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Purpose: The aim of this work was to investigate the ability of a biological oxygen enhancement ratio-weighted dose, D OER , to describe acute skin toxicity variations observed in mice after proton pencil beam scanning irradiations with changing doses and beam time structures., Methods and Materials: In five independent experiments, the right hind leg of a total of 621 CDF1 mice was irradiated previously in the entrance plateau of a pencil beam scanning proton beam. The incidence of acute skin toxicity (of level 1.5-2.0-2.5-3.0-3.5) was scored for 47 different mouse groups that mapped toxicity as function of dose for conventional and FLASH dose rate, toxicity as function of field dose rate with and without repainting, and toxicity when splitting the treatment into 1 to 6 identical deliveries separated by 2 minutes. D OER was calculated for all mouse groups using a simple oxygen kinetics model to describe oxygen depletion. The three independent model parameters (oxygen-depletion rate, oxygen-recovery rate, oxygen level without irradiation) were fitted to the experimental data. The ability of D OER to describe the toxicity variations across all experiments was investigated by comparing D OER -response curves across the five independent experiments., Results: After conversion from the independent variable tested in each experiment to D OER , all five experiments had similar MDD OER 50 (D OER giving 50% toxicity incidence) with standard deviations of 0.45 - 1.6 Gy for the five toxicity levels. D OER could thus describe the observed toxicity variations across all experiments., Conclusions: D OER described the varying FLASH-sparing effect observed for a wide range of conditions. Calculation of D OER for other irradiation conditions can quantitatively estimate the FLASH-sparing effect for arbitrary irradiations for the investigated murine model. With appropriate fitting parameters D OER also may be able to describe FLASH effect variations with dose and dose rate for other assays and endpoints., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Preclinical studies have shown a preferential normal tissue sparing effect of FLASH radiation therapy with ultra-high dose rates. The aim of the present study was to use a murine model of acute skin toxicity to investigate the biologic effect of varying dose rates, time structure, and introducing pauses in the dose delivery., Methods and Materials: The right hind limbs of nonanaesthetized mice were irradiated in the entrance plateau of a pencil beam scanning proton beam with 39.3 Gy. Experiment 1 was with varying field dose rates (0.7-80 Gy/s) without repainting, experiment 2 was with varying field dose rates (0.37-80 Gy/s) with repainting, and in experiment 3, the dose was split into 2, 3, 4, or 6 identical deliveries with 2-minute pauses. In total, 320 mice were included, with 6 to 25 mice per group. The endpoints were skin toxicity of different levels up to 25 days after irradiation., Results: The dose rate 50 , which is the dose rate to induce a response in 50% of the animals, depended on the level of skin toxicity, with the higher toxicity levels displaying a FLASH effect at 0.7-2 Gy/s. Repainting resulted in higher toxicity for the same field dose rate. Splitting the dose into 2 deliveries reduced the FLASH effect, and for 3 or more deliveries, the FLASH effect was almost abolished for lower grades of toxicity., Conclusions: The dose rate that induced a FLASH effect varied for different skin toxicity levels, which are characterized by a differing degree of sensitivity to radiation dosage. Conclusions on a threshold for the dose rate needed to obtain a FLASH effect can therefore be influenced by the dose sensitivity of the used endpoint. Splitting the total dose into more deliveries compromised the FLASH effect. This can have an impact for fractionation as well as for regions where 2 or more FLASH fields overlap within the same treatment session., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Oxygen dynamics may be important for the tissue-sparing effect observed at ultra-high dose rates (FLASH sparing effect). This study investigated the correlation between local instantaneous dose rate and radiation-induced oxygen pressure reduction during proton pencil beam scanning (PBS) irradiations of a sample and quantified the oxygen consumption g-value., Materials and Methods: A 0.2 ml phosphorescent sample (1 μM PtG4 Oxyphor probe in saline) was irradiated with a 244 MeV proton PBS beam. Four irradiations were performed with variations of a PBS spot pattern with 5 × 7 spots. During irradiation, the partial oxygen pressure (pO 2 ) was measured with 4.5 Hz temporal resolution with a phosphorometer (Oxyled) that optically excited the probe and recorded the subsequently emitted light. A calibration was performed to calculate the pO 2 level from the measured phosphorescence lifetime. A fiber-coupled scintillator simultaneously measured the instantaneous dose rate in the sample with 50 kHz sampling rate. The oxygen consumption g-value was determined on a spot-by-spot level and using the total pO 2 change for full spot pattern irradiation., Results: A high correlation was found between the local instantaneous dose rate and pO 2 reduction rate, with a correlation coefficient of 0.96-0.99. The g-vales were 0.18 ± 0.01 mmHg/Gy on a spot-by-spot level and 0.17 ± 0.01 mmHg/Gy for full spot pattern irradiation., Conclusions: The pO 2 reduction rate was directly related to the local instantaneous dose rate per delivered spot in PBS deliveries. The methodology presented here can be applied to irradiation