Abstract Background and purpose Radiation-induced lymphopenia is associated with adverse outcomes in thoracic malignancies. FLASH radiotherapy delivers radiation over a timescale of hundreds of milliseconds, potentially reducing the fraction of irradiated circulating lymphocytes. In this study, we investigated whether FLASH mitigates lymphopenia after thoracic irradiation delivered with protons or photons. Materials and methods C57BL/6 mice received three 13.5-Gy whole-heart fractions at 48-hour intervals using FLASH or standard dose-rate proton irradiation at the University of Pennsylvania (n=15), with photon validation at Queen’s University Belfast (n=72). Leukocytes and CD4 + T cells, CD8 + T cells, B cells, and NK cells were quantified by hemocytometer and flow cytometry. A continuous-time Markov model simulated lymphocyte trafficking, dose accumulation, and post-irradiation recovery. Results FLASH attenuated leukocyte depletion across both proton and photon irradiation modalities. In the proton cohort, white blood cell counts were significantly higher after FLASH at D1, D3, D7, and D14; CD4 + T cells and NK cells were preserved through D14, while CD8 + T cell sparing persisted through D21. Photon FLASH preserved CD45 + leukocytes at D1, D3, D7, and D21, with sustained CD8 + sparing at D21. Modeling showed that FLASH shifted the lymphocyte dose distribution toward lower exposures, increasing the proportion of lymphocytes receiving <1 Gy from 2.4% to 16.4%, and reduced the proportion of lymphocytes repeatedly irradiated across all three fractions from 36.3% at standard dose rate to 9.18%, despite similar median cumulative doses. The spleen contributed substantially to cumulative lymphocyte dose, and marrow-entering lymphocytes displayed a more high-dose-enriched distribution after FLASH irradiation. Conclusion FLASH consistently mitigated radiation-induced lymphopenia for proton and photon modalities, with durable CD8 + T cell preservation. These findings support a kinetic mechanism and provide a rationale for combining FLASH radiotherapy with immune-sparing planning and immunotherapy.

The list of antibodies used for the immunofluorescence and the western blot assay.

The TaqMan primer list for RT–qPCR.

Detection of oral mucositis as observed after staining with 1% Toluidine Blue.

Estimation of saliva flow in male C57BL/6 mice following irradiation with a single dose of 16 Gy of proton irradiation.

Objective.Proton radiotherapy requires accurate quality assurance (QA) of beam range and output. Conventional QA methods using water phantoms and ionization chambers are time-consuming and may suffer from dose-rate dependence, limiting their use for FLASH radiation therapy at ultra-high dose rates (UHDRs). This work evaluated a multi-layer Faraday cup (MLFC) as a detector for proton therapy QA, capable of high-resolution energy verification, beam charge measurements, and operation at both conventional and UHDR.Approach.A commercial 128-layer MLFC made of thin copper layers separated by Kapton and equipped with a MicroHexTMenergy filter was used to measure conventional and UHDR proton pencil beams from a IBA ProteusPlus system. A TOPAS Monte Carlo model was developed to benchmark depth-charge readout. Energy calibration of the pencil beams was performed by Gaussian fitting charge peaks and comparing to nominal ranges in water. Delivered charge was determined by integrating the collected charge across all MLFC channels and comparing it with a reference Faraday cup. Its application to UHDR FLASH radiotherapy was evaluated using spread-out Bragg Peak Conformal FLASH plan deliveries.Main results.The MLFC provided a reproducible high-resolution energy calibration within 1 mm water equivalent depth resolution, with the MicroHexTMfilter broadening charge deposition peaks and improving measurement precision and accuracy. Integrated charge was linear with monitor units and agreed with Faraday cup measurements within 5% for all measured energies (100-228 MeV). The 4 kHz sampling rate enabled spot-by-spot energy and charge output measurements at UHDR.Significance.The MLFC enables simultaneous energy and charge output verification in a compact, dose-rate-independent detector, providing a practical solution for routine QA in both conventional and FLASH proton therapy.

