Background and Aims

FLASH proton pencil-beam-scanning (p-PBS) showed 10-40% reduction in mouse skin fibrosis when delivered as a single uninterrupted high dose fraction. Clinical p-PBS treatment usually requires multiple beams separated by minutes to allow gantry repositioning, with some overlap of beams inside and outside the tumor. This study addressed whether the pause in delivery between overlapping beams affects the FLASH sparing effect in one model of normal tissue damage.

This study aimed to experimentally simulate a beam overlap area in a mouse skin model and evaluate the impact of the time-interruption between beams on the FLASH sparing effect on skin toxicity.

Methods

The right hind leg of 10-week-old female C57Bl/6j mice was irradiated using a ProBeam PBS Gantry system (Varian) at conventional (1Gy/s) or FLASH (100Gy/s) average field dose rate. We scored the skin toxicity for 8 weeks. Treatment was delivered in 0,1, or 2 beams with interruptions of 2 minutes. Each beam remained in the same position so that there was full overlap of beams administered.

Results

Single beam delivery confirmed a benefit for p-PBS-FLASH in this model, at 30 and 35 Gy. At 35 Gy, an interruption of 2 minutes (2 x 17.5 Gy) still demonstrated FLASH normal tissue sparing effect (p<0.001). Two interruptions (3 x 11.6 Gy) abrogated the sparing effect. At 30 Gy, where the single beam sparing effect was less striking than 35 Gy, 1 or 2 interruptions of 2 minutes abrogated the FLASH effect.

Conclusions

Our results suggest that the FLASH normal tissue sparing effect in areas of beam overlap can be compromised by interruptions to the total delivery time. Future research on the FLASH effect should study the time gap between overlapping beams and the spatial arrangement of delivered beams. The effect of multibeam needs to be studied on different organs of interest.

Background and Aims

FLASH radiotherapy (FLASH-RT) is a new technique, involving treatment of tumours at ultra-high dose rates, which has been shown to reduce normal tissue from radiation-induced toxicity, whilst equalling the anti-tumour effect of conventional dose rate radiotherapy (CONV-RT). Here, we performed a dose-response study in mice comparing the effect of FLASH-RT versus CONV-RT on skin and lung toxicity as well as tumour response in a lung cancer xenograft model.

Methods

Two human tumours xenografted in CD1-nude mice and one syngeneic tumour xenografted in C57BL/6J mice were used for the comparative determination of the antitumor response. Mice were treated using a 6 MeV electron linear accelerator once xenografts reached 80 mm³ with FLASH-RT or CONV-RT, both at a single dose of 20 Gy or at a fractionated dose of 30 Gy (3 x 10 Gy). Acute and late radiation effects were quantified in healthy C57BL/6J mice by skin toxicity scoring, lung CT-scan imaging and histopathological analysis, after hemithorax irradiation in the dose range of 10 to 30 Gy in a single fraction.

Results

We found that FLASH-RT and CONV-RT showed similar efficacy with regards to growth delay/control of lung cancer cells transplanted into immunocompromised and immunocompetent mice. No differences were observed between the treatments with single dose of 20 Gy and fractionated dose of 30 Gy, for both, FLASH and CONV irradiations. No macroscopic signs of cutaneous lesions were observed after 30 Gy hemithorax FLASH-RT, although we observed hair depigmentation restricted to the irradiated area. In contrast, mice exposed to 20 or 25 Gy CONV-RT developed severe cutaneous lesions and earlier hair depigmentation. Both, lung CT-scan imaging and histopathological analysis, demonstrated lower inflammation after FLASH-RT compared to CONV-RT.

Conclusions

In this study, the results showed that FLASH-RT reduces radiation-induced skin and lung toxicity, while showing equivalent tumour response as CONV-RT.

Background and Aims

Rotating anode x-ray tubes can sufficiently handle the input power and heat-loading requirements to reach FLASH dose rates at short source-to-surface distances. This study presents the dosimetric characterization and implementation of a FLASH-capable x-ray tube for an in vivo study of radiation skin toxicity in mice.

Methods

A high-capacity rotating anode x-ray tube and 75kW generator were utilized for FLASH study, operating at 150kVp with 0.025mmCu added filtration. A variety of wild-type mice (FVBN, C57BL6) were irradiated to the hind flank to 28, 35, or 40Gy at FLASH (>40Gy/s) or conventional (CONV; <1Gy/s) dose rates. Output and in vivo dose measurements were performed using EBT3 film and thermoluminescent dosimeters. Visible skin toxicities were assessed weekly using a skin scoring scale. Skin samples were harvested at 9-days post-treatment for TGFB1 and LRG6 assay. Samples for H&E and trichrome were harvested at 27- and 56-days post-treatment.