at ultra-high dose rates with modifications in the experimental setup., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Objective: A favorable effect of ultra-high dose rate (FLASH) radiation on normal tissue-sparing has been indicated in several preclinical studies. In these studies, the adverse effects of radiation damage were reduced without compromising tumor control. Most studies of proton FLASH investigate these effects within the entrance of a proton beam. However, the real advantage of proton therapy lies in the Spread-out Bragg Peak (SOBP), which allows for giving a high dose to a target with a limited dose to healthy tissue at the entrance of the beam. Therefore, a clinically relevant investigation of the FLASH effect would be of healthy tissues within a SOBP. Our study quantified the tissue-sparing effect of FLASH radiation on acute and late toxicity within an SOBP in a murine model., Material/methods: Radiation-induced damage was assessed for acute and late toxicity in the same mice following irradiation with FLASH (Field dose rate of 60 Gy/s) or conventional (CONV, 0.34 Gy/s) dose rates. The right hindleg of unanesthetized female CDF1 mice was irradiated with single-fraction doses between 19.9-49.7 Gy for CONV and 30.4-65.9 Gy for FLASH with 5-8 mice per dose. The leg was placed in the middle of a 5 cm SOBP generated from a mono-energetic beam using a 2D range modulator. Acute skin toxicity quantified by hair loss, moist desquamation and toe separation was monitored daily within 29 days post-treatment. Late toxicity of fibrotic development measured by leg extendibility was monitored biweekly until 30 weeks post-treatment., Results: Comparison of acute skin toxicity following radiation indicated a tissue-sparing effect of FLASH compared to conventional single-fraction radiation with a mean protection ratio of 1.40 (1.35-1.46). Fibrotic development similarly indicated normal tissue sparing with a 1.18 (1.17-1.18) protection ratio. The acute skin toxicity tissue sparing was similar to data from entrance-beam irradiations of Sørensen et al. (4)., Conclusion: Full dose-response curves for acute and late toxicity after CONV and FLASH radiation were obtained. Radiation within the SOBP retains the normal-tissue-sparing effect of FLASH with a dose-modifying factor of 40% for acute skin damage and 18% for fibrotic development., Competing Interests: BS and PP are co-inventors on a patent-application application number 63257211 and EFS ID: 44064136. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kristensen, Poulsen, Kanouta, Rohrer, Ankjærgaard, Andersen, Johansen, Simeonov, Weber, Grau and Sørensen.)

Background: Dosimetry in pre-clinical FLASH studies is essential for understanding the beam delivery conditions that trigger the FLASH effect. Resolving the spatial and temporal characteristics of proton pencil beam scanning (PBS) irradiations with ultra-high dose rates (UHDR) requires a detector with high spatial and temporal resolution., Purpose: To implement a novel camera-based system for time-resolved two-dimensional (2D) monitoring and apply it in vivo during pre-clinical proton PBS mouse irradiations., Methods: Time-resolved 2D beam monitoring was performed with a scintillation imaging system consisting of a 1 mm thick transparent scintillating sheet, imaged by a CMOS camera. The sheet was placed in a water bath perpendicular to a horizontal PBS proton beam axis. The scintillation light was reflected through a system of mirrors and captured by the camera with 500 frames per second (fps) for UHDR and 4 fps for conventional dose rates. The raw images were background subtracted, geometrically transformed, flat field corrected, and spatially filtered. The system was used for 2D spot and field profile measurements and compared to radiochromic films. Furthermore, spot positions were measured for UHDR irradiations. The measured spot positions were compared to the planned positions and the relative instantaneous dose rate to equivalent fiber-coupled point scintillator measurements. For in vivo application, the scintillating sheet was placed 1 cm upstream the right hind leg of non-anaesthetized mice submerged in the water bath. The mouse leg and sheet were both placed in a 5 cm wide spread-out Bragg peak formed from the mono-energetic proton beam by a 2D range modulator. The mouse leg position within the field was identified for both conventional and FLASH irradiations. For the conventional irradiations, the mouse foot position was tracked throughout the beam delivery, which took place through repainting. For FLASH irradiations, the delivered spot positions and relative instantaneous dose rate were measured., Results: The pixel size was 0.1 mm for all measurements. The spot and field profiles measured with the scintillating sheet agreed with radiochromic films within 0.4 mm. The standard deviation between measured and planned spot positions was 0.26 mm and 0.35 mm in the horizontal and vertical direction, respectively. The measured relative instantaneous dose rate showed a linear relation with the fiber-coupled scintillator measurements. For in vivo use, the leg position within the field varied between mice, and leg movement up to 3 mm was detected during the prolonged conventional irradiations., Conclusions: The scintillation imaging system allowed for monitoring of UHDR proton PBS delivery in vivo with 0.1 mm pixel size and 2 ms temporal resolution. The feasibility of instantaneous dose rate measurements was demonstrated, and the system was used for validation of the mouse leg position within the field., (© 2024 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.)