F-PRT reduces radiation-induced damage to the oral cavity compared to S-PRT.

Immunofluoresecnce of TUNEL and AQP5 after 10 days post irradiation of PRT.

The beam profile, as acquired with the film for irradiation with 16 Gy proton.

Histopathological analysis of submandibular gland tissues post-28 days of irradiation with S-PRT/F-PRT.

The percent difference in body weight of mice after irradiation with a hypofractionated regime of 8 Gy x 3.

BACKGROUND:Ultra-high dose rate radiotherapy elicits a biological effect (FLASH), which has been shown to reduce toxicity while maintaining tumor control in preclinical radiobiology experiments. FLASH depends on the dose rate, with evidence that higher dose rates drive increased normal tissue sparing. The pattern of dose delivery also has significance for conformal proton FLASH delivered via pencil beam scanning (PBS) given its unique spatio-temporal distribution of dose deposition. PURPOSE:In PBS, the machine-generated log file contains information on the spatio-temporal pattern of PBS delivery measured by the segmented ionization chambers in the treatment nozzle. The spot position and monitor unit (MU) obtained from log files have previously been used to reconstruct the treatment dose by Monte Carlo (MC) simulations. The incorporation of spot timing allows reconstruction of the 3D temporal dose distribution. The log-based dose and dose rate can have a role in quality assurance (QA) and FLASH treatment verification if the reconstruction can be shown to be accurate in spatial and temporal domains of dose deposition. Thus, the objective of this study is to validate the accuracy of dose rate reconstruction using input data from machine log files of PBS delivery. By analyzing the delivered spot timing, position, and MU extracted from the logs, we aim to evaluate the reliability and precision of the log data for dose and dose rate reconstruction. METHODS:FLASH PBS spread-out Bragg peak (SOBP) treatment fields were delivered using a cyclotron accelerated proton beam. This method involves a patient and field-specific conformal energy modulator (CEM) to achieve a SOBP at the tumor site. Log files record spot positions and the delivered MU with timing information at 250 µs resolution. To validate timing information, a 9.9 mm diameter parallel plate ionization chamber was positioned at various locations within the SOBP. An electrometer sampling at 20 kHz recorded the time-resolved ionization current collected by the ionization chamber. These measurements were used to determine spot dose, dose rate, duration, and transition times. Disparities between the measured and logged spot map MU and timing were determined. Dose average and PBS dose rates were compared between the measurement and log-based MC simulations. RESULTS:There was a good agreement between the measured dwell time and transition time and the logged information across various detector positions. The median disparities for inter-spot dwell time range from -0.041  to 0.024 ms. Differences between logged and planned spot positions are minimal, measuring less than 1.08 mm in the x direction and 1.15 mm in the y direction, consistent with prior studies and the spatial resolution of the PBS nozzle ionization chamber. Delivered MU were within 1.9% of the planned MU. Measured dose and dose rates are consistent with simulated outcomes derived from MC simulation. CONCLUSION:We validated the precision and accuracy of PBS log file data through measurements and MC simulations. These findings support the use of log files in MC calculations as one part of patient-specific quality assurance (PSQA) and dose rate delivery verification for conformal proton FLASH radiotherapy with SOBP.

