)47Sc production measurements within the INFN-LNL REMIX (Research on Emerging Medical radIonuclides from X-section) project. The analysis consists in the modeling, using the nuclear reaction code TALYS2, of the relevant 47Sc cross section, and includes the production of its main contaminants, starting from the long-lived 46Sc. Since a large variety of pre-equilibrium and level-density models can be selected within TALYS, we describe statistically the results by means of the interquartile band and the Min/Max excursion obtainable from all considered models3. Then we optimize the level density parameters (starting from the existing data for 46Sc) to obtain a better agreement between measurements and theoretical cross-section4. The new, preliminary data for 47Sc and contaminants, from the REMIX project, allow to refine the optimization to obtain more reliable cross sections and from there the evaluation of yields, radionuclidic purities and dosimetric impact of contaminants. Finally, from these results we will discuss the feasibility of production routes employing enriched titanium targets.

1 C. Müller et al., Br J Radiol, 91: 20180074, 2018. 2 S. Goriely, S. Hilaire, and A. J. Koning. Astron. Astrophys., 487:767, 2008. 3 A. Colombi et al. Nucl. Technol., 208:4, 735-752, 2022. doi:10.1080/00295450.2021.1947122. 4 F. Barbaro et al. Phys. Rev. C, 104:044619, 2021.

Comparison of natGd(p,x), 155Gd(p,n) and 159Tb(p,5n) reaction modeling for 155Tb production

ABSTRACT. In the field of radiopharmaceutical production, terbium has gained the attention of the community since it is the only element in the periodic table with four isotopes (149Tb, 152Tb, 155Tb and 161Tb) that can be used for both imaging and therapy1. In particular 155Tb is a Auger-electron emitter and has γ rays suitable for SPECT (Single Photon Emission Computed Tomography) imaging. In this work we investigate and compare different nuclear reactions for the production of 155Tb using medium-low energy proton beams on natGd, 155Gd, and 159Tb targets, contributing to the activities of the INFN-LNL REMIX (Research on Emerging Medical radIonuclides from the X-sections) project. The considered production routes are analyzed with the nuclear reaction code TALYS2 performing initially a statistical analysis3 to take into account the variability of the models for the level density and preequilibrium processes implemented in the code. In a subsequent step, we perform an optimization of the calculated cross sections considering specific combinations of the models and varying their parameters, in order to improve the agreement between the cross sections and the data4. This analysis is carried out for the reactions with natGd and 159Tb since many experimental data are available for these production routes, and it is tentatively applied to the 155Gd(p,n)155Tb reaction, whose cross sections have not been measured yet. After an inspection of the complex decay schemes of all the Tb isotopes produced in the reactions, the extended Bateman's equations are solved to finally calculate the yields and purities for optimal irradiation conditions, in view of possible dosimetric studies and clinical applications.

1 C. Müller et al. J. Nucl. Med, 53(12):1951–1959, 2012 2 S. Goriely, S. Hilaire, and A. J. Koning. Astron. Astrophys., 487:767, 2008 3 A. Colombi et al. Nucl. Technol., 208:4, 735–752, 2022, doi: 10.1080/00295450.2021.194712 4 F. Barbaro et al. Phys. Rev. C, 104:044619, 2021.

2D dosimetry for evaluation of in vitro clonogenic survival following exposure to spatially fractionated GRID radiation field
PRESENTER: Delmon Arous

ABSTRACT. Background: Spatially fractionated (GRID) irradiation is an approach to deliver high local radiation doses in an ’on-off’ pattern. The radiobiolgical effects from GRID needs to be better characterized and understood to promote clinical applications. The purpose of the current work was to develop a framework to evaluate in vitro effects of GRID.

Materials and methods: A549 lung cancer cells cultured in vitro in T25 cm2 flasks were irradiated using 220 kV X-rays with an open field or through a tungsten GRID collimator with periodical 5 mm openings and 10 mm blockings. Delivered doses were 2, 5, and 10 Gy. A novel approach for image segmentation was used to locate the centroid of surviving colonies in scanned images of the cell flasks. Gafchromic film dosimetry (GFD) and FLUKA Monte Carlo simulations (MC) were employed to map the dose distribution in the flasks at each surviving colony centroid. Fitting the linear-quadratic (LQ) function to open field survival data, the expected survival level at a given dose level was obtained. The expected survival level was then mapped together with observed levels in the GRID-irradiated flasks.

Results: GFD and FLUKA MC gave similar dose distributions, with a mean peak-to-valley dose ratio of about 5. LQ-parameters for open field irradiation gave α=0.16±0.04 Gy-1 and β=0.001±0.004 Gy-2. Using the image segmentation method, the surviving colony distribution in the cell flasks gave a pattern qualitatively resembling the GRID collimator outline. The mean absolute percentage deviation between predicted and observed survival in the (peak;valley) dose regions was (8;10) %, (4;41) %, and (3;138) % for 2, 5 and 10 Gy, respectively.

Conclusion: A framework for mapping of surviving colonies following GRID irradiation together with predicted survival levels from homogeneous irradiation was presented. For the given cell line, our findings indicate that GRID irradiation, especially at high peak doses, causes reduced survival compared to an open field configuration.

Italian dosimetry audit: service for radiotherapy centres and a new study for non-reference conditions
PRESENTER: Alessia Embriaco

ABSTRACT. The Italian National Institute of Ionizing Radiation Metrology (ENEA-INMRI) and the Italian Association of Medical Physics (AIFM) offer a certified audit service to radiotherapy (RT) centres for dosimetry in photon beams. Audits are provided for photon beams in the range 6-18 MV including flattening filter free beams, CyberKnife and TomoTherapy. Thermoluminescent dosimeters consisting of a set of TLD chips embedded in a PMMA waterproof holder are used. The dosimeter signal is the average value of 20 or 10 TLD chips depending on the beam uniformity at the measurement point. Dosimeters are calibrated at ENEA-INMRI in terms of absorbed dose to water in a reference 60Co gamma beam. Correction factors accounting for energy dependence, signal reproducibility and response stability are applied to evaluate absorbed dose. For the audit, measurements are performed in reference conditions according to the international dosimetry protocols [1-3]. For each beam, irradiation of two dosimeters with 2 Gy is required. Audit results are evaluated in terms of the normalized error En [4]: it is satisfactory if |En| ≤ 1.0. Distribution of En scores for all irradiated dosimeters shows that 99.4% of values are in the range [-1.0,1.0]. As for the single unsatisfactory result, data from the form filled in by the RT centre allowed to identify an error in the dosimeter positioning. In non-reference condition, a technical protocol for verification of dose delivery in VMAT treatments is being developed. VMAT delivery simulating head-neck treatments with Simultaneous Integrated Boost on two different target volumes while sparing the parotid glands and the spinal cord to the proper dose constrains have been planned. A uniform dose (within 2%) was required in the planning phase on a region with dimensions comparable to sensitive volume of the detector used to measure the delivered dose in a cylindrical water phantom. TLD measurement accuracy is evaluated by comparison to a reference ionization chamber. From preliminary results, it emerges that dosimeters with 10 TLD chips are suitable for dose measurements also in non-reference conditions. The reference dosimetry audit was successfully performed for the 38 RT centres and 84 photon beams. Meanwhile, other measurements will be planned to evaluate different VMAT plan configuration.

Simulation of microgravity with random positioning machine induces promoting effects in Triple Negative Breast Cancer cells
PRESENTER: Marco Calvaruso

ABSTRACT. Space environment is a harsh place to host human life and exposes the crew to many physical challenges. The absence of gravity (known as microgravity, mg) affects many sides of human biology and the human body requires to adopt several mechanisms of adaptation as a countermeasure to restrain the effects induced by gravity unloading [1]. The interest towards mg is not only speculative but it could help to predict potential long-term alterations and to develop countermeasure to manage healthy problems in space. Microgravity can be reproduced in vitro with the help of microgravity simulators and simulated microgravity (s-mg) is applied in many fields of medical research, including cancer biology [2,3]. In our study, we aimed to determine in vitro the effects that simulated microgravity is able to induce in a model of Triple Negative Breast Cancer (TNBC), an aggressive form of tumor. In fact, the role played by mg in cancer progression still remains an open debate [4]. The investigation on the effects induced by s-mg in neoplastic cells is not related to the need to cure cancer in space, instead, it’s aimed to clarify if the absence of gravity is associated with the acquisition of mg-related phenotypes both in morphology and at molecular level which may help to highlight novel cancer biomarkers. For our analysis we used a random positioning machine (RPM) which is able to counterbalance gravity forces by rotating along the x, y and z axis. In particular, we investigated and compared the effects played by s-mg at two different timepoints (24 and 72 hours), analyzing cell viability by MTT test and the change of expression of some genes, driving proliferation, survival, cell death, cancer stemness, and metastasis, in the human TNBC MDA-MB-231 cell line by qRT-PCR. Our biological findings demonstrated that s-mg can sustain some key aspects of TNBC development and dissemination. To date, results about this field of investigation are controversial, in fact, both pro- and anti-tumoral roles of microgravity are reported in the literature when different cancer settings are analyzed (e.g: lymphomas, gliomas and pancreatic cancer) [5-7]. Hence, our results may help to shed the light on the potential involvement of gravity unloading as a stressor whose role requires a deeper knowledge to understand its effects in tumor development and maintenance.