Results

The maximum dose rates were 91.3 ± 3.3 and 93.8 ±4.9Gy/s, as measured in the same position by EBT3 and TLD, respectively. The average delivered dose rate was 86.7 ± 3.9Gy/s in our treatment setup in a 10x20mm^2 area. Differences in skin toxicity scoring were significant between FLASH and CONV dose rates (p<0.01; paired t-test). Histology assessment shows higher frequency of ulceration and more severe hyperplasia and fibrosis in CONV-irradiated mice than FLASH-irradiated mice at 35Gy. All endpoints were comparable between dose rates at 28 and 43Gy. Immunofluorescence assays remain in progress and will be presented.

Conclusions

Rotating anode x-ray tubes are capable of inducing FLASH normal tissue sparing effects at comparable doses to prior proton and electron FLASH experiments. In contrast, doses are delivered in a single pulse, suggesting that average dose rate may be the determining factor to the induction of FLASH effects, rather than pulse structure. Tumor control studies remain a topic of further research.

Background and Aims

Background: FLASH proton radiotherapy is evidenced to alleviate radiation-related toxicities in normal skin tissue compared to Standard radiotherapy. Aim: To investigate the transcriptomic changes induced by FLASH proton radiotherapy (F-PRT) that could be responsible for the protection of normal epithelial tissues by radiation-induced toxicities as have been previously shown by us and others.

Methods

Methods: C57BL/6J mice received 30 Gy of F-PRT or S-PRT to the hind leg at respective dose rates of 69-124 Gy/sec or 0.39–0.65 Gy/sec. RNA sequencing was performed using full-thickness leg skin at 5 days after radiation revealing major pathways regulated by F-PRT and S-PRT. In an endeavor to identify the full repertoire of cells and gene expression profiles that are involved in the sparing effects of FLASH PRT, we expanded our studies to include single-cell RNA sequencing (sc-RNA seq) and examined additional time points such as Day 2 and Day 10 after radiation. Single-cell transcriptome libraries were generated on a 10X Genomics Chromium system. Datasets were acquired from cell samples derived and sequenced from pooled skin samples of three mice per group. Skin from the sequenced mice was also embedded for spatial analysis of gene expression.

Results

Results: RNA sequencing revealed that F-PRT uniquely upregulates almost four times more genes compared to S-PRT (F-PRT-uniquely upregulated 489 genes vs S-PRT-uniquely upregulated 129 genes). Also, F-PRT uniquely downregulated 178 genes, compared to the 125 genes uniquely downregulated by S-PRT. GO analysis demonstrates that the keratinization and apoptosis pathways are uniquely upregulated by S-PRT, whereas F-PRT uniquely upregulates genes involved in vascular development pathway. During submission of the abstract, analysis of sc-RNA seq samples was pending.

Conclusions

Conclusion: Our comprehensive studies inform on the transcriptomic profiling of skin cell populations that are affected by F-PRT vs S-PRT; this insight will further spur discoveries on the biology of FLASH radiotherapy effects.

Background and Aims

A well-known pathological outcome of incidental exposure of the heart to radiotherapy (RT), in patients undergoing thoracic RT, is the radiation-induced myocardial fibrosis (RIMF) leading to pericarditis, cardiomyopathy, and in certain cases, congestive heart failure. We have recently demonstrated that delivery of FLASH proton RT (F-PRT; >70 Gy/s) ameliorates inflammation, late-stage toxicity, and primarily fibrosis, compared to Standard Proton RT (S-PRT; <1 Gy/s). Our aim is to investigate if F-PRT reduces heart injury and mitigates RIMF compared to S-PRT, in a preclinical mouse model of focal heart irradiation.

Methods

RNA sequencing (RNA-seq), immunofluorescence (IF) staining, cytokine analysis, and 2D-echocardiography were used in this study.

Results

To test the potential differences between the F-PRT and S-PRT on PRT-induced cardiotoxicity and on the onset of RIMF, we performed RNA-seq analysis on F-PRT and S-PRT-irradiated hearts and NR (mock-irradiated) at 3 weeks post a single dose of 40 Gy. Reactome pathway analysis on the 100 mostly upregulated genes in F-PRT-treated hearts showed enrichment in DNA repair pathways, while S-PRT-treated hearts revealed enrichment mainly in inflammatory signaling pathways. Analysis of the inflammatory status of heart lysates at 3 weeks post 40 Gy, revealed that F-PRT-treated hearts presented significantly lower levels of TNF-a, compared to S-PRT-treated hearts. Moreover, we found that S-PRT significantly increased the levels of TGF-b1 compared to the F-PRT-treated hearts, aligning with the inflammation at this time point. Preliminary data on 2D echocardiography analysis shows a better myocardial function in F-PRT-treated hearts compared to the S-PRT group.

Conclusions

Our preliminary findings suggest that, in contrast to the F-PRT, the S-PRT modality causes a persistent inflammatory environment with the overexpression of inflammatory cytokines and profibrotic factors in the irradiated cardiac apex. Further investigation will provide valuable information on the sparing effects of F-PRT with potential application to the clinic in the near future.