Background: The spatial and temporal dose rate distribution of pencil beam scanning (PBS) proton therapy is important in ultra-high dose rate (UHDR) or FLASH irradiations. Validation of the temporal structure of the dose rate is crucial for quality assurance and may be performed using detectors with high temporal resolution and large dynamic range., Purpose: To provide time-resolved in vivo dose rate measurements using a scintillator-based detector during proton PBS pre-clinical mouse experiments with dose rates ranging from conventional to UHDR., Methods: All irradiations were performed at the entrance plateau of a 250 MeV PBS proton beam. A detector system with four fiber-coupled ZnSe:O inorganic scintillators and 20 μs temporal resolution was used for dose rate measurements. The system was first characterized in terms of precision and stem signal. The detector precision was determined through repeated irradiations with the same field. The stem signal contribution was quantified by irradiating two of the detector probes alongside a bare fiber (fiber without a coupled scintillator). Next, the detector system was calibrated against an ionization chamber (IC) with all four detector probes and the IC placed in a water bath at 2 cm depth. A scan pattern covering 9.6 × 9.6 cm was used. Multiple irradiations with different requested nozzle currents provided instantaneous dose rates at the detector positions in the range of 7-1270 Gy/s. The correspondence of the detector signal (in Volts) to the instantaneous dose rate (in Gy/s) was found. The instantaneous dose rate was calculated from the beam current and the spot-to-detector distance assuming a Gaussian beam profile at distances up to 8 mm from the spot. Afterwards, the calibrated system was used in vivo, in mouse experiments, where mouse legs were irradiated with a constant dose and varying field dose rates of 0.7-87.5 Gy/s. The instantaneous dose rate was measured for each delivered spot and the delivered dose was determined as the integrated instantaneous dose rate. The spot dose profile and PBS dose rate map were calculated. The dose contamination to neighbouring mice were measured together with the upper limit of the dose to the mouse body., Results: The detectors showed high precision with ≤0.4% fluctuations in the measured dose. The stem signal exceeded 10% for spots <5 mm from the optical fiber and >18 mm from the scintillator. It contributed up to 0.2% to the total dose, which was considered negligible. All four detectors showed a non-linear relation between signal and instantaneous dose rate, which was modelled with a polynomial response function. In the mouse experiments, the measured scintillator dose showed 1.8% fluctuations, independent of the field dose rate. The in vivo measured spot dose profile had tails that deviated from a Gaussian profile with measurable dose contributions from spots up to 85 mm from the detector. Neighbour mouse irradiation contributed ∼1% of the total mouse dose. The upper limit of the mouse body dose was 6% of the mouse leg dose., Conclusions: A fiber-coupled inorganic scintillator-based detector system can provide high precision in vivo measurements of the instantaneous dose rate if correction for the non-linear dose rate dependency is applied., (© 2022 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.)

Purpose: Preclinical studies indicate a normal tissue sparing effect when ultra-high dose rate (FLASH) radiation is used, while tumor response is maintained. This differential response has promising perspectives for improved clinical outcome. This study investigates tumor control and normal tissue toxicity of pencil beam scanning (PBS) proton FLASH in a mouse model., Methods and Materials: Tumor bearing hind limbs of non-anaesthetized CDF1 mice were irradiated in a single fraction with a PBS proton beam using either conventional (CONV) dose rate (0.33-0.63 Gy/s field dose rate, 244 MeV) or FLASH (71-89 Gy/s field dose rate, 250 MeV). 162 mice with a C3H mouse mammary carcinoma subcutaneously implanted in the foot were irradiated with physical doses of 40-60 Gy (8-14 mice per dose point). The endpoints were tumor control (TC) assessed as no recurrent tumor at 90 days after treatment, the level of acute moist desquamation (MD) to the skin of the foot within 25 days post irradiation, and radiation induced fibrosis (RIF) within 24 weeks post irradiation., Results: TCD 50 (dose for 50% tumor control) was similar for CONV and FLASH with values (and 95% confidence intervals) of 49.1 (47.0-51.4) Gy for CONV and 51.3 (48.6-54.2) Gy for FLASH. RIF analysis was restricted to mice with tumor control. Both endpoints showed distinct normal tissue sparing effect of proton FLASH with MDD 50 (dose for 50% of mice displaying moist desquamation) of <40.1 Gy for CONV and 52.3 (50.0-54.6) Gy for FLASH, (dose modifying factor at least 1.3) and FD 50 (dose for 50% of mice displaying fibrosis) of 48.6 (43.2-50.8) Gy for CONV and 55.6 (52.5-60.1) Gy for FLASH (dose modifying factor of 1.14)., Conclusions: FLASH had the same tumor control as CONV, but reduced normal tissue damage assessed as acute skin damage and radiation induced fibrosis., Competing Interests: Conflict of interest statement This study presented in this manuscript is partly funded by Varian. Two co-authors are co-inventors on a patent-application filled with application number 63257211 and EFS ID: 44064136, which contains parts of the data included in the manuscript., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Key factors in FLASH treatments are the ultra-high dose rate (UHDR) and the time structure of the beam delivery. Measurement of the time structure in pencil beam scanning (PBS) proton FLASH treatments is challenging for many types of detectors since high temporal resolution is needed. In this study, a fast scintillator detector system was developed and used to measure the individual spot durations as well as the time when the beam moves between two positions (transition duration) during PBS proton FLASH and UHDR treatments. The spot durations were compared with machine log-file recordings., Methods: A detector system based on inorganic scintillating crystals was developed. The system consisted of four detector probes made of a sub-millimeter ZnSe:O crystal that was coupled via an optical fiber to an optical reader with 50 kHz sampling rate. The detector system was used in two experiments, both performed with a PBS proton beam with 250 MeV beam energy and 215 nA requested nozzle beam current. The sampling rate enabled multiple measurements during each spot delivery and during the beam transition between spots. First, the detector was tested in a phantom experiment, where a total of 305 scan sequences were delivered to the four detectors. The number of spots delivered without beam interruption in a single scan sequence ranged from one to 35. The spot duration and transition duration were measured for each individual spot. Secondly, the detector system was used in vivo in preclinical experiments with FLASH irradiation of mouse legs placed in the entrance plateau of the beam. A single detector was placed 1 cm downstream of the irradiated mouse leg. The mouse dose ranged from 30.5 to 44.2 Gy and the field consisted of 35 spots. The spot durations as well as the mean dose rate (field dose divided by the measured field duration) for each mouse were determined using the detector and then compared with the corresponding log files., Results: The phantom experiment showed that the logged total duration of an uninterrupted spot sequence was consistently shorter than the measured duration with a difference of -0.252 ms (95% confidence interval: [-0.255, -0.249 ms]). This corresponded to 0.05%-0.07% of the spot sequence duration in the mice experiments. For individual spots, the mean ± 1SD difference between logged and measured spot duration was -0.39 ± 0.05 ms for the first spot in a sequence, 0.13 ± 0.04 ms for the last spot in a sequence, and -0.0017 ± 0.09 ms for the intermediate spots in a sequence. The measured spot transition durations were 0.20 ± 0.04 ms (5.1 mm horizontal steps) and 0.50 ± 0.04 ms (5.0 mm vertical steps). For the mouse experiments, the mean dose rate calculated from the measured field duration was 84.1-92.5 Gy/s. It agreed with log files with a root mean square difference of 0.02 Gy/s., Conclusions: Fiber-coupled scintillator detectors were designed with sufficient temporal resolution to measure the spot and transition duration during PBS proton UHDR deliveries. Their small volume makes them feasible for in vivo use in preclinical FLASH studies. The logged spot durations were in excellent agreement with measurements but showed small systematic errors in the logged duration for the first and last spot in a sequence., (© 2022 American Association of Physicists in Medicine.)

Background and Purpose: Preclinical studies indicate a normal tissue sparing effect using ultra-high dose rate (FLASH) radiation with comparable tumor response. Most data so far are based on electron beams with limited utility for human treatments. This study validates the effect of proton FLASH delivered with pencil beam scanning (PBS) in a mouse leg model of acute skin damage and quantifies the normal tissue sparing factor, the FLASH factor, through full dose response curves., Materials and Methods: The right hind limb of CDF1 mice was irradiated with a single fraction of proton PBS in the entrance plateau of either a 244 MeV conventional dose rate field or a 250 MeV FLASH field. In total, 301 mice were irradiated in four separate experiments, with 7-21 mice per dose point. The endpoints were the level of acute moist desquamation to the skin of the foot within 25 days post irradiation., Results: The field duration and field dose rate were 61-107 s and 0.35-0.40 Gy/s for conventional dose rate and 0.35-0.73 s and 65-92 Gy/s for FLASH. Full dose response curves for five levels of acute skin damage for both conventional and FLASH dose rate revealed a distinct normal tissue sparing effect with FLASH: across all scoring levels, a 44-58% higher dose was required to give the same biological response with FLASH as compared to the conventional dose rate., Conclusions: The normal tissue sparing effect of PBS proton FLASH was validated. The FLASH factor was quantified through full dose response curves., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)