Recommendations for optimizing radiation protection of patients and health workers: results of the European Project MEDIRAD
PRESENTER: Alessandra Palma

ABSTRACT. MEDIRAD - Implications of Medical Low Dose Radiation Exposure is a European research project, funded by EURATOM under the Horizon 2020 programme which involved 35 institutions from 14 EU states with the aim of increasing the scientific basis and clinical practice of radiation protection (RP) in the medical field. In particular, the project aimed to improve the understanding and evaluation of the health effects of exposure to low doses of ionizing radiation resulting from diagnostic and therapeutic applications (off-target exposures), to optimize practices to reduce doses and to develop dose assessment tools that can be used in clinical practice and to develop of a series of science-based recommendations to ensure adequate and better RP for patients and staff. Specifically, the work package 6 of the project foresaw: -drafting of recommendations (RECOs) based on scientific results of the project; these RECOs are mainly addressed to institutional bodies and scientific associations to drive research on ionizing radiation towards an improvement of the RP of patients and health workers; -wide stakeholder involvement at European and international level to discuss and share the RECOs; -dissemination of MEDIRAD RECOs The scientific basis for the RECOs stems from the research developed in the course of the MEDIRAD project. In order to achieve a sufficient degree of consensus, MEDIRAD engaged in a substantial dialogue with relevant stakeholders in Europe and internationally. The MEDIRAD Stakeholder Forum, which underpinned this dialogue, included representatives from 86 organisations who were invited to express their views on issues to be considered as priority and to comment on the draft formulation of MEDIRAD recommendations. Among the four RECOs developed, the ISS contribution was mainly focused on RECO1, especially on the aspects related to the GDPR Directive [1] compliance. This RECO aims to facilitate the development of large scale multinational epidemiological studies by proposing for example guidelines to help European countries implement European regulatory requirements on ethics and encouraging harmonization of regulatory practice notably through the collection of experience gathered through the EURATOM research projects. A synthetic excursus of the RECOs together with a deep description of RECO 1 “GDPR and Medical Radiation Protection Research” will be presented. The four RECOs are available at

Retrospective dosimetry using components of mobile phones
PRESENTER: Michael Discher

ABSTRACT. Retrospective dosimetry refers to dose assessment of individuals after a radiological incident or in situations when conventional personal dosimeters were not available or sufficient (ICRU, 2019, Bailiff et al., 2016). In daily life, a mobile phone is usually carried closely to a body and is a perfect candidate to fulfil the requirement as a fortuitous dosimeter. Consequently, different components of a mobile phone have been investigated by different research groups and the dosimetric properties were examined. There are systematic investigations available using different materials which are found on or inside a phone, such as glass extracted from the display or from its covering, Al2O3 ceramics extracted from the circuit board or silicate-based filler materials found in chip cards (i.e. SIM cards). This paper gives a brief overview of the different luminescence materials available from a phone and highlights their dosimetric properties by analysing the strengths, weaknesses, potentials and challenges of the different luminescence methods.

ICRU, 2019. International Commission on Radiation Units and Measurements, Methods for initial-phase assessment of individual doses following acute exposure to ionizing radiation. ICRU Report 94. J. ICRU 19 (1), 1-162. Bailiff I.K., Sholom S., McKeever S.W.S., Retrospective and emergency dosimetry in response to radiological incidents and nuclear mass-casualty events: A review. Radiation Measurements. Volume 94, November 2016, 83-139.

Validation and automation of a novel biological dosimetry method combining dicentric chromosome and micronucleus assays
PRESENTER: Clarice Patrono

ABSTRACT. In large-scale radiological and nuclear emergencies the early individual dose assessment is of primary importance to rapidly identify overexposed individuals requiring medical intervention. In this regard, we are validating a novel biological dosimetry method combining the dicentric chromosome assay (DCA) and the micronucleus test (MN) in a single protocol [1]. The method was optimized within the BioPhyMeTRE project “Novel biological and physical methods for triage in radiological and nuclear (R/N) emergencies” ( [2], modifying key steps (i.e. colcemid treatment and fixation) in order to obtain an adequate number of good quality metaphases and binucleated cells for triage procedure, both in control and in irradiated samples. Validation of the combined biological protocol was performed by comparing it with the standard methods for DCA and MN in terms of numbers of dicentrics and micronuclei induced by 60Co gamma rays at the following doses: 0.5, 1, 2, 3 Gy. The reproducibility of the method is under evaluation: dicentric and micronuclei yields in irradiated blood samples from healthy donors are being compared, taking into account inter-individual variability in response. The production of dose-response curves for the combined protocol is ongoing, on a panel of 10 doses in the range of 0.25–5.0 Gy, according to IAEA recommendations. Moreover, the automation of the scoring for the combined protocol has been performed by the Metafer 4 Scanning Platform (MetaSystems): for the analysis a single classifier called SIF (Search Information File) is used, which combines three different search/analysis algorithms by executing them in an ordered sequence (micronucleus analysis, metaphases search and automated metaphases capture).

Acknowledgements: the BioPhyMeTRE Project is fully funded by Science for Peace and Security NATO Programme (Grant G5684).

1Testa, A., Palma, V., Patrono, C. (2019) Radiat Prot Dosimetry, 186(1), 9-11. 2Testa, A., Patrono, C., Palma, V., Kenzhina, L., Mamyrbayeva, A., Biyakhmetova, D., et al. (2020) Il nuovo cimento C, 43(6), 1-8.

Experimental determination of concentration factors of Mn, Zn and I in the phytoplankton species Phaeodactylum Tricornutum

ABSTRACT. During normal operation of nuclear facilities, small and controlled amounts of radioactive elements are released into the environment. The resulting dose to humans is estimated using ecosystem models, and one category of model parameters is concentration factors (CFs), which relate the concentration of an element in organism, under equilibrium conditions, to the concentration in the surrounding medium. Many nuclear facilities, in particular nuclear power plants, are located at the sea and hence the marine ecosystem is important. The primary producers in the marine ecosystem are phytoplankton, and hence their CFs are central for correct dose predictions.

In this work, we have identified elements with phytoplankton CFs that need to be better known, due to their relatively large impact on the dose assessment as well as scarcity and spread of literature data. These elements were Mn, Zn and I. Their phytoplankton CFs were experimentally determined in this work. Cultures of the diatom species Phaeodactylum Tricornutum were grown in saline and brackish seawater samples (from Skagerrak and the Baltic Sea, respectively) with additions of nutrients and radioactive isotopes of the concerned elements (Mn-54, Zn-65 and I-131). The CFs were determined by relating the activity concentration in the seawater to the activity in the phytoplankton.

Based on the present data, our conservative estimates (the highest of the values obtained for saline and brackish water respectively) of these CFs are (relative standard deviations in parenthesis) 7 200 L/kg (36%) for Mn, 9 100 L/kg for Zn (45%) and 33 L/kg (38%) for I, all phytoplankton masses referring to fresh weight. The passive adsorption of radionuclides on senescent cells was also quantified and found to be much lower than the active uptake.

Our results are in general similar to or lower than the lowest published values, which may be due to the particular phytoplankton species considered, phytoplankton being a diverse organism category.

Development of a real time reporting system for dose assessment of operators in abnormal events in nuclear medicine therapy: the SIREN project
PRESENTER: Sveva Grande

ABSTRACT. The SIREN Project (2020-2022) aims to develop procedures for reporting abnormal events in a Nuclear Medicine Therapy Unit by the implementation of an Internet of Things (IoT) dose monitoring system and a mobile application. The involvement of the operators in all phases, from the design of the IoT system and app up to their final test, is expected to contribute significantly to a more positive attitude of the staff towards incident reporting and proactive actions and in a general increase in incident prevention and preparedness. This seemed to be a necessary step to increase the perception of the usefulness of reporting or reduce the fear of the consequences. Face-to-face interviews were carried out with 24 staff in different roles in the Unit (radiation protection experts, physicians, physicists, nuclear medicine technicians and nurses). The interviews with the staff were aimed at collecting a list of needs and expectations with respect to the reporting system, the attitude towards radiation protection devices as well as opinions on the abnormal events considered most frequent and at highest risk of accident. Based on this list, a mobile application has been designed and is under development. For the dose monitoring system, the type, number, and location of dose detectors will be based on the dose levels estimated at different distances from the patient and at different times after radiopharmaceutical administration. These estimates were obtained from a literature review and simulations (obtained with ResRad Build) of the dose distribution in the inpatient room under abnormal event scenarios, such as patient's emesis or catheter leakage. There was a good response from the staff with being involved in the design of the app and of the dosimetry system. This involvement is expected to increase the users’ acceptance and the availability to use it.

