Abstract Body

A re-emergence of research implementing radiation delivery at ultra-high dose rates (UHDR) has triggered intense interest in the radiation sciences and has opened a new field of investigation in radiobiology. Much of the promise of UHDR irradiation involves the FLASH effect, an in vivo biological response observed to maintain anti-tumor efficacy without the normal tissue complications associated with standard dose rates. The FLASH effect has been validated primarily, using intermediate energy electron beams able to deliver high doses in a very short period of time (<200 ms), but has also been found with photon and proton beams. The clinical implications of this new area of research are highly significant, as FLASH radiotherapy (FLASH-RT) has the potential to enhance the therapeutic index, opening new possibilities for eradicating radio-resistant tumors without toxicity. As pioneers in this field, my group has developed a multidisciplinary research team focused on investigating the mechanisms and clinical translation of the FLASH effect. I will tell the story of the recent discovery of the FLASH effect and will review the recent results obtained with electron beams at ultra-high dose rate.

Abstract Body

A re-emergence of research implementing radiation delivery at ultra-high dose rates (UHDR) has triggered intense interest in the radiation sciences and has opened a new field of investigation in radiobiology. Much of the promise of UHDR irradiation involves the FLASH effect, an in vivo biological response observed to maintain anti-tumor efficacy without the normal tissue complications associated with standard dose rates. The FLASH effect has been validated primarily, using intermediate energy electron beams able to deliver high doses in a very short period of time (<200 ms), but has also been found with photon and proton beams. The clinical implications of this new area of research are highly significant, as FLASH radiotherapy (FLASH-RT) has the potential to enhance the therapeutic index, opening new possibilities for eradicating radio-resistant tumors without toxicity. As pioneers in this field, my group has developed a multidisciplinary research team focused on investigating the mechanisms and clinical translation of the FLASH effect. I will tell the story of the recent discovery of the FLASH effect and will review the recent results obtained with electron beams at ultra-high dose rate.

Abstract Body

Toward single dose radiotherapy: Dream or reality?

Radiation oncology has been entrenched and remains dependent on advancements in technology. The capability to safely generate high energy beam modalities, characterize beam time signatures, perform accurate dosimetry and deliver image-guided treatment plans has been the cornerstone of the field. Over the years, the ultimate success of radiotherapy has been reliant on interdisciplinary contributions from physics, chemistry and biology, but has moved forward conservatively, constrained by the very technologies that have now ushered in precision driven stereotactic approaches such as SBRT/SABR and SRS. However, ultra-high dose rate FLASH radiotherapy (FLASH-RT), overlooked for over 40 years, has now triggered a renaissance in the field, aimed at evaluating if/how dose rate modifications can be garnered for therapeutic gain. This so called “FLASH effect” has been defined and validated in vivo, and provides a heretofore unforeseen capability to minimize normal tissue complications while maintaining isoefficient tumor control. The potential promise of affording curative, dose escalation via FLASH-RT was immediately recognized, as dose limiting toxicities have and always will dictate the maximum tolerated dose that can be applied to any given tissue bed.

In this regard, photon and particle FLASH radiotherapy have the potential to transform healthcare, and dovetail nicely into current trends toward hypofractionation and possibly single dose therapy. This talk will highlight unpublished data regarding the response of the adult and juvenile rodent brain to fractionated FLASH-RT, and try to link the known and possible physico-chemical and biological mechanisms that might help us translate these findings to the clinic. If/how that laudable goal can be accelerated or even achieved through forthcoