Background and Aims

Using glioblastoma as model, the aim of the present study was to investigate the role of G2/M arrest in tumor response to FLASH-RT and to characterize Damage Associated Molecular Patterns (DAMPs) that might amplify anti-tumor immunogenic response.

Methods

In vitro, GL261, H454 and PDGC2159 GBM and HaCat normal cells were synchronized (or not) in G2/M phase using a CDK1 (9uM) or PLK1 (25nM) inhibitor 24hours before 20Gy FLASH-RT (2.10³Gy/s, 2 pulses of 10Gy, 100Hz) or CONV-RT (~0.1-0.2Gy/s, 10Hz) with eRT6 (Jorge, 2019). Calreticulin, HSPA5, ATP, HMGB1, DNA release, micronuclei formation and cGAS-STING-type I IFN response were investigated. In vivo, murine GL261 and PDGC2159 GBM cells were orthotopically grafted to C57Bl6 and Swiss nude mice. Mice were treated with a single dose of 10Gy delivered Whole-Brain either with FLASH (≥10⁷Gy/s, 1 pulse) or CONV-RT (~0.1-0.2Gy/s). Tumor control, normal brain toxicity, immune response and in situ vaccination were evaluated.

Results

In vitro, the level of micronuclei positive cells was similar after FLASH and CONV (40% vs 0% in non-RT) and HMGB1 mRNA level was enhanced (+1.8fold) in FLASH vs CONV irradiated samples. G2/M blockade significantly increased micronuclei formation (+20%), and cGAS mRNA level (+2.33fold) in FLASH vs CONV irradiated samples. Other markers were not modified. In vivo experiments are ongoing.

Conclusions

These preliminary results support a G2/M-dependent release of DAMPs after FLASH irradiation that might trigger downstream immune response. Experiments are ongoing to characterize this response along with anti-tumor efficacy and normal toxicity in immune-deficient/competent mice.

Background and Aims

Normal tissue-sparing property of FLASH-RT has been shown in various studies, including a dose-escalating trial with single-dose FLASH-RT (25-41Gy) in cat-patients. Results prompted us to design this prospective, randomized clinical phase-III-trial in cat-patients with spontaneous tumors, to compare single-high-dose FLASH-RT to a standard of care (SOC); with tumour control-rate at 1 year as primary endpoint (hypothesis= 95% with FLASH-RT versus 71% for SOC, alpha=0.05 and beta=0.2, 29 cats needed).

Methods

Ethic’s approval was obtained (ZH204/18) and cats with T1-T2 N0 carcinomas of the nasal planum were randomly assigned to 2 arms of electron radiation. Arm 1 used 10x4.8Gy (90%IDL), delivered in one week with a 6MeV linear accelerator, dose rate of 600MU/min. Arm 2 used 1x30Gy (89%IDL) with eRT6/Oriatron, delivered in 20ms using 3 pulses, instantaneous dose rate of 6.3x10⁶Gy/s (mean dose rate 1700Gy/s).

Results

While acute side effects were mild to moderate and similar in both arms, the trial was prematurely stopped due an excess of maxillary bone necrosis which occurred 9-12 months after RT in 3/7 cats treated with FLASH-RT (43%), as compared to 0/9 cats in SOC. Regarding the primary endpoint, all cats were free of tumor progression at 1 year in both arms, but one tumor progression occurred later in FLASH-RT arm. Overall survival rates were similar in both arms, 690 days for SOC and 680 days for FLASH.

Conclusions

When compared to SOC, 1x30Gy-FLASH was beyond the maximal tolerated dose, causing severe late toxicity without better tumor control.

Acknowledgments: Krebsliga, KFS-4438-02-2018: Phase III clinical trial on cat patients

Background and Aims

Ultra-high dose rate (UHDR) irradiation produces the FLASH effect (anti-tumor effect without normal tissue toxicity) at average dose rates above 100 Gy/s. Two physico-chemical scenarios were proposed as a mechanistic basis for the FLASH effect following water radiolysis: 1. Altered radical-radical reactions and 2. Depletion of O₂ by free radicals. To investigate these questions, we determined primary radiolytic yields (G°-values) of hydrogen peroxide and hydroxyl radicals. G(H₂O₂) was also determined in low O₂ condition (1%), intermediate levels (4%) and atmospheric conditions (21%) following homogenous phase. O₂ depletion was also measured.

Methods

Scavenging methods were used to estimate radiolytic yields of H₂O₂ in water samples. Hydroxyl radicals production was estimated using EPR spin trapping with DMPO. O₂ measurements were performed using OxyLite probe.

Results

When UHDR and CONV-irradiation were compared, similar primary yields of H₂O₂ were found and EPR measurements suggested no differences in HO· production. However, G(H₂O₂) was significantly lower after irradiation at UHDR in samples equilibrated at 4%. O₂ measurements resulted in similar but low depletion with both modalities at intermediate and atmospheric O₂ conditions, whereas at low O₂ level, oxygen depletion was lower at UHDR.