Tomato hairy roots accumulating anthocyanins as a test bed for space food anti-oxidant properties

ABSTRACT. In the near future, NASA plans to launch the Artemis program with the aim to look further into human life in deep space. Ionizing radiation is considered the main hazard in spaceflights both to humans and plants, due to the generation of cell metabolic stress leading to free radical species. The issue of counteracting overproduction of free radicals is crucial for survival in space outposts and opens the way to the idea of the ‘antioxidant space fresh food’. As a consequence, there is great interest in the development of plants producing natural antioxidant food that may be introduced into space crews’ diet. Among flavonoids, anthocyanins are well-known as health-promoting and chronic-diseases-preventing molecules due to antioxidant, anti-inflammatory, anti-proliferative and anti-neurodegenerative functions. Here, tomato hairy root cultures engineered to synthesize anthocyanins, normally not accumulated by commercially available tomato crops, were used as a test bed for the design of bio-fortified whole plants aimed at agrospace applications. This model allowed to profile the accumulation of 5 major anthocyanins in the engineered tomato cells. A detailed analysis on phenylpropanoids was carried out by LC-HRMS. The antioxidant properties of the engineered tomato cells were evaluated upon ex-vivo high dose radiation, as a potent pro-oxidant stimulus (i.e. 2 kGy, administered through the Calliope 60Co gamma irradiation facility, at ENEA). Significantly higher free radicals scavenging activity and lower accumulation of reactive oxygen species were found in the engineered tomato model with respect to control, as demonstrated by Electron Spin Resonance Spectroscopy. Moreover, both UV-VIS spectra and photoluminescence analysis demonstrated that polyphenols content and folding of tomato soluble protein were not significantly affected by the pro-oxidant stimulus. These results may have significance in the engineering of whole tomato plants that can benefit space agriculture.

Long 222-220Rn time series at Campi Flegrei caldera (southern Italy)

ABSTRACT. A large study on long-term monitoring of radon time series data (10 years from 2011) at sites in the Campi Flegrei volcanic area (Naples, Italy) is presented. Measurements were performed with the RaMonA system which is based on measurement of the alpha particles of the daughters of radon (222Rn) and thoron (220Rn). The time series are drawn with powerful mathematical methods (hybrid methods) to capture and interpret radon trends and anomalies due to volcanic-seismic events. The investigations on the gas composition of the monitoring area were carried out in relation to the characteristics of the time series studied. A particular novelty is that the radon, and here also the thoron, signal was studied in connection with the fumarolic tremors recorded in a site of the Campi Flegrei area. The results are also compared with the following indicators: the cumulative of background seismicity; the maximum vertical deformation acquired by GPS networks; the temperature-pressure of the hydrothermal system estimated based on gas geo-indicators. The 222-220Rn trends and the results of the obtained anomalies confirm recent studies that have shown a new and long unrest phase in that area. The comparisons also suggest that the extension of the area affected by current Campi Flegrei crisis is larger than the area of seismicity and of intense hydrothermal activity from which the radon stations are 1–4 km away. This unrest phase has greatly influenced public opinion in Italy in recent years. The results of the study represent an absolute novelty in the study of a such calderic area and mark a significant step forward in the use and interpretation of the radon, and mainly for the rarely studied thoron signal4. In fact, although thoron comes only from the most surface layer (half-life 55.6 s), so the characteristics of its time series are strictly connected to the shallow phenomena, this study provides good evidence that the gas is related to the carrier effect of CO2 within the caldera. The hydrothermal alterations, induced by the increase in temperature and pressure of the caldera system, occur in the surface soils and significantly influence thoron’s power of exhalation from the surface layer.

The first map of the radon potential of the Campania region and the radiological characterization of natural building materials

ABSTRACT. For a better understanding of the 222Rn risk associated with soils and building materials in the Campania region, extensive research has been done. The first potential map of the Radon distribution in the Campania region was created, using the kriging geostatistical interpolation method using the QGIS® software. The response variable is the activity concentration of indoor radon and the proxy variables used as predictors are: geology, faults, soil permeability, meteorological/climatic parameters, 238U distribution, emanation coefficients and radon exhalation rate, 226Ra in building materials and gamma dose rate in buildings. The obtained map highlighted the areas with a high Radon potential in correspondence with the main volcanic centers and the fault network of the region [1]. This map is a useful tool for identifying radon risk areas and assessing the level of radon risk for the population. The characterization of the natural radioactivity content was also carried out in thirty-one natural construction materials representative of the Campania region, using gamma spectrometry to determine the activity concentrations of 226Ra, 232Th and 40K. Radiation hazard indices commonly used to assess human radiation exposure were calculated. The comparison of the results with the limit values established by Directive 2013/59/Euratom and by UNSCEAR 2000 demonstrates the high content of natural radioactivity and the risk of exposure to radiation from some samples of volcanic origin. The emitted fraction and exhalation rate of 222Rn and 220Rn (thoron) were also measured. The different materials show interesting differences in their radioactivity content and their ability to generate radon [2].

[1] Sabbarese C. et al., (2021). The first radon potential map of the Campania region (southern Italy). Applied Geochemistry 126 (3), 104890. [2] Sabbarese C. et al., (2020). Radiological characterization of natural building materials from the Cam-pania region (Southern Italy). Construction and Building Materials, 268 (6), 121087.

Quantification of the hydrated electron by scavengers and Geant4-DNA simulation

ABSTRACT. This study aims at better evaluating the indirect effects of water radiolysis on the ionizing radiation damage mechanisms during radiotherapy treatment. Under irradiation of a cell, ionizing radiations will generate radicals, which will yield damages to biomolecules, including proteins and DNA. Our body is mostly made up of water (~65 – 70%), proteins (~20%), lipids (~10%), with DNA accounting for only 0.1%. When an ionizing radiation enters a cell, it can either interact directly with a biomolecule (direct effect) or interact with water, resulting in the formation of reactive species by radiolysis of water. These reactive species will then react more or less quickly with the surrounding biomolecules, inducing damage by indirect effect. Given the proportion of water, indirect effects will play a very important role in the phenomena occurring under irradiation in the cell. The precise quantification of water radiolysis species is therefore essential for understanding the mechanisms of damage formation to biomolecules. Regardless of the ionizing radiation (accelerated ion, electron, X-ray or gamma), reactive species generated by radiolysis of water are about the same: hydroxyl radical HO•, hydrogen atom H•, hydrogen peroxide H2O2, and hydrated electron eaq-.

In this poster, I will present the production yield kinetics of hydrated electron eaq¬ , on time scales from nanosecond to microsecond, determined by scavenging. Experiments were realized with 6 MeV X-rays (ICANS – Strasbourg), 2 MeV protons (ACACIA – Strasbourg) and 1 MeV electrons (AERIAL – Strasbourg). Experimental data of reactive species have been compared to Monte Carlo simulation results produced with the Geant4-DNA software [1], an extension of Geant4 that was developed to simulate physical, chemical and biological effects of ionizing radiation on DNA.

Coralyne radiosensitizes human lung A549 adenocarcinoma cells by upregulation of CDKN1A expression

ABSTRACT. Introduction

Protoberberine alkaloids and their derivatives are used as antitumor agents in traditional medicine and have been investigated nowadays. Coralyne is a synthetic analogue of protoberberines alkaloids. Structure of coralyne is related to the isoquinoline four rings, but their arrangement in contrast to other protoberberines alkaloids is fully aromatic, which makes the structure flat. This alterations in their chemical structures significantly affect the biological properties of coralyne. For this purpose in our study we analyse effects of coralyne and IR on cell cycle progression and expression of selected genes and proteins implicated in cell cycle regulation in human lung A549 adenocarcinoma cells. A549 cells were treated with 1–25 μM coralyne for 24 hours and then irradiated with 2 Gy of gamma-rays using a 6 MV Medical Linear Accelerator Artiste (Siemens). 24 h after irradiation cells were harvested and proceed for cell cycle analysis and genes and proteins expression.

Results and conclusion The results showed that treatment of cells with different concentration of coralyne alone did not affect the cell cycle progression. Exposure to 2 Gy of ionizing radiation alone resulted in an enhanced accumulation of A549 cells in the G2/M phase. This effect was significantly reduced when cells were pretreated with coralyne in a dose 25 µM for 24 h before irradiation with 2 Gy, suggesting that coralyne can eliminate the radiation-induced G2/M arrest what effectively increases A549 cells radiosensitivity. We investigated the expression of three genes related to cell cycle progression in cells: cyclin B1-interacting protein 1 (CCNB1IP1), cyclin-dependent kinase inhibitor 1 (CDKN1A) andcyclin D-binding Myb-like transcription factor 1 (DMTF1). We have observed that the combined treatment of A549 cells with coralyne and IR strongly upregulated CDKN1A expression. The level of CDKN1A protein was significantly increased only in cells treated with 25 μM coralyne and 2 Gy of IR.

