4Din vivodosimetry for a FLASH electron beam using radiation-induced acoustic imaging.

Objective. The primary goal of this research is to demonstrate the feasibility of radiation-induced acoustic imaging (RAI) as a volumetric dosimetry tool for ultra-high dose rate FLASH electron radiotherapy (FLASH-RT) in real time. This technology aims to improve patient outcomes by accurate measurements ofin vivodose delivery to target tumor volumes.Approach. The study utilized the FLASH-capable eRT6 LINAC to deliver electron beams under various doses (1.2 Gy pulse-1to 4.95 Gy pulse-1) and instantaneous dose rates (1.55 × 105Gy s-1to 2.75 × 106Gy s-1), for imaging the beam in water and in a rabbit cadaver with RAI. A custom 256-element matrix ultrasound array was employed for real-time, volumetric (4D) imaging of individual pulses. This allowed for the exploration of dose linearity by varying the dose per pulse and analyzing the results through signal processing and image reconstruction in RAI.Main Results. By varying the dose per pulse through changes in source-to-surface distance, a direct correlation was established between the peak-to-peak amplitudes of pressure waves captured by the RAI system and the radiochromic film dose measurements. This correlation demonstrated dose rate linearity, including in the FLASH regime, without any saturation even at an instantaneous dose rate up to 2.75 × 106Gy s-1. Further, the use of the 2D matrix array enabled 4D tracking of FLASH electron beam dose distributions on animal tissue for the first time.Significance. This research successfully shows that 4Din vivodosimetry is feasible during FLASH-RT using a RAI system. It allows for precise spatial (∼mm) and temporal (25 frames s-1) monitoring of individual FLASH beamlets during delivery. This advancement is crucial for the clinical translation of FLASH-RT as enhancing the accuracy of dose delivery to the target volume the safety and efficacy of radiotherapeutic procedures will be improved.

Multi-Institutional Audit of FLASH and Conventional Dosimetry With a 3D Printed Anatomically Realistic Mouse Phantom.

PURPOSE: We conducted a multi-institutional dosimetric audit between FLASH and conventional dose rate (CONV) electron irradiations by using an anatomically realistic 3-dimensional (3D) printed mouse phantom. METHODS AND MATERIALS: A computed tomography (CT) scan of a live mouse was used to create a 3D model of bony anatomy, lungs, and soft tissue. A dual-nozzle 3D printer was used to print the mouse phantom using acrylonitrile butadiene styrene (∼1.02 g/cm3) and polylactic acid (∼1.24 g/cm3) simultaneously to simulate soft tissue and bone densities, respectively. The lungs were printed separately using lightweight polylactic acid (∼0.64 g/cm3). Hounsfield units (HU), densities, and print-to-print stability of the phantoms were assessed. Three institutions were each provided a phantom and each institution performed 2 replicates of irradiations at selected anatomic regions. The average dose difference between FLASH and CONV dose distributions and deviation from the prescribed dose were measured with radiochromic film. RESULTS: Compared with the reference CT scan, CT scans of the phantom demonstrated mass density differences of 0.10 g/cm3 for bone, 0.12 g/cm3 for lung, and 0.03 g/cm3 for soft tissue regions. Differences in HU between phantoms were <10 HU for soft tissue and bone, with lung showing the most variation (54 HU), but with minimal effect on dose distribution (<0.5%). Mean differences between FLASH and CONV decreased from the first to the second replicate (4.3%-1.2%), and differences from the prescribed dose decreased for both CONV (3.6%-2.5%) and FLASH (6.4%-2.7%). Total dose accuracy suggests consistent pulse dose and pulse number, although these were not specifically assessed. Positioning variability was observed, likely due to the absence of robust positioning aids or image guidance. CONCLUSIONS: This study marks the first dosimetric audit for FLASH using a nonhomogeneous phantom, challenging conventional calibration practices reliant on homogeneous phantoms. The comparison protocol offers a framework for credentialing multi-institutional studies in FLASH preclinical research to enhance reproducibility of biologic findings.

Acute Hypoxia Does Not Alter Tumor Sensitivity to FLASH Radiation Therapy.