Conclusions

These observations suggest that initial radiation chemistry is similar in both modalities: Similar yields of radicals ‘‘escaping’ ’track recombination after ionization. However, irradiation at UHDR produces less H₂O₂ at intermediate O₂ levels following initial chemistry events, supporting occurrence of scenario 1. O₂ depletion hypothesis is not favored by results obtained in pure water.

Acknowledgement: The study is funded by FNS Synergia grant (FNS CRS II5_186369)

Background and Aims

In our work we thought to compare the effects of conventional (CONV) vs ultra-high dose rate (UHDR) by quantifying DNA strand breaks (SB) after irradiation of plasmid-DNA (pBR322) under various oxygen concentrations.

Methods

Supercoiled pBR322 was irradiated dry or in water using a 6 MeV FLASH-validated electrons beam, with increasing doses (1-100 Gy) and dose per pulse (0.01 Gy/s (CONV), 5.0*10² to 5.6*10⁶ Gy/s (UHDR)) and at atmospheric (21%), physoxic (4%) and hypoxic (0.5%) oxygen level. The increase of relaxed (R) and linear (L) plasmid forms after irradiation was quantified by agarose gel electrophoresis and used to compute single and double SB yields.

Results

Dry, atmospheric conditions cause similar yields of SB in CONV and UHDR. Aqueous conditions shows higher SB yields as expected. Physoxia induces radioprotection compare to atmospheric condition: 50% of R at 10 Gy (4% O₂) vs 2 Gy (21% O₂), but no difference relative to dose rate. Hypoxia revealed higher SB yields than physoxia in CONV (50% of R at 6 Gy) but 2x less SB in UHDR for doses >30 Gy (see figure for L).

Conclusions

First results in dry condition suggest that direct effects are not involved in FLASH. In aqueous condition, 4% oxygen mimicking healthy tissues shows no difference between UHDR and CONV, while 0.5% oxygen mimicking tumors shows less damages in UHDR. These results are opposite to the preclinical results showing the FLASH effect. Thus, plasmid irradiation might be useful to understand DNA damage at UHDR but seems barely relevant to investigate the FLASH effect at the biological level.

Background and Aims

FLASH radiation modalities deliver ultra-high dose rates with high dose per pulse and a low number of pulses. In this work, we investigate the impact of electron beams with different doses per pulse (from 0.01 to 40 Gy) on water radiolysis using the Geant4-DNA code.

Methods

The single-track simulation mode is extended to multiple electron tracks delivered in the same pulse to obtain the desired dose to the target volume. This extension allows us to simulate the chemical stage up to hours after radiation exposure.

Results

The G values of hydroxyl radicals (^•OH), hydrated electrons (e⁻_aq) and hydrogen radicals (H^•) decrease earlier in time as the dose per pulse increases. In oxygenated water, a shorter lifetime of the reactive oxygen species (ROS) – superoxide radical (O₂^•-) and hydroperoxyl radical (HO₂^•) – is obtained for higher doses per pulse, which reduces the level of hydrogen peroxide (H₂O₂). These observations are compared with the available experimental data.

Conclusions

We found that the significant reduction in ROS yield results from the high concentration of species generated with high doses per pulse.

Background and Aims

In this study, we investigated the effects of tumor oxygen tension on the anti-tumor efficacy of ultra-high-dose-rate (FLASH) radiotherapy (RT).

Methods

U87 glioblastoma cells were xenografted in Swiss Nude mice and irradiated using a single 20-Gy dose administered at UHDR (2 pulses, 100 Hz, 1.8 µs pulse width, 0.01 s delivery) or CONV (~ 0.1 Gy/s) dose rates with the Oriatron/eRT6 (PMB, CHUV) under normoxia, hypoxia (vascular clamp), and hyperoxia (carbogen breathing). In situ oxygen tension was measured during and following irradiation using an OxyLite probe. Tumor growth was monitored using caliper measurements and tumor were sampled for RNA and protein profiling (GIF, UNIL). Metabolic analysis and ROS measurements were performed in vitro using Seahorse XF96 Analyzer and CellROX.

Results

Surprisingly, the anti-tumor efficacy of FLASH-RT was not affected by hypoxia in this U87 xenograft model, whereas hypoxia induced radioresistance with CONV-RT. Genomic profiling revealed a decrease in hypoxia signaling in the FLASH-treated compared to the CONV-treated and control tumors 24h post-RT. Oxidative metabolism was also altered in response to FLASH-RT. Real-time tumor oxygen readout, ROS levels, and metabolic testing at different oxygen tensions and timepoints post-RT are in progress.

Conclusions

FLASH-RT anti-tumor efficacy does not seem to be affected by hypoxia supporting a differential role for oxygen signaling between FLASH and CONV-RT and opening new venues for clinical application of FLASH-RT in a subset of highly radiation resistant tumors.

Acknowledgement: The study is funded by SNF Synergia grant (FNS CRS II5_186369)