Adverse Outcome Pathways and Linkages to Genomic Signatures Relevant to Ionizing Radiation Injury

ABSTRACT. A large body of data on the effects of radiation on gene expression has been generated over the past three decades. The data has allowed for an understanding of events at the molecular-level and has shown a level of consistency in response despite the vast formats and experimental procedures being used across institutions. However, clarity on how the accumulated data is applied to regulatory decision-making is needed. An approach to bridge this gap is the adverse outcome pathway (AOP) framework. AOPs represent an illustrative framework, characterizing a stressor associated with a sequential set of causally linked key events (KEs) at different levels of biological organization, beginning with a molecular initiating event (MIE) and culminating in an adverse outcome (AO). Launched by the Organisation for Economic Cooperation and Development (OECD), AOPs enable the union of existing biological knowledge to essential KEs that initiate a path to disease, and are utilized in both chemical and ecological risk assessment. Here, we demonstrate the potential application of the AOP framework within the field of ionizing radiation illustrating a transcriptomic gene-informed radiation-related AOP to lung cancer (the AO). Radiation-induced gene alterations and pathways found within the AOP have been characterized, however, there is a need to apply the gene signatures and pathways within KEs identified and to elucidate causal gene linkages and pathway interactions. This approach not only enhances the AOP under development, but also provides a means for the data to be used to inform regulatory-decision making and highlights important research gaps. Accordingly, through systemic mining of the literature and publicly available databases using the software ‘DistillerSR’, we selected radiation-specific transcriptomic studies and data within these KEs at the molecular, cellular, organ, and population level. Gene signatures and pathways were then associated to each KE and an evidence map was constructed of consistent gene responses and areas that require directed research by Reactome and GeneMANIA. This approach allowed for the identification of previously described genes and pathways (e.g. DNA repair, inflammation) associated with the new identified associate events (AEs), KEs, and novel characterization of radiation-induced gene signatures enabling AOP enhancement.

Radiotherapy induced alteration in cytokine levels are mitigated by TSA in C57BL/6 mice
PRESENTER: Teena Haritwal

ABSTRACT. Secondary malignancies caused by exposure to radiation during radiotherapy in the course of treatment of cancer, have become a clinically significant issue. Hematopoietic stem cells (HSCs) are fast-dividing cells that can become cancerous if exposed to whole-body radiation during cancer treatment. There is an increased chance of developing secondary cancers such as chronic myeloid leukemia (CML), Acute myeloid leukemia (AML), and Acute non-lymphocytic leukemia(ANLL). Trichostatin A is a very potent histone deacetylase inhibitor (HDACi) at a very low concentration. It has anti-cancer activity too. We hypothesized that administering TSA shortly after exposure to radiation will enhance the acetylation status of genes, lowering the risk of subsequent cancers by reducing apoptosis and pro-and anti-inflammatory responses. To detect various cytokine levels C57Bl/6 mice were used and TSA was administered 1 and 24h after 5 Gy of gamma radiation (60Co) exposure and various cytokine levels were measured after 2 and 24h post-irradiation. For apoptotic studies, TUNEL assay was performed on the 2nd, 4th and 8th day after radiation exposure. Radiation-induced changes in the levels of IL-2, IL-5, TNF-α, GM-CSF, and apoptosis were mitigated by TSA administration. Therefore, our study suggests that all of these cytokines increase the recovery of hematopoietic stem cells, reduce the number of apoptotic cells in the bone marrow and hasten the recovery of a variety of blood parameters. The JAK-STAT pathway, as well as numerous anti and pro-apoptotic proteins, are being studied to validate the mitigation of probabilities of radiation-induced leukemia by TSA.

Verification of the accuracy of delivered dose by the treatment planning system for high-dose-rate skin brachytherapy through thermoluminescence dosimetry
PRESENTER: Francesco Manna

ABSTRACT. Brachytherapy (BT) is a radiotherapy technique that allows to deliver dose through a radioactive source placed inside or next to the tumor. Due to the characteristic dose fall-off beyond the tumor, BT leads to an higher sparing of the surrounding tissues compared to conventional external beam radiation therapy. Despite BT is a technique widely implemented in the clinical setting, the dosimetric issue represents an interesting research field. In fact, the commercially available Treatment Planning Systems used to carry out the treatment plan are provided with a dose calculation algorithm (AAPM TG-43) affected by some limitations and approximations: it assumes that the patient is totally made of water neglecting tissue heterogeneities and intersource dose attenuation, leading to uncertainties affecting dose delivery. The aim of the presented work is to investigate the dosimetric accuracy of the TPS (Oncentra Brachy by Elekta) for high-dose-rate skin BT performing thermoluminescence dosimetry by the use of dosimeters LiF:Mg,Ti (TLD-100) provided by Harshaw Chemical Company. The work was carried out at the Istituto Nazionale Tumori IRCCS Fondazione G. Pascale (Naples) and at “LaRa” Radioactive Laboratory (Certified ISO 9001) of the Physics Department Ettore Pancini, University Federico II, were the TLD analyzing system is installed. The radioactive source used for treatments is Iridum-192 (Ir-192). The TLDs used were first characterized through the measurement of the individual sensibility factor with a 6 MV photon beam. Then, to use them as dosimeters, the calibration factor (CF) that converts the thermoluminescent signal into dose value was determined. The method implemented consists in an indirect calibration: the CF for the photon energy of Ir-192 was obtained by linear interpolation between the CF corresponding to 250 kV X-rays, Cs-137 and Co-60. Superficial and anthropomorphic phantoms have been used to simulate the treatment and verify the delivered dose. Dose comparisons have been performed by simultaneously measurements obtained with gafchromic EBT3 films. The preliminary results will be presented.

Experimental and Monte Carlo simulation study of Linear Energy Transfer with heavy ions beams (4He, 16O and 12C) for future applications in ion-beam therapy
PRESENTER: Giada Petringa

ABSTRACT. In the present hadrontherapy scenario, there is a growing interest in exploring the capabilities of different ion species other than protons and carbons. The possibility of using different ions paves the way for new radiotherapy approaches, such as the multi-ions treatment, where radiation could vary according to the target volume, shape, depth and histologic characteristics of the tumour. In this work, the study and understanding of biological-relevant quantities were extended for the case of the 4He ion, 12C and 16O. Monte Carlo based algorithms in the dose and track-averaged LET (Linear Energy Transfer) calculations were validated also for the case of a mixed field characterized by the presence of secondary ions from both target and projectile fragmentation1. The simulated dose and track averaged LETs were compared with the corresponding dose and frequency mean values of the lineal energy, yD and yF, derived from experimental microdosimetric spectra. Three microdosimetric experimental campaigns were carried out at the Laboratori Nazionali del Sud of Istituto Nazionale di Fisica Nucleare (INFN-LNS, Catania, I) using the silicon microdosimeter MicroPlus probe. The MicroPlus probe is an array of 3D right parallelepiped shape sensitive volumes (diodes) with area 30 um X 30 um, fabricated using silicon on insulator wafers with an active layer of 10 um thickness. The LET distribution was evaluated in both irradiation conditions: with a pristine Bragg Beak and modulated peak (obtained by using a proper ridge filter). The beam energy was 62 MeV/n in all the investigated cases. The obtained results verified with the statistical analysis based on the chi-squared goodness of fit test, the total averaged LET quantities obtained with the Monte Carlo calculations, including all the contributions of secondary ions, find a remarkable agreement with the microdosimetric experimental data, within their respective uncertainties.

Dose-response effects of Lu-177-octreotide and Lu-177-octreotate in neuroblastoma-bearing mice
PRESENTER: Arman Romiani

ABSTRACT. Background: Patients with aggressive neuroblastoma (NB) have a 5-year survival rate of only 40-50%, indicating the need for novel and improved treatment options. Since NBs overexpress somatostatin receptors, radiolabelled somatostatin analogues have a potential treatment option. In our previous biodistribution studies with Lu-177-octreotate, mice xenografted with human NB cell lines were shown to have high uptake in tumor tissue compared to risk organs. The aim of this work was to compare the biodistribution of Lu-177-octreotide with Lu-177-octreotate and to study their therapeutic effects in CLB-BAR xenografted mice. Methods: Female nude BALB/c mice (5-6 weeks old) were s.c. injected on their flank with CLB-BAR cells. Biodistribution of 1.5 or 15 MBq Lu-177-octreotide was investigated at 1h, 24h and 168h. For the therapeutic study animals were divided into groups and administered with 15, 30 or 60 MBq Lu-177-octreotide or Lu-177-octreotate. Other mice received fractionated administration with 1x15, 2x7.5, and 3x5 MBq Lu-177-octreotate.Treatment effects were studied by tumour volume measurements. Results from both studies were compared with those from sham treated mice (i.v. saline injections). The tumour volume was measured with a caliper twice a week and the mice were killed when the tumour mass exceeded 10 % of the body weight. Results: Comparable biodistribution profiles were observed after administration with Lu-177-octreotide or Lu-177-octreotate. Treatment with Lu-177-octreotide displayed a clear dose-response relation and a clear anti-tumour effect was observed. No clear dose-response relationship was observed after treatment with Lu-177-octreotate. Although the mice that received 60 MBq showed a better response, the tumour volume was not significantly lower than other groups. Furthermore, fractionation with 3x5 MBq Lu-177-octreotate displayed a more profound effect on the tumour volume. Discussion and conclusion: The mice that received Lu-177-octreotide showed a clearer dose-response relationship with a stronger anti-tumour effect in comparison with Lu-177-octreotate. Pronounced anti-tumour effects following fractionated administration merits our thoughts regarding saturation effects and further studies will investigate this theory.