BACKGROUND:To maximize the potential benefit of the FLASH sparing effect during treatment, normal tissue regions would ideally be irradiated only briefly, typically for a couple of hundred milliseconds. Achieving such fast proton irradiation involves a mono-energetic beam at the highest cyclotron energy and the use of 3D-printed conformal energy modulators (CEM). In ConformalFLASH, a dedicated snout is mounted on the nozzle, containing the CEM, a range shifter, and an aperture. PURPOSE:Demonstrate that ConformalFLASH irradiation using a coarse 3D-printed CEM, defined by a geometry with spike resolution > 0.5 mm in any dimension, is fulfilling existing clinical dose standards. The CEM is robust to printing errors and can be reliably manufactured with unmodified commercially available 3D printers. METHODS:Monte-Carlo simulations were conducted to define the 3D-printing specifications of the CEM. A variety of CEMs were then printed according to specifications. CT scans were acquired, and in-beam measurements were performed for each part, using the FLASH beam properties, the FLASH snout, and dosimetry detectors. RESULTS:Considering the proposed ConformaFLASH setup choice, it was possible to design coarse CEM that are both robust and easily printable using commercial technology. Over several measured cases, the 3D-printed CEM yields clinical-grade proton dose distributions. This confirms the irradiation set-up and the CEM manufacturing specifications as predefined through Monte Carlo simulations. CEM CT scans reinforce further the dosimetric results, to provide additional evidence of 3D printing quality. CONCLUSIONS:The dose distribution obtained through carefully specified CEM proves robust to production errors typically occurring in commercial 3D printing. The robustness opens the way to simplified manufacturing of relatively complex parts. Owing to the beam configuration, the snout, and the range shifter, the CEM was able to generate clinical-quality dose distributions. The integration of the FLASH snout with its elements on the nozzle of the proton therapy system represents an important step forward in comparison to existing state-of-the-art, facilitating easier preclinical and future clinical trial investigations.

Radiation therapy manages pancreatic cancer in various settings; however, the proximity of gastrointestinal (GI) luminal organs at risk (OARs) poses challenges to conventional radiation therapy. Proton beam therapy (PBT) may reduce toxicities compared to photon therapy. This consensus statement summarizes PBT's safe and optimal delivery for pancreatic tumors. Our group has specific expertise using PBT for GI indications and has developed expert recommendations for treating pancreatic tumors with PBT. Computed tomography (CT) simulation: Patients should be simulated supine (arms above head) with custom upper body immobilization. For stomach/duodenum filling consistency, patients should restrict oral intake within 3 hours before simulation/treatments. Fiducial markers may be implanted for image guidance; however, their design and composition require scrutiny. The reconstruction field-of-view should encompass all immobilization devices at the target level (CT slice thickness 2-3 mm). Four-dimensional CT should quantify respiratory motion and guide motion mitigation. Respiratory gating is recommended when motion affects OAR sparing or reduces target coverage. Treatment planning: Beam-angle selection factors include priority OAR-dose minimization, water-equivalent-thickness stability along the beam path, and enhanced relative biological effect consideration due to the increased linear energy transfer at the proton beam end-of-range. Posterior and right-lateral beam angles that avoid traversing GI luminal structures are preferred (minimizing dosimetric impacts of variable anatomies). Pencil beam scanning techniques should use robust optimization. Single-field optimization is preferable to increase robustness, but if OAR constraints cannot be met, multifield optimization may be used. Treatment delivery: Volumetric image guidance should be used daily. CT scans should be acquired ad hoc as necessary (at minimum every other week) to assess the dosimetric impacts of anatomy changes. Adaptive replanning should be performed as required. Our group has developed recommendations for delivering PBT to safely and effectively manage pancreatic tumors.