PURPOSE: Tumor hypoxia is a major cause of treatment resistance, especially to radiation therapy at conventional dose rate (CONV), and we wanted to assess whether hypoxia does alter tumor sensitivity to FLASH. METHODS AND MATERIALS: We engrafted several tumor types (glioblastoma [GBM], head and neck cancer, and lung adenocarcinoma) subcutaneously in mice to provide a reliable and rigorous way to modulate oxygen supply via vascular clamping or carbogen breathing. We irradiated tumors using a single 20-Gy fraction at either CONV or FLASH, measured oxygen tension, monitored tumor growth, and sampled tumors for bulk RNAseq and pimonidazole analysis. Next, we inhibited glycolysis with trametinib in GBM tumors to enhance FLASH efficacy. RESULTS: Using various subcutaneous tumor models, and in contrast to CONV, FLASH retained antitumor efficacy under acute hypoxia. These findings show that in addition to normal tissue sparing, FLASH could overcome hypoxia-mediated tumor resistance. Follow-up molecular analysis using RNAseq profiling uncovered a FLASH-specific profile in human GBM that involved cell-cycle arrest, decreased ribosomal biogenesis, and a switch from oxidative phosphorylation to glycolysis. Glycolysis inhibition by trametinib enhanced FLASH efficacy in both normal and clamped conditions. CONCLUSIONS: These data provide new and specific insights showing the efficacy of FLASH in a radiation-resistant context, proving an additional benefit of FLASH over CONV.

Construction and dosimetric characterization of a motorized scanning-slit system for electron FLASH experiments.

BACKGROUND: Beam scanning is a useful technique for the treatment of large tumors when the primary beam size is limited, which is the case with radiation beams used in FLASH radiotherapy. PURPOSE: To optimize beam scanning as a dose delivery method for FLASH radiotherapy, it is necessary to first understand the effects of beam scanning on the FLASH effect. To do so, biological FLASH experiments need to be done using defined beam parameters with beam scanning and compared to the situation without beam scanning. In this regard, we propose implementation of a simple slit scanning system with an electron FLASH beam to obtain a scanned radiation field that closely resembles a static field. METHODS: A pulsed electron linear accelerator (linac) was used in combination with a scanning slit system in order to simulate a scanned electron beam. Three configurations that produced homogeneous lateral profiles and high enough doses per pulse for FLASH experiments were established. The optimal scanning parameters were found for each configuration by examining the flatness of the obtained lateral dose profiles. Using the optimal scanning parameters, the scanned FLASH beams were dosimetrically characterized and compared to non-scanned open field beam. RESULTS: A final electron FLASH beam scanning configuration was found for a 1 mm wide slit at a distance of 350 mm from the linac and a 2 mm wide slit at distances of 350 and 490 mm from the linac. The lateral profiles for these final configurations were found to have a homogeneity that is comparable to the open field profiles. The percentage depth dose (PDD) values found for these final configurations closely matched (by a few percentage) the PDD of the open field beam. CONCLUSIONS: Three electron FLASH beam scanning configurations achieved by the motorized slit system were found to produce radiation fields similar to a non-scanned open field electron beam. These final configurations can therefore be used in future biological FLASH experiments to compare to non-scanned beam experiments in order to optimize beam scanning as a technique permitting the treatment of larger tumors with FLASH radiotherapy.

Antitumor Effect by Either FLASH or Conventional Dose Rate Irradiation Involves Equivalent Immune Responses.