Isotope labelled ferritin – an alternative drug delivery system
PRESENTER: Lukasz Cheda

ABSTRACT. Cancers are the second most common cause of death worldwide. Each year, they cause the death of nearly 10 million people. The high diversity of neoplastic diseases and the existence of metastases, characteristic for malignant tumours, makes finding a universal diagnostic and therapeutic method a difficult and so far unresolved problem. Current research is focused on the development of methods enabling detection of smaller metastases and diagnosis of cancers at early stages of growth, as well as more effective and safer therapies. This is expected to reduce mortality and improve patient welfare. The aim of our studies is to assess the applicability of ferritins as an alternative delivery system of isotopes for tumour diagnosis. Ferritins are proteins with a cage-like structure and they are responsible mainly for the storage and transport of iron in the organism. Our experiments with ferritin molecules labelled with the copper-64 isotope indicate highest concentration of the compound in the tumour tissue 12-24 h after injection. Therefore, we decided to perform experiments with other radionuclides selected based on their half-life and characteristics of the emitted ionizing radiation, i.e.: iodine-131 and lutetium-177. We have carried out an optimization procedure of ferritin labelling experiments and gathered extensive data on biodistribution of the radioconjugates. The obtained results indicate the possible use of radioisotope labelled ferritins for diagnosis and treatment of tumours, especially when combined with molecular imaging techniques such as PET or SPECT. The research is founded by the National Science Centre, PRELUDIUM 18 grant no. UMO-2019/35/N/ST4/01987 and the European Research Council Starting Grant McHAP no. 715048. The contribution of L. Cheda was realized within the “Operational Project Knowledge Education Development 2014-2020” grant no. POWR.03.02.00-00-I009/17-00, co-financed by the European Social Fund.

From design to in vitro evaluation of potential radioprotectors
PRESENTER: Vojtěch Chmil

ABSTRACT. Purpose: The risk of radiation exposure, which is increasing due to recent events, highlights the need for new radioprotectants. The aim of this work was to design, synthesize and determine the toxicity and radioprotective effects of a series of 1-(2-hydroxyethyl)piperazine derivatives in vitro. The substances were in silico modeled and in-house synthesized. Materials and methods: First, a docking study toward Bcl-2 anti-apoptotic protein was performed and the most suitable structures were designed and synthesized. 10 human cell lines, 9 cancerous and 1 non-cancerous were used for in vitro evaluation of toxicity of concentrations 10 µM and 100 µM by the WST-1 proliferation assay. MTT test was used to assess half inhibitory concentration (IC50) and maximum tolerated concentration (MTC) in A-549 lung cancer cell line. The radioprotective effect was tested by flow cytometry with Annexin V/propidium iodide staining on the MOLT-4 T-lymphoblastic leukemia cell line, which was treated by 100µM concentration of tested compounds 1 hour before irradiation by gamma source 60Co. Results: Tested compounds showed MTC ranging from 0.002 to 6.25 mM and IC50 from 0,04 to > 25 mM, nevertheless the least toxic substance was considerably unstable. Several compounds significantly increased survival fraction (SF) ranging from 51 to 65 % in the MOLT-4 cell line after 1 Gy irradiation compared to non-treated irradiated control (SF=40 %) and non-treated non-irradiated control (SF=92 %). An overview of the impact on viability will be given. Conclusion: The results suggest that some of the 1-(2-hydroxyethyl)piperazine derivatives have promising radioprotective potential. Currently, the structure of the best substance is being improved to more pronounced radioprotectivity and lower toxicity. In parallel, the putative mechanism of action is investigated and indications are that it might involve inhibition of the intrinsic apoptotic pathway.

Challenges and opportunities of ultra-high dose rate dosimetry for FLASH radiotherapy

ABSTRACT. Preclinical studies have shown that FLASH radiotherapy (RT) may substantially improve normal tissue sparing while maintaining high tumour control probability compared to conventional dose-rate RT [1]. However, the clinical translation of FLASH RT requires challenges related to dosimetry and beam monitoring of ultra-high dose rate (UHDR) beams to be addressed. Active detectors currently in use suffer from saturation effects under UHDR regimes and passive detectors able to measure on site in a reasonable time are not available [2]. There is a significant interest in identifying the most reliable experimental approach for UHDR dosimetry. In this presentation, the main challenges coming from the peculiar beam parameters characterizing UHDR beams for FLASH RT will be discussed. These challenges vary considerably depending on the accelerator type. A detailed status of the current technology will be presented, with the aim of discussing the detector features and their performance characteristics and/or limitations in UHDR regimes [3]. Further developments for established detectors will be also reported together with novel solutions currently under investigation, based on new prototypes of solid-state detectors, scintillators, modified ionization chambers and portable calorimeters, with a view to predicting future directions in terms of dosimetric procedures. In particular, a description of the new approaches for dosimetry and beam monitoring, developed in the framework of the Italian INFN FRIDA (FLASH Radiotherapy with hIgh Dose rate particle beAms) collaboration will be provided, discussing recently achieved results.

1 V. Favaudon et al., Science Translational Medicine, 6(245), 245ra93 (2014). 3 M. McManus M., SCIENTIFIC REPORTS, vol. 10, ISSN: 2045-2322 (2020). 2 F. Romano et al., Medical Physics, in-press, doi: 1002/mp.15649

Monitoring of the long-term trends in parameters of electromagnetic radiation in urban environment of Warszawa

ABSTRACT. The highest density of radio communication antennas and electromagnetic radiation (EMR) with the most complex frequency spectrum may be found in the urban environment. The aim was to identify and assess the trends in long-term changes of parameters of EMR emitted by urban radiocommunication systems in Warszawa (capital city of Poland). The frequency spectrum of EMR was measured and analyzed in order to determine the components of exposure in particular locations, and monitoring of the time-variability of the level of EMR exposure (using exposimeters – multi frequency-narrow-band electric field data recorders – collecting characteristics of exposure to EMR harmonized with the frequencies of typical urban radiocommunication systems: mobile phones (downlink signal transmission from the base station to the terminal: LTE 800, GSM 900, DCS/LTE 1800, UMTS/LTE 2100, LTE 2600) and radio and television transmitters (FM, TV VHF/UHF), local network of communication between devices and access to the Internet (Wi-Fi 2.4/5 GHz). It has been shown that the level of exposure in a given place depends mainly on the impact from the nearest EMR transmitters – mobile phone base station in the typical urban location. A comparative analysis of the results of EMR over years did not show any significant changes in the total level of exposure in Warszawa during the last 20 years. However, changes in the composition of dominating frequency components have been documented - related to the migration of radiocommunication services into new technologies and frequency bands (such as migration of analog TV into the digital service, or introduction of new mobile phone systems – 3G, 4G, LTE). Currently, the observed dominating exposure is associated with the use of mobile internet access (LTE) and to a lesser extent from voice calls (GSM). The results of the EMR monitoring in urban environment correlate with reports regarding changes in the structure of telecommunication services usage.

Funding: the National Programme “Improvement of safety and working conditions” (the scientific research and development funded by the National Center for Research and Development/No II.PB.16)

Study of potentially renal cancerous effect of uranium in genetically-engineered mouse models: UKCAN project
PRESENTER: Yann Guéguen

ABSTRACT. Exposure of uranium compounds can occur in several situations including nuclear fuel processing, military activities, and natural exposure. Uranium (U) is a radio element with known radiological (α-emitting radionuclide) and chemical (as a heavy metal) toxicities, which accumulates preferentially in kidneys. Renal carcinogenic effect in U-exposed population is suspected and need additional epidemiological or experimental studies. Since the process of initiation and propagation of cancer can be particularly long, we have chosen to use two genetically-engineered mouse models (GEMM) predisposed to its development to investigate the link between uranium exposure and the risk of renal cancer. The project aims to: • Assess the relevance of non-invasive imaging tool (ultrasonography, metabolic cages) and innovative biomarkers in urines to determine the incidence and grade of tumors • Study of benign and malignant renal tumors in GEMM models as a function of time and dose of uranium exposure through histo- and immunohistology • Determine the renal oncogenesis and carcinogenesis biological mechanisms and cancer hallmarks (initiation, promotion, proliferation) in GEMM by clinical biochemistry, molecular biology. • Analyze the uranium content in targets organs and excreta’s by ICP-MS Our preliminary studies aim to correlate specifically histology to ultrasound echography in order to follow-up endogenous renal tumors and determine their characteristics (type, number, volume). The phenotypic characterization is done at regular time intervals up to the age of 1 year (bodyweight, diuresis, ultrasonography). The evolution of renal injuries and masses leading to tumors as small as 200 µm are detected and quantified. Histological analyses showed delimited areas of different tumor types in GEMM. Immunostaining for CAIX, CK7, PS6 or VIM protein allows the identification, localization, and quantification of tumors in renal tissue. Immunostainings are in progress to complete the phenotypic characterization of RCC. The comparison of the increase, or not, of tumor effects in these respective models will provide experimental knowledge to analyze the possible link between uranium exposure and the development of renal cancer.