BACKGROUND AND PURPOSE:The normal tissue sparing afforded by FLASH radiotherapy is being intensely investigated for potential clinical translation. Here, we studied the effects of FLASH proton radiotherapy (F-PRT) in the reirradiation setting, with or without hypofractionation. Chronic toxicities in three murine models of normal tissue toxicity including the intestine, skin, and bone were investigated. MATERIALS AND METHODS:In studies of the intestine, single-dose irradiation was performed with 12 Gy of standard proton RT (S-PRT), followed by a second dose of 12 Gy of F-PRT or S-PRT. Additionally, a hypofractionation scheme was applied in the reirradiation setting (3 x 6.4 Gy of F-PRT or S-PRT, given every 48 hrs). In studies of skin/bone of the murine leg, 15 Gy of S-PRT was followed by hypofractionated reirradiation with F-PRT or S-PRT (3 x 11 Gy). RESULTS:Compared to reirradiation with S-PRT, F-PRT induced less intestinal fibrosis and collagen deposition that was accompanied by significantly increased survival rate, demonstrating its protective effects on intestinal tissues in the reirradiation setting. In previously irradiated leg tissues, reirradiation with hypofractionated F-PRT created transient dermatitis that fully resolved in contrast to reirradiation with hypofractionated S-PRT. Lymphedema was also alleviated after a second course of radiation with F-PRT, along with significant reductions in the accumulation of fibrous connective tissue in the skin, compared to mice reirradiated with S-PRT. The delivery of a second course of fractionated S-PRT induced tibial fractures in 83.3% of the mice, whereas only 20% of mice reirradiated with F-PRT presented with fractures. CONCLUSION:These studies provide the first evidence of the sparing effects of F-PRT in the setting of hypofractionated reirradiation. The results support FLASH as highly relevant to the reirradiation regimen where it exhibits significant potential to minimize chronic complications for patients undergoing RT.

Proton FLASH radiation therapy (RT) is an emerging technique that offers highly conformal doses similar to conventional intensity modulated proton therapy but with the added potential benefit of protecting organs at risk through the FLASH-sparing effect. This review examines recent advancements in proton FLASH-RT, including transmission beams (TB), single-energy Bragg peak, single-energy spread-out Bragg peak, hybrid FLASH, and multiple-energy spread-out Bragg peak. These proton FLASH technologies are discussed in detail, highlighting their advantages, limitations, and dosimetric comparisons with intensity modulated proton therapy and other FLASH techniques. Although TB achieves dose conformity through multifield optimization, it also has unnecessary exit doses. In contrast, single-energy Bragg peak and single-energy spread-out Bragg peak offer improved organ at risk protection and superior target conformity at the cost of using range compensators and/or ridge filters. Additionally, hybrid FLASH-RT combines TB and Bragg peak methods to target the tumor core and edges separately, whereas multiple-energy spread-out Bragg peak FLASH leverages ultra-fast energy switching. Despite these advancements, only nonconformal TB FLASH-RT has been applied clinically with single fields for palliative RT because of the complexity of other methods and uncertainties about the FLASH effect. This review summarizes the technical details of these FLASH-RT methods and discusses their utilization across various anatomical sites.

FLASH radiotherapy holds promise for treating solid tumors given the potential lower toxicity in normal tissues but its therapeutic effects on tumor immunity remain largely unknown. Using a genetically engineered mouse model of medulloblastoma, we show that FLASH radiation stimulates proinflammatory polarization in tumor macrophages. Single-cell transcriptome analysis shows that FLASH proton beam radiation skews macrophages toward proinflammatory phenotypes and increases T cell infiltration. Furthermore, FLASH radiation reduces peroxisome proliferator-activated receptor-γ (PPARγ) and arginase 1 expression and inhibits immunosuppressive macrophage polarization under stimulus-inducible conditions. Mechanistically, FLASH radiation abrogates lipid oxidase expression and oxidized low-density lipid generation to reduce PPARγ activity, while standard radiation induces reactive oxygen species-dependent PPARγ activation in macrophages. Notably, FLASH radiotherapy improves infiltration and activation of chimeric antigen receptor (CAR) T cells and sensitizes medulloblastoma to GD2 CAR-T cell therapy. Thus, FLASH radiotherapy reprograms macrophage lipid metabolism to reverse tumor immunosuppression. Combination FLASH–CAR radioimmunotherapy may offer exciting opportunities for solid tumor treatment. Fan and colleagues show that FLASH radiation promotes proinflammatory polarization of tumor-associated macrophages, which in turn increases T cell influx in medulloblastoma and synergizes with CAR-T cell therapy.