Subacute Cutaneous Toxicity with Single-Fraction Electron FLASH RT in Yorkshire Swine

Abstract

Information regarding acute/subacute skin toxicity of electron FLASH radiation therapy (RT) is limited. We evaluated short-term safety of electron FLASH for human trials by investigating subacute toxicity compared to conventional dose-rate RT (CONV) in the Yorkshire pig, an animal model known to closely approximate human skin and routinely used for toxicity studies. Two healthy 50 kg pigs underwent CT imaging for RT treatment planning with field visualization via BBs and tattoos on each dorsolateral flank. Each target received a single fraction of 20, 25 or 30 Gy with FLASH and CONV on opposing sides delivered using a dedicated mobile linear accelerator. FLASH dose rates ranged from 164-245 Gy/sec (12 pulses delivered over 0.122 sec) while the CONV dose rate was set at 0.18 Gy/sec. Doses were verified using thermo- and optically stimulated luminescent dosimeters, and Gafchromic films. We obtained baseline and weekly images up to 98 days post-RT (D98) for blinded toxicity grading by 3 expert radiation oncologists using the modified RTOG radiation dermatitis (RD) scale. We measured erythema and pigmentation indices on those timepoints using a handheld spectrophotometer. We also obtained punch biopsies of targets and non-irradiated controls on D10 and D30 for RNA sequencing and two 6-marker multiplex immunofluorescence analyses of inflammation, immune response, and fibrosis. FLASH and CONV data were compared using repeated measures ANOVA and transcriptomic analyses using DESeq2. All RT targets developed peak median grade 4 (ulceration, hemorrhage, or necrosis) RD by D84 regardless of FLASH or CONV delivery. However, FLASH targets developed peak RD later than CONV targets after 20 Gy (D84 vs D63), 25 Gy (D84 vs D49) and 30 Gy (D63 vs D42). FLASH induced qualitatively lower mean pigmentation and erythema indices than CONV for all 3 doses. Similarly, peak mean pigmentation indices occurred later with FLASH vs CONV for 20 Gy (D84 vs D63), 25 Gy (D84 vs D49) and 30 Gy (D77 vs D63). However, peak mean erythema indices occurred on the same day for FLASH and CONV (D63 for 20 Gy and D42 for 25 and 30 Gy). Transcriptomic analyses revealed significantly upregulated signals for wound healing (including TGF-beta, cell adhesion and extracellular matrix receptor interaction) and leukocyte infiltration with 20 Gy CONV mostly by D10, while FLASH upregulated those pathways only after 25 or 30 Gy, or by D30, or never at all. Preliminary immunofluorescence data showed FLASH may induce less T cell infiltrate and TGF-beta-expressing macrophages than CONV. Single-fraction electron FLASH resulted in delayed onsets of both subacute cutaneous toxicity and wound healing with leukocytic infiltration signaling than dose-matched CONV based on both subjective and objective metrics of skin injury. Our findings suggest further investigations of optimal dose of electron FLASH for safe clinical translation is warranted, and we have a dose-finding study currently underway.