The cancer suppression mechanisms of elephants: An in vitro study to shed light on Peto's paradox

ABSTRACT. The risk for cancer between mammalian species is not related to body size and species life span. This lack of correlation is known in evolutionary biology as Peto's Paradox. In elephants, the largest land mammals, the answer to this paradox might lie in the redundancy of the tumour suppressor gene TP53 gene. The presence of 20 TP53 copies and the more recently discovered LIF6 pseudogene might suggest that the cancer defence of elephants is mediated by an enhanced apoptotic response to DNA damage, removing potentially cancerous cells at an early stage. However, the redundancy in tumour suppressor genes cannot resolve Peto’s paradox completely in elephants, since they should have developed a trade-off between the aggressive elimination of damaged cells and senescence, resulting in depletion of their stem cell pool. Here, the first results of a joint project are presented, highlighting what implications Peto’s paradox could have for radiation protection and radiation therapy strategies. Blood samples were collected from elephants by experienced wildlife veterinarians in the Zoo of Naples (Italy) and private game reserves with free-roaming elephants in South Africa. After transport to the laboratories, the elephant blood samples were irradiated and apoptosis (Annexin V-FITC/PI) and DNA repair response (γ-H2AX foci) was compared to human samples. In addition, comparative next generation sequencing was performed on human and elephant blood samples, to investigate which specific pathways are up- or downregulated after radiation exposure. The results of the apoptosis assay illustrate that elephant cells go into apoptosis at much higher rates than human cells, even after exposure to doses as low as 0.125 Gy photons. While no statistically significant difference could be observed in the number of DNA DSBs at 1 hour post-irradiation, the 24 hours result confirm that elephant lymphocytes have lower numbers of residual DNA DSBs compared to human lymphocytes. The results confirm the working mechanisms of the tumour suppressor gene and striking differences in DNA repair capacity between human and elephant cells. It is envisaged that this project could rapidly advance the development of new strategies for the prevention of radiation-induced cancers or the sensitization of cancer cells to radiotherapy.

A Monte Carlo simulation for estimation of percentage depth dose distribution using the PRAGUE detection system prototype
PRESENTER: Alma Kurmanova

ABSTRACT. The interaction of radiation with matter is an inherently stochastic phenomenon involving complicated analytical functions which are near-impossible and time-consuming to solve. At the same time, the needs of the nuclear and particle physics community for large-scale, accurate, and comprehensive simulations of particle detectors are on the rise. In the past decades, several Monte Carlo based simulation toolkits such as Geant4, PENELOPE, EPOTRAN, and TOPAS have been developed to meet these growing demands, with each suited to a specific scenario. TOPAS [1] is a simple, yet robust, toolkit based on Geant4 and offering all its versatility but adapted to advanced radiotherapy simulations. Considering its capabilities, it was used for analyzing a new detection system PRAGUE (Proton RanGe measurement Using silicon carbidE) - SiC detectors in stack configuration operated with conventional and ultra-high dose beams. Experimental setups were reproduced in the simulation space to obtain the theoretical dose profiles and compared with measured data. The results from simulation agree well with experimental data, with chi-square test analysis indicating consistency between the two at a significance level of 0.05.

Extracellular vesicle mediated bystander effects in ionising radiation induced leukaemia
PRESENTER: Eric Rutten

ABSTRACT. Extracellular vesicles (EVs) are a class of small biologically active lipid-membrane enclosed vesicles, comprised of microvesicles and exosomes. They are an important component of intercellular communications, capable of shuttling proteins, lipids, lncRNA and miRNA cargoes from cell to cell (1). Cancer cells are very active in EV production (2), and tumour-derived EVs carry a high proportion of oncogenic miRNAs and proteins (3,4,5,6), suggesting that they participate to the remodelling of their immediate environment, mainly promoting growth. Acute myeloid leukaemia (AML) is one of the most common secondary cancers post ionising radiation (IR) exposure. EVs can possibly propagate radiation effects, e.g. DNA damage via the bystander effect (7), and are implicated in radiation-induced AML (rAML). The gene PU1/Sfpi1 exhibits a dose dependent deletion of a single allele post IR exposure in the mouse CBA mouse model with a proportion of mice developing rAML (8,9). To investigate the role of EVs in rAML, a tailored transgenic CBA mouse model was used, engineered with mCh and GFP fluorescent markers integrated next to Sfpi1 on chromosome 2 alleles, as such, loss of mCh or GFP signal is indicative of a preleukaemic clonal expansion (10). Mice were either mocked or 3 Gy X-irradiated at 10-12 weeks of age and blood samples were analysed by flow cytometry for a period of 12 months (or until death if before this period) for monitoring allele deletion. A distinct, radiation and pro-oncongenic miRNA signature was found in mice with pronounced levels of leukaemic clonal expansion. Dysregulated miRNA included mmu-miR-761 and mmu-miR-29b, among others. Further experiments on murine bone marrow derived primary cells (macrophages, mesenchymal stem cells, and haematopoetic stem cells) demonstrated a conserved miRNA profile post X-irradiation, such as an upregulated level of mmu-miR-582-5p, previously found upregulated in rAML (8), as well as other miRNA related to the regulation of lysine degradation. In parallel, the direct impact of EVs on bystander cells was also assayed, wherein the human myelomonocytic cell line CESS was irradiated, its EVs labelled and coincubated with human T-lymphocytes, which were subsequently sorted into EV-positive and EV-negative populations. The levels of DNA and chromosomal damage were measured by yH2AX and dicentrics respectively. The role of EVs in radiation-induced carcinogenesis will be discussed.

Imidazolyl Ethanamide Pentandioic Acid as a mitigator of Acute Radiation Syndrome in a Total Body Irradiation juvenile rat model

ABSTRACT. Introduction: Acute ionizing radiation exposure at doses of 2 to 6 Gy in humans results in Hematopoietic Acute Radiation Syndrome (H-ARS). The disease manifests through severe depletion of hematopoietic stem cells and reduced blood cell counts, leading to increased susceptibility to infections, hemorrhages, and premature death. Imidazolyl Ethanamide Pentandioic Acid (IEPA; Myelo001), an orally administrable small molecule drug with a good safety profile, mitigated myelosuppression in adult mice after sublethal and lethal (≤LD50/30) total body irradiation (TBI). The radiation effects after TBI exposure are reported to be age-dependent. The data presented here tested two doses of IEPA to determine the drug’s survival and hematological efficacy in a special juvenile WAG/RijCmcr rat population. Methods: Juvenile male rats (5-6 weeks old, n=16-25/group) were irradiated at 6.25 Gy TBI (X-RAD 320 Precision, 320 kVp, 13 mA, 1.73 Gy/min). Rats received vehicle (sterile water) or IEPA (10 or 60 mg/kg p.o.) at 24h, 48h, and 72h post-TBI. Peg-G-CSF (s.c.) was used as a positive control, given at 0.55 mg/kg once 24h post-TBI. Endpoints included 30-day survival, body weight, peripheral blood counts on days 3, 7, 10, 15, and 21, with each rat bled twice using a staggered collection method during the study, and a terminal bleed of the survivors on day 30. Bone marrow was analyzed via fluorescence-activated cell sorting (FACS) and Colony Forming unit assay (CFU-G, CFU-M, CFU-GM). Results: TBI resulted in 84% survival (n=21/25) in IEPA 10 mg/kg-treated (IEPA10) rats as compared to 72% survival (n=18/25) in the vehicle-treated group and 92% survival (n=23/25) after Peg-G-CSF treatment. In contrast, IEPA 60 mg/kg (IEPA60) treatment showed a 56% survival (n=9/16). A small positive increase in body weight was observed in IEPA10 versus the vehicle group. In contrast to the vehicle group, the IEPA10-treated rats showed a slower decline, reduced nadir, and an accelerated recovery of neutrophils, monocytes, and red blood cells. In addition, IEPA10 led to improvement of bone marrow cells with significantly increased CFU-GM populations. Conclusion: Dose optimization of IEPA shows survival and hematological efficacy for the lower 10 mg/kg dose at LD30/30 in the juvenile rat TBI model. Funding: NIH-NIAID contract 75N93020C00005.

14:30-15:15 Session Keynote Speaker
The role of extracellular vesicles in mediating acute and late radiation effects in the bone marrow

ABSTRACT. Extracellular vesicles (EVs) are membrane-bound structures released by the cells in the extracellular compartment. EVs are very heterogeneous in size and their internal cargo. The major role of EVs is intercellular communication, which is achieved by molecular mediators carried by EVs able to modify the function and/or fate of recipient cells. These mediators can be nucleic acids (mRNAs and diverse miRNAs as well as other small or long non-coding RNAs, DNA), proteins, lipids and small molecular weight metabolites, which by being protected by the double membrane layer preserve their integrity and biological activity in the extracellular space. This complex cargo allows for the transmission of “information packets”, which is a much more efficient way of signalling between cells than transmission of individual “information units”. Bone marrow is a particularly radiosensitive organ, where radiation damage of the stem cell compartment can be significantly modulated by signals received from the microenvironment. Thus, intercellular signalling is a key mechanism in modulating radiation damage in the bone marrow. A growing number of evidence shows that EVs play an important role in the manifestation of ionizing radiation-induced acute and late bone marrow damage. This presentation aims to give an overview on how ionizing radiation influences EV release and uptake by the different cellular subpopulations in the bone marrow. We will review how EVs by transmitting radiation-induced signals to non-targeted cells within the hematopoietic system are able to increase the level of radiation damage, or in contrary, mitigate certain aspects of radiation damage. We will shortly refer to the role of EVs in radiation leukemogenesis as well.