PURPOSE: The capability of ultrahigh dose rate FLASH radiation therapy to generate the FLASH effect has opened the possibility to enhance the therapeutic index of radiation therapy. The contribution of the immune response has frequently been hypothesized to account for a certain fraction of the antitumor efficacy and tumor kill of FLASH but has yet to be rigorously evaluated. METHODS AND MATERIALS: To investigate the immune response as a potentially important mechanism of the antitumor effect of FLASH, various murine tumor models were grafted either subcutaneously or orthotopically into immunocompetent mice or in moderately and severely immunocompromised mice. Mice were locally irradiated with single dose (20 Gy) or hypofractionated regimens (3 × 8 or 2 × 6 Gy) using FLASH (≥2000 Gy/s) and conventional (CONV) dose rates (0.1 Gy/s), with/without anti-CTLA-4. Tumor growth was monitored over time and immune profiling performed. RESULTS: FLASH and CONV 20 Gy were isoeffective in delaying tumor growth in immunocompetent and moderately immunodeficient hosts and increased tumor doubling time to >14 days versus >7 days in control animals. Similar observations were obtained with a hypofractionated scheme, regardless of the microenvironment (subcutaneous flank vs ortho lungs). Interestingly, in profoundly immunocompromised mice, 20 Gy FLASH retained antitumor activity and significantly increased tumor doubling time to >14 days versus >8 days in control animals, suggesting a possible antitumor mechanism independent of the immune response. Analysis of the tumor microenvironment showed similar immune profiles after both irradiation modalities with significant decrease of lymphoid cells by ∼40% and a corresponding increase of myeloid cells. In addition, FLASH and CONV did not increase transforming growth factor-ß1 levels in tumors compared with unirradiated control animals. Furthermore, when a complete and long-lasting antitumor response was obtained (>140 days), both modalities of irradiation were able to generate a long-term immunologic memory response. CONCLUSIONS: The present results clearly document that the tumor responses across multiple immunocompetent and immunodeficient mouse models are largely dose rate independent and simultaneously contradict a major role of the immune response in the antitumor efficacy of FLASH. Therefore, our study indicates that FLASH is as potent as CONV in modulating antitumor immune response and can be used as an immunomodulatory agent.

Multi-Institutional Audit of FLASH and Conventional Dosimetry with a 3D-Printed Anatomically Realistic Mouse Phantom.

We conducted a multi-institutional audit of dosimetric variability between FLASH and conventional dose rate (CONV) electron irradiations by using an anatomically realistic 3D-printed mouse phantom. A CT scan of a live mouse was used to create a 3D model of bony anatomy, lungs, and soft tissue. A dual-nozzle 3D printer was used to print the mouse phantom using acrylonitrile butadiene styrene ($~1.02 g/cm^3$) and polylactic acid ($~1.24 g/cm^3$) simultaneously to simulate soft tissue and bone densities, respectively. The lungs were printed separately using lightweight polylactic acid ($~0.64 g/cm^3$). Hounsfield units (HU) and densities were compared with the reference CT scan of the live mouse. Print-to-print reproducibility of the phantom was assessed. Three institutions were each provided a phantom, and each institution performed two replicates of irradiations at selected mouse anatomic regions. The average dose difference between FLASH and CONV dose distributions and deviation from the prescribed dose were measured with radiochromic film. Compared to the reference CT scan, CT scans of the phantom demonstrated mass density differences of $0.10 g/cm^3$ for bone, $0.12 g/cm^3$ for lung, and $0.03 g/cm^3$ for soft tissue regions. Between phantoms, the difference in HU for soft tissue and bone was <10 HU from print to print. Lung exhibited the most variation (54 HU) but minimally affected dose distribution (<0.5% dose differences between phantoms). The mean difference between FLASH and CONV from the first replicate to the second decreased from 4.3% to 1.2%, and the mean difference from the prescribed dose decreased from 3.6% to 2.5% for CONV and 6.4% to 2.7% for FLASH. The framework presented here is promising for credentialing of multi-institutional studies of FLASH preclinical research to maximize the reproducibility of biological findings.

The sparing effect of FLASH-RT on synaptic plasticity is maintained in mice with standard fractionation.

Long-term potentiation (LTP) was used to gauge the impact of conventional and FLASH dose rates on synaptic transmission. Data collected from the hippocampus and medial prefrontal cortex confirmed significant inhibition of LTP after 10 fractions of 3 Gy (30 Gy total) conventional radiotherapy. Remarkably, 10x3Gy FLASH radiotherapy and unirradiated controls were identical and exhibited normal LTP.

Proton and alpha radiation-induced mutational profiles in human cells.