This project has received funding from the Euratom Research and Training Programme 2014-2018 under grant agreement No 662287 (CONCERT) and Horizon-EURATOM under grant agreement 101061037 (PIANOFORTE).

15:15-15:45 Session Invited Speaker
Low doses ionizing radiation and vascular diseases

ABSTRACT. The relationship between high dose ionising radiation and cardiovascular diseases is well established. In contrast, after moderate doses (0,5-5Gy) the relationship is suggestive and weak after low doses (<0,5 Gy). However evidence is emerging after low-level exposure to ionizing radiation that the risk of diseases of the circulatory system could increase and is one of the most important matters currently facing radiological protection (Gillies et al, 2017). In the present framework recommended by the ICRP, no account is taken of any risk of CVD (e.g., heart disease and stroke) consequent to exposure to low doses or low doserates. Epidemiological studies, mainly occupational ones, suggest that the risk of CVD following low-level exposure is increased, but the results are not always consistent and causative associations cannot yet be made because of unresolved interpretational issues. Another difficulty for epidemiologists is the lack of data related to the biological mechanisms associated with the observed findings. This is complicated by uncertainty on what are the doses to the relevant organs/tissues (e.g., brain, heart) that increase the incidence of CVD. A better understanding of the underlying biological and molecular mechanisms is needed. If one proves that there is an increased risk of CVD following low-dose exposure, it may have a considerable impact on current low-dose health risk estimates. Experimental studies on cardiovascular system indicate different responses based on the target, the dose and the dose-rate. The response of low doses on macrovascular or microvascular diseases is different based on cellular and molecular microenvironnement. For instance, in the context of atherosclerosis, a chronic inflammatory disease, low doses modulate inflammatory profile and decreases the lesion sizes depending of the dose-rate in mice models (Mancuso et al, 2015; Legallic et al, 2015; Ebrahimian et al, 2017). Whereas in the context of ischemia low doses increase post-ischemic nevascularisation (Heissig et al, 2002; Ministro et al, 2016). All of the current data suggests that all depends on the target and that more epidemiological and experimental research is required before final conclusions.

15:45-16:15 Session Oral Presentations
Early detection of subclinical left ventricular dysfunction by speckle-tracking echocardiography after radiation therapy for breast cancer: results from the MEDIRAD EARLY HEART study
PRESENTER: Médéa Locquet

ABSTRACT. Background: Radiotherapy (RT) for breast cancer (BC) can lead to an excess risk of cardiovascular (CV) diseases arising many years after treatment2,3. Early detection of subclinical CV changes post-RT could prove beneficial for asymptomatic patients by investigating early subclinical left ventricle (LV) dysfunction.

Objective: Within the European MEDIRAD project, the multicenter EARLY HEART prospective cohort aimed to assess the impact of RT-related cardiac exposure on subclinical LV dysfunction occurring 6 months and 24 months after RT based on measurements of myocardial function and wall deformation by speckle-tracking echocardiography4.

Methods: The study included chemotherapy naïve BC women aged 40-75 years treated adjuvant RT. Multi-atlas based segmentation was used to determine whole heart and LV doses (Dmean for mean dose)5,6,7. Echocardiography was performed at baseline, 6 and 24 months after RT. Subclinical LV dysfunction was defined with two approaches: a relative decrease in global longitudinal strain (GLS) >15% compared to baseline value (GLS-based LV dysfunction)8; a decline in LV ejection fraction ≥10% from baseline to a final value less than 53% post-RT (EF-based LV dysfunction)9.

Results: The analyzed population included 186 BC women (mean age 57.5±7.9 years, 64% left-sided BC). Six and 24 months after RT, 14% and 13% of patients respectively presented GLS-based LV dysfunction and 4% and 13% respectively with EF-based LV dysfunction. Significant dose-response relationships were observed with the risk of GLS-based LV dysfunction, 6 months after RT (Odds Ratio OR=1.74 (1.20-2.61) for Dmean whole heart, OR=1.46 (1.17-1.87) for Dmean LV), and remained significant in the same range 24 months after RT. For EF-based LV dysfunction, there was no association with cardiac doses at 6 months, but a significant dose-response relationship was observed at 24 months: OR=1.50 (1.20–1.87) for Dmean whole heart, OR=1.91 (1.31–2.77) for Dmean LV.

Conclusion: These results highlighted that all cardiac doses were strongly associated with the occurrence of subclinical LV dysfunction arising 6 and 24 months after BC RT. Whether measurements of GLS at baseline and 6 months after RT combined with cardiac doses can early predict efficiently subclinical events occurring 24 months after RT remains to be investigated.

Development of new radioprotective substances: An untold story.

ABSTRACT. Purpose: The classification of chemical radioprotectant includes compounds with the capacity to mitigate the biological effects of ionizing radiation. The research conducted in this regard tries to discover and develop non-toxic active chemical compounds able to protect the affected individuals from various types of post-radiation damage. The present work evaluates a group of 1-(2-hydroxyethyl)piperazine derivatives as potential and affordable radioprotective agents. Material and methods: Novel compounds were synthesized based on 1-(2-hydroxyethyl)piperazine derivatives and characterized using nuclear magnetic resonance and high-resolution mass spectrometry, followed by cytotoxicity and radioprotective evaluation in vitro (i.e. cell lines) and in vivo (i.e. an animal experimental model). Results: In the present study, 19 compounds were tested, with compounds 3e and 8 showing a radioprotective effect that, although comparable with the other molecules tested, displayed a greater tolerance at higher concentrations in vitro. The derivate 3e prolonged mouse survival. There was also a significant lymphocyte count increment on day 30, erythrocytes on day 7, and platelets on day 12 after irradiation. On the other hand, compound 8 resulted in greater radioprotective and survival values in the tested mice after 30 days of irradiation. It must be noted that this compound is structurally different from the other derivatives, as it contains no aromatic moiety, has low toxicity, and it is highly soluble. Conclusion: The overall design of this study included in silico study, in-house synthesis, physicochemical analysis, in vitro/vivo toxicity testing, and radioprotective effect evaluation. The most promising compounds were selected based on their toxicological and physicochemical profile, which suggested an interesting structural activity-relationship in the improved radioprotective effect of the tested compounds. Taken together, the present study reports the synthesis and biological characterization of a novel series of (4-alkylpiperazin-1-yl)-3-phenoxypropan-2-ol compound derivatives as promising radioprotective agents.

16:15-16:45Coffee Break
16:45-17:45 Session YIA ERRS: Young Investigator Award ERRS
Microbeam radiation therapy induces a sparing effect of normal tissue cells through increased expression of ROS scavengers
PRESENTER: Sandra Bicher

ABSTRACT. Microbeam radiation therapy (MRT) employs spatially fractionated radiation with 25 - 100 µm peak and 400 - 800 µm valley regions. The dose in peak regions reaches up to several hundred Gy, whereas the dose in valley regions stays below the tissue tolerance dose. One hypothesis of an increased therapeutic window of MRT is the influence of intercellular communication.

The influence of intercellular communication after BB and MRT was analyzed by clonogenic cell survival, DNA damage repair, ROS production, and ROS gene expression were evaluated in tumor cell lines A549 and LN18 and normal tissue cell line MRC5.

MRT showed a reduced cell survival in A549 (p< 0.01) and LN18 (p< 0.05) and a higher cell survival in MRC5 (p< 0.01) compared to BB. In all three cell lines a significant increase in DNA double strand breaks was observed 30 min after MRT compared to BB (A549 p < 0.0001, LN18 p< 0.0001, MRC5 p< 0.001). However, 24 h after MRT the significantly higher damage remained in A549 and LN18 (p< 0.0001), whereas no significant difference was detected in MRC5 cells. The Amplex Red assay for measuring ROS production showed a significantly higher H2O2 production in LN18 and MRC5 cell lines 24 h after MRT compared to BB (LN18 p< 0.05, MRC5 p< 0.001). On RNA level, a significant increase in ROS scavengers SOD2 (p< 0.05) and PER3 (p< 0.01) was detected after MRT compared to BB. However, in the tumor cell line A549, PER3 was significantly upregulated after BB compared to MRT (p< 0.05), but no change was detected in LN18.

Our results demonstrate a normal tissue sparing after MRT in vitro. MRT caused less DNA double strand breaks and led to an increased survival of normal tissue cells and a reduced survival of tumor cells with a possible relation to an increased expression of ROS scavengers compared to BB. This diverse effect widens the therapeutic window of MRT, making it a promising novel radiotherapy approach.