Ionizing radiation is known to be DNA damaging and mutagenic, however less is known about which mutational footprints result from exposures of human cells to different types of radiation. We were interested in the mutagenic effects of particle radiation exposures on genomes of various human cell types, in order to gauge the genotoxic risks of galactic cosmic radiation, and of certain types of tumor radiotherapy. To this end, we exposed cultured cell lines from the human blood, breast and lung to fractionated proton and alpha particle (helium nuclei) beams at doses sufficient to considerably affect cell viability. Whole-genome sequencing revealed that mutation rates were not overall markedly increased upon proton and alpha exposures. However, there were modest changes in mutation spectra and distributions, such as the increases in clustered mutations and of certain types of indels and structural variants. The spectrum of mutagenic effects of particle beams may be cell-type and/or genetic background specific. Overall, the mutational effects of repeated exposures to proton and alpha radiation on human cells in culture appear subtle, however further work is warranted to understand effects of long-term exposures on various human tissues.

Modeling of scavenging systems in water radiolysis with Geant4-DNA.

PURPOSE: This paper presents the capabilities of the Geant4-DNA Monte Carlo toolkit to simulate water radiolysis with scavengers using the step-by-step (SBS) or the independent reaction times (IRT) methods. It features two examples of application areas: (1) computing the escape yield of H2O2 following a 60Co γ-irradiation and (2) computing the oxygen depletion in water irradiated with 1 MeV electrons. METHODS: To ease the implementation of the chemical stage in Geant4-DNA, we developed a user interface that helps define the chemical reactions and set the concentration of scavengers. The first application area example required two computational steps to perform water radiolysis using NO2- and NO3- as scavengers and a 60Co irradiation. The oxygen depletion computation technique for the second application area example consisted of simulating track segments of 1 MeV electrons and determining the radio-induced loss and gain of oxygen molecules. RESULTS: The production of H2O2 under variable scavenging levels is consistent with the literature; the mean relative difference between the SBS and IRT methods is 7.2 % ± 0.5 %. For the oxygen depletion 1 µs post-irradiation, the mean relative difference between both methods is equal to 9.8 % ± 0.3 %. The results in the microsecond scale depend on the initial partial pressure of oxygen in water. In addition, the computed oxygen depletions agree well with the literature. CONCLUSIONS: The Geant4-DNA toolkit makes it possible to simulate water radiolysis in the presence of scavengers. This feature offers perspectives in radiobiology, with the possibility of simulating cell-relevant scavenging mechanisms.

Elucidating the neurological mechanism of the FLASH effect in juvenile mice exposed to hypofractionated radiotherapy.

BACKGROUND: Ultrahigh dose-rate radiotherapy (FLASH-RT) affords improvements in the therapeutic index by minimizing normal tissue toxicities without compromising antitumor efficacy compared to conventional dose-rate radiotherapy (CONV-RT). To investigate the translational potential of FLASH-RT to a human pediatric medulloblastoma brain tumor, we used a radiosensitive juvenile mouse model to assess adverse long-term neurological outcomes. METHODS: Cohorts of 3-week-old male and female C57Bl/6 mice exposed to hypofractionated (2 × 10 Gy, FLASH-RT or CONV-RT) whole brain irradiation and unirradiated controls underwent behavioral testing to ascertain cognitive status four months posttreatment. Animals were sacrificed 6 months post-irradiation and tissues were analyzed for neurological and cerebrovascular decrements. RESULTS: The neurological impact of FLASH-RT was analyzed over a 6-month follow-up. FLASH-RT ameliorated neurocognitive decrements induced by CONV-RT and preserved synaptic plasticity and integrity at the electrophysiological (long-term potentiation), molecular (synaptophysin), and structural (Bassoon/Homer-1 bouton) levels in multiple brain regions. The benefits of FLASH-RT were also linked to reduced neuroinflammation (activated microglia) and the preservation of the cerebrovascular structure, by maintaining aquaporin-4 levels and minimizing microglia colocalized to vessels. CONCLUSIONS: Hypofractionated FLASH-RT affords significant and long-term normal tissue protection in the radiosensitive juvenile mouse brain when compared to CONV-RT. The capability of FLASH-RT to preserve critical cognitive outcomes and electrophysiological properties over 6-months is noteworthy and highlights its potential for resolving long-standing complications faced by pediatric brain tumor survivors. While care must be exercised before clinical translation is realized, present findings document the marked benefits of FLASH-RT that extend from synapse to cognition and the microvasculature.

Design and validation of a dosimetric comparison scheme tailored for ultra-high dose-rate electron beams to support multicenter FLASH preclinical studies.

BACKGROUND AND PURPOSE: We describe a multicenter cross validation of ultra-high dose rate (UHDR) (>= 40 Gy/s) irradiation in order to bring a dosimetric consensus in absorbed dose to water. UHDR refers to dose rates over 100-1000 times those of conventional clinical beams. UHDR irradiations have been a topic of intense investigation as they have been reported to induce the FLASH effect in which normal tissues exhibit reduced toxicity relative to conventional dose rates. The need to establish optimal beam parameters capable of achieving the in vivo FLASH effect has become paramount. It is therefore necessary to validate and replicate dosimetry across multiple sites conducting UHDR studies with distinct beam configurations and experimental set-ups. MATERIALS AND METHODS: Using a custom cuboid phantom with a cylindrical cavity (5 mm diameter by 10.4 mm length) designed to contain three type of dosimeters (thermoluminescent dosimeters (TLDs), alanine pellets, and Gafchromic films), irradiations were conducted at expected doses of 7.5 to 16 Gy delivered at UHDR or conventional dose rates using various electron beams at the Radiation Oncology Departments of the CHUV in Lausanne, Switzerland and Stanford University, CA. RESULTS: Data obtained between replicate experiments for all dosimeters were in excellent agreement (±3%). In general, films and TLDs were in closer agreement with each other, while alanine provided the closest match between the expected and measured dose, with certain caveats related to absolute reference dose. CONCLUSION: In conclusion, successful cross-validation of different electron beams operating under different energies and configurations lays the foundation for establishing dosimetric consensus for UHDR irradiation studies, and, if widely implemented, decrease uncertainty between different sites investigating the mechanistic basis of the FLASH effect.

Comparing radiolytic production of H2O2 and development of Zebrafish embryos after ultra high dose rate exposure with electron and transmission proton beams.

The physico-chemical and biological response to conventional and UHDR electron and proton beams was investigated, along with conventional photons. The temporal structure and nature of the beam affected both, with electron beam at ≥1400 Gy/s and proton beam at 0.1 and 1260 Gy/s found to be isoefficient at sparing zebrafish embryos.

Ultra-high dose rate radiation production and delivery systems intended for FLASH.

Higher dose rates, a trend for radiotherapy machines, can be beneficial in shortening treatment times for radiosurgery and mitigating the effects of motion. Recently, even higher doses (e.g., 100 times greater) have become targeted because of their potential to generate the FLASH effect (FE). We refer to these physical dose rates as ultra-high (UHDR). The complete relationship between UHDR and the FE is unknown. But UHDR systems are needed to explore the relationship further and to deliver clinical UHDR treatments, where indicated. Despite the challenging set of unknowns, the authors seek to make reasonable assumptions to probe how existing and developing technology can address the UHDR conditions needed to provide beam generation capable of producing the FE in preclinical and clinical applications. As a preface, this paper discusses the known and unknown relationships between UHDR and the FE. Based on these, different accelerator and ionizing radiation types are then discussed regarding the relevant UHDR needs. The details of UHDR beam production are discussed for existing and potential future systems such as linacs, cyclotrons, synchrotrons, synchrocyclotrons, and laser accelerators. In addition, various UHDR delivery mechanisms are discussed, along with required developments in beam diagnostics and dose control systems.

Dose- and Volume-Limiting Late Toxicity of FLASH Radiotherapy in Cats with Squamous Cell Carcinoma of the Nasal Planum and in Mini Pigs.

PURPOSE: The FLASH effect is characterized by normal tissue sparing without compromising tumor control. Although demonstrated in various preclinical models, safe translation of FLASH-radiotherapy stands to benefit from larger vertebrate animal models. Based on prior results, we designed a randomized phase III trial to investigate the FLASH effect in cat patients with spontaneous tumors. In parallel, the sparing capacity of FLASH-radiotherapy was studied on mini pigs by using large field irradiation. EXPERIMENTAL DESIGN: Cats with T1-T2, N0 carcinomas of the nasal planum were randomly assigned to two arms of electron irradiation: arm 1 was the standard of care (SoC) and used 10 × 4.8 Gy (90% isodose); arm 2 used 1 × 30 Gy (90% isodose) FLASH. Mini pigs were irradiated using applicators of increasing size and a single surface dose of 31 Gy FLASH. RESULTS: In cats, acute side effects were mild and similar in both arms. The trial was prematurely interrupted due to maxillary bone necrosis, which occurred 9 to 15 months after radiotherapy in 3 of 7 cats treated with FLASH-radiotherapy (43%), as compared with 0 of 9 cats treated with SoC. All cats were tumor-free at 1 year in both arms, with one cat progressing later in each arm. In pigs, no acute toxicity was recorded, but severe late skin necrosis occurred in a volume-dependent manner (7-9 months), which later resolved. CONCLUSIONS: The reported outcomes point to the caveats of translating single-high-dose FLASH-radiotherapy and emphasizes the need for caution and further investigations. See related commentary by Maity and Koumenis, p. 3636.