Single cell profiling identifies a differential transcriptomic response to photon versus proton irradiation in salivary gland organoids
PRESENTER: Davide Cinat

ABSTRACT. An increasing number of head and neck cancer patients is being treated with proton therapy. Besides its physical advantages over conventional photon-based radiotherapy, little is known about the biological response to protons of healthy salivary gland tissue. To investigate and compare the potential differences of photon and proton irradiation, we used a salivary gland organoid model and performed ATAC and single-cell RNA sequencing to identify transcriptomic changes at the single cell level. Our analysis showed the presence of a population of epithelial stem cells (EpSC) enriched in gene expression related to tissue and organ development. Interestingly, photon and proton irradiation (IR) led to a similar reduction of the EpSC population 2 days after IR. However, proton IR samples maintained a higher self-renewal capacity compared to photons, measured as secondary organoid forming efficiency (OFE). In line with this observation, we found that several transcription factors, highly upregulated in the EpSC population of non-irradiated organoids, were significantly downregulated upon photon IR but not protons. Furthermore, while photon IR induced a higher expression of genes related to stress and cell death, proton IR led to an enrichment of cell cycle related genes, such as CDKN1A, CDKN2B and JUNB, and tissue development related genes such as SOX9 and EPCAM. Lastly, in both EpSC and salivary duct cells, we observed a deregulation of genes related to mitochondrial function upon photons compared to protons. This correlated with a slower recovery of the mitochondrial membrane potential detected by flow cytometry at later timepoints. Mitochondria play a crucial role in the maintenance of stem cell self-renewal and fate decision, and their dysfunction in the long term may affect salivary gland EpSC function. Our study suggests that photons lead to a higher EpSC functional decline compared to protons that provide new possibilities for future therapy improvements.

Ionizing radiation effect on the extracellular vesicle cargo
PRESENTER: Ilona Csordás

ABSTRACT. Background: Although ionizing radiation (IR) induced bystander effect (RIBE) is long-identified, the exact mechanisms behind the phenomenon have not been fully explored. Extracellular vesicles (EV) are able to induce RIBE, possibly due to their IR-altered cargo. miRNAs as key gene expression regulators carried by EVs, participate in the development of RIBE, but their incorporation methods into vesicles are not yet fully understood. In this study we examined and compared alterations in miRNA profile, and proteins packaging miRNAs into EVs after IR, in bone marrow cells (BMC) and bone marrow-derived EVs (BMC-EVs). Materials and methods: Male CBA mice were total body irradiated with 0.1 and 3Gy and BMCs and BMC-EVs were isolated after 24h. 14 miRNAs were analyzed by qPCR. The concentration of miRNA packaging hnRNPA2B1 and hnRNPQ proteins were analyzed by Western Blot (WB), their cellular localization was investigated by confocal microscopy. Bioinformatic tools were used to calculate the effects of altered miRNA expression on signaling pathways, and to link RNA binding proteins to miRNAs. Results: In BMCs the expression of 6 out of 14 miRNAs, while in BMC-EVs 9 out of 14 miRNAs was significantly altered by IR. Changes of miRNA profiles in EVs did not follow the pattern observed in BMC cells, indicating the existence of selective miRNA sorting. Pathway enrichment analysis revealed that differentially expressed miRNAs in EVs were involved in several cellular pathways, such as radiation-response related signaling pathways, support the role of EVs in RIBE. hnRNP protein levels were also modified both in BMC and EV after IR: A2B1 was increased in BMC while decreased in EVs, Q was not altered in BMC, but was elevated in EVs. Changes in the level of particular miRNAs carrying recognition motifs for hnRNP proteins followed the quantitative changes of their respective binding partner protein in EVs. For example miR-93, the most significantly decreased miRNA in EV, binds to hnRNPA2B1, which was also decreased in EVs. Conclusion: Different miRNA profiles in BMC and BMC-EVs, along with a strong correlation between the level of hnRNP proteins and the concentration of their binding miRNAs in EV, support the existence of selective miRNA packaging mechanisms. Our results also indicate, that IR affects mechanism of miRNA sorting and incorporation into EVs. Funding: The Euratom research and training programme 2014-18 (CONCERT 662287), H2020 (EURAMED rocc-n-roll, 899995)

Development of a IORT Treatment Planning System using a GPU-based fast Monte Carlo
PRESENTER: Gaia Franciosini

ABSTRACT. Intra Operative Radiation Therapy (IORT) is a technique that, after the surgical tumor removal, delivers a dose of ionizing radiation (4-12 MeV electrons beam) directly to the surgery bed. During IORT treatments the beam is passively collimated by means of a PMMA hollow tube and whenever needed, temporarily beam modifiers are used to protect the healthy tissues surrounding the target. The use of high intensity pulses of electrons makes IORT the current best candidate for the first implementation of the FLASH effect into clinic. An important IORT limitation is the lack of a Treatment Planning System (TPS) capable of coping with the very limited amount of time available after the surgery (~ 1 min) to obtain both the new imaging of the surgical field, which has undergone substantial morphological modification, and the TPS computation. In this contribution, exploiting the new 3D real-time echographic imaging acquisition provided by the SIT company1, we investigate the efficiency achievable in IORT and IORT-FLASH breast cancer treatment using a GPU-based (Graphics Processing Unit) fast Monte Carlo (MC) called FRED (Fast particle thErapy Dose evaluator)2,3 for treatment planning. The FRED MC has been developed to allow a fast optimization of the TPS while keeping the dose release accuracy typical of a MC tool. Using FRED we have simulated in detail the LIAC HWL mobile IORT accelerator produced by SIT, and we have developed an optimization tool that, starting from the CT imaging, explores different treatment configurations. We have then combined the FRED simulation with a simple modeling of the FLASH effect. The tumour coverage and the dose absorbed by the organs at risk have been compared, carrying out a quantitative analysis adopting Dose Volume Histograms. The results demonstrate the potential of FRED as a tool for treatment planning and of the FLASH effect in IORT treatments.

A systematic review of radiation biomarkers for the advancement of human space exploration.
PRESENTER: Nicholas Rajan

ABSTRACT. Radiation biomarkers are an emerging and rapidly developing area of research, with potential applications in predicting individual radiation susceptibility, predicting severity of normal tissue injury among patients, assessing and monitoring of tumor response to radiation therapy as well as in estimating dose to accidentally radiation-exposed individuals. In this study we utilize the knowledge gathered from various fields about radiation response-related biomolecules to identify biomarkers for space radiation in view of long-term human space exploration. This comprehensive search for radiation biomarkers was divided into ‘biomarkers of radiation exposure’ and ‘biomarkers of radiation susceptibility/sensitivity’ based on time parameters and complexity of the topic (i.e. molecular influencers post-radiation exposure and molecules controlling susceptibility/ sensitivity prior and post-radiation). The strategy for the current study is adapted from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and 2020 statement (1) and a guide for conducting a systematic review and meta-analysis (2). The search plan not only considers publications (study articles, literature reviews, systematic reviews, and meta-analyses) but also takes databases, repositories, and registers containing relevant information into account. Biomolecules such as genes, transcripts (coding and noncoding), proteins, as well as epigenetic and possible epitranscriptomic modifications related to radiation response events are all taken into consideration. This research presents a state-of-the art report on scientific evidence on biomarkers of response to ionizing radiation. The compiled list of recommended biomarkers could undoubtedly improve crew health risk assessment in order to better forecast the health implications of ionizing radiation exposure in astronauts and as a potential tool for biodosimetry.

Abstract acknowledgements: SCK CEN - European Space Agency (ESA) - BIOMOON project: ESA RFP/3-17004

References: 1. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: An updated guideline for reporting systematic reviews. BMJ. 2021;372. 2. Tawfik GM, Dila KAS, Mohamed MYF, Tam DNH, Kien ND, Ahmed AM, et al. A step by step guide for conducting a systematic review and meta-analysis with simulation data. Trop Med Health. 2019;47(1):1–9.


ABSTRACT. Approximately 50 million people globally suffer from Alzheimer’s disease (AD) and no effective cure exists for this neurodegenerative disorder up to now. The NEutron Capture-enhanced Treatment of neurotoxic Amyloid aggRegates project (NECTAR) aims to develop, test, and prove the feasibility, safety, and effectiveness of a novel and revolutionary approach based on Capture-Enhanced Neutron Irradiation (CENI) for damaging and subsequent disintegration of amyloid-beta (Aβ) aggregates. The boron neutron capture technology (BNCT) radiation field consists of a mixture of qualities and, thus, the radiobiological effect of BNCT treatment consists of components with different linear energy transfer (LET) characteristics. In this study, we are Modelling the mixed beam of the BNCT reaction on human microglial HMC3 cells using different proportions of alfa particles (high LET) and X-rays (low LET) to evaluate and quantify possible neurotoxic and inflammatory effects induced by this treatment. HMC3 cells were irradiated with different doses of α- (0.5-2.0 Gy), X-ray-radiation (0.5-2.0 Gy) alone as well as mixed radiation (0.5 α + 0.5 X; 1.0 α + 1.0 X; 1.5 α + 0.5 X; 0.5 α + 1.5 X Gy). We characterized the formation of DNA double-strand breaks in cells exposed to various irradiation modalities by quantifying γ-H2AX foci. Corresponding experiments exhibited a significant decrease in cell viability and survival of HMC3 cells after exposure to more than 1.0 Gy of α and X-ray alone or mixed beam. Gene expression of two cytokines, IL-6 and IL-1β, was measured by qRT-PCR at 3 and 6 hrs after irradiation. We observed an increase in gene expression of IL-1β in cells irradiated with α-particles and mixed irradiation 6 hrs post-irradiation; no significant changes in cells irradiated with X-rays alone were observed. Studies will be extended to neuronal cells as well.

This project has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No. 964934.