Technical note: Validation of an ultrahigh dose rate pulsed electron beam monitoring system using a current transformer for FLASH preclinical studies.

PURPOSE: The Oriatron eRT6 is a linear accelerator (linac) used in FLASH preclinical studies able to reach dose rates ranging from conventional (CONV) up to ultrahigh (UHDR). This work describes the implementation of commercially available beam current transformers (BCTs) as online monitoring tools compatible with CONV and UHDR irradiations for preclinical FLASH studies. METHODS: Two BCTs were used to measure the output of the Oriatron eRT6 linac. First, the correspondence between the set nominal beam parameters and those measured by the BCTs was checked. Then, we established the relationship between the total exit charge (measured by BCTs) and the absorbed dose to water. The influence of the pulse width (PW) and the pulse repetition frequency (PRF) at UHDR was characterized, as well as the short- and long-term stabilities of the relationship between the exit charge and the dose at CONV and UHDR. RESULTS: The BCTs were able to determine consistently the number of pulses, PW, and PRF. For fixed PW and pulse height, the exit charge measured from BCTs was correlated with the dose, and linear relationships were found with uncertainties of 0.5 % and 3 % in CONV and UHDR mode, respectively. Short- and long-term stabilities of the dose-to-charge ratio were below 1.6 %. CONCLUSIONS: We implemented commercially available BCTs and demonstrated their ability to act as online beam monitoring systems to support FLASH preclinical studies with CONV and UHDR irradiations. The implemented BCTs support dosimetric measurements, highlight variations among multiple measurements in a row, enable monitoring of the physics parameters used for irradiation, and are an important step for the safety of the clinical translation of FLASH radiation therapy.

Implementation and validation of a beam-current transformer on a medical pulsed electron beam LINAC for FLASH-RT beam monitoring.

PURPOSE: To implement and validate a beam current transformer as a passive monitoring device on a pulsed electron beam medical linear accelerator (LINAC) for ultra-high dose rate (UHDR) irradiations in the operational range of at least 3 Gy to improve dosimetric procedures currently in use for FLASH radiotherapy (FLASH-RT) studies. METHODS: Two beam current transformers (BCTs) were placed at the exit of a medical LINAC capable of UHDR irradiations. The BCTs were validated as monitoring devices by verifying beam parameters consistency between nominal values and measured values, determining the relationship between the charge measured and the absorbed dose, and checking the short- and long-term stability of the charge-absorbed dose ratio. RESULTS: The beam parameters measured by the BCTs coincide with the nominal values. The charge-dose relationship was found to be linear and independent of pulse width and frequency. Short- and long-term stabilities were measured to be within acceptable limits. CONCLUSIONS: The BCTs were implemented and validated on a pulsed electron beam medical LINAC, thus improving current dosimetric procedures and allowing for a more complete analysis of beam characteristics. BCTs were shown to be a valid method for beam monitoring for UHDR (and therefore FLASH) experiments.

Commissioning of an ultra-high dose rate pulsed electron beam medical LINAC for FLASH RT preclinical animal experiments and future clinical human protocols.

PURPOSE: To present the acceptance and the commissioning, to define the reference dose, and to prepare the reference data for a quality assessment (QA) program of an ultra-high dose rate (UHDR) electron device in order to validate it for preclinical animal FLASH radiotherapy (FLASH RT) experiments and for FLASH RT clinical human protocols. METHODS: The Mobetron® device was evaluated with electron beams of 9 MeV in conventional (CONV) mode and of 6 and 9 MeV in UHDR mode (nominal energy). The acceptance was performed according to the acceptance protocol of the company. The commissioning consisted of determining the short- and long-term stability of the device, the measurement of percent depth dose curves (PDDs) and profiles at two different positions (with two different dose per pulse regimen) and for different collimator sizes, and the evaluation of the variability of these parameters when changing the pulse width and pulse repetition frequency. Measurements were performed using a redundant and validated dosimetric strategy with alanine and radiochromic films, as well as Advanced Markus ionization chamber for some measurements. RESULTS: The acceptance tests were all within the tolerances of the company's acceptance protocol. The linearity with pulse width was within 1.5% in all cases. The pulse repetition frequency did not affect the delivered dose more than 2% in all cases but 90 Hz, for which the larger difference was 3.8%. The reference dosimetry showed a good agreement within the alanine and films with variations of 2.2% or less. The short-term (resp. long-term) stability was less than 1.0% (resp. 1.8%) and was the same in both CONV and UHDR modes. PDDs, profiles, and reference dosimetry were measured at two positions, providing data for two specific dose rates (about 9 Gy/pulse and 3 Gy/pulse). Maximal beam size was 4 and 6 cm at 90% isodose in the two positions tested. There was no difference between CONV and UHDR mode in the beam characteristics tested. CONCLUSIONS: The device is commissioned for FLASH RT preclinical biological experiments as well as FLASH RT clinical human protocols.

Small Molecule Responses to Sequential Irradiation with Neutrons and Photons for Biodosimetry Applications: An Initial Assessment.

Mass casualty exposure scenarios from an improvised nuclear device are expected to be far more complex than simple photons. Based on the proximity to the explosion and potential shielding, a mixed field of neutrons and photons comprised of up to approximately 30% neutrons of the total dose is anticipated. This presents significant challenges for biodosimetry and for short-term and long-term medical treatment of exposed populations. In this study we employed untargeted metabolomic methods to develop a biosignature in urine and serum from C57BL/6 mice to address radiation quality issues. The signature was developed in males and applied to samples from female mice to identify potential sex differences. Thirteen urinary (primarily amino acids, vitamin products, nucleotides) and 18 serum biomarkers (primarily mitochondrial and fatty acid ß oxidation intermediates) were selected and evaluated in samples from day 1 and day 7 postirradiation. Sham-irradiated groups (controls) were compared to an equitoxic dose (3 Gy X-ray equivalent) from X rays (1.2 Gy/min), neutrons (∼1 Gy/h), or neutrons-photons. Results showed a time-dependent increase in the efficiency of the signatures, with serum providing the highest levels of accuracy in distinguishing not only between exposed from non-exposed populations, but also between radiation quality (photon exposures vs. exposures with a neutron component) and in between neutron-photon exposures (5, 15 or 25% of neutrons in the total dose) for evaluating the neutron contribution. A group of metabolites known as acylcarnitines was only responsive in males, indicating the potential for different mechanisms of action in baseline levels and of neutron-photon responses between the two sexes. Our findings highlight the potential of metabolomics in developing biodosimetric methods to evaluate mixed exposures with high sensitivity and specificity.

Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures.

The consideration of how a given technique affects results of experimental measurements is a must to achieve correct data interpretation. This might be challenging when it comes to measurements on biological systems, where it is unrealistic to have full control (e.g. through a software replica) of all steps in the measurement chain. In this work we address how the effectiveness of different radiation qualities in inducing biological damage can be assessed measuring DNA damage foci yields, only provided that artefacts related to the scoring technique are adequately considered. To this aim, we developed a unified stochastic modelling approach that, starting from radiation tracks, predicts both the induction, spatial distribution and complexity of DNA damage, and the experimental readout of foci when immunocytochemistry coupled to 2D fluorescence microscopy is used. The approach is used to interpret γ-H2AX data for photon and neutron exposures. When foci are reconstructed in the whole cell nucleus, we obtain information on damage characteristics "behind" experimental observations, as the average damage content of a focus. We reproduce how the detection technique affects experimental findings, e.g. contributing to the saturation of foci yields scored at 30 minutes after exposure with increasing dose and to the lack of dose dependence for yields at 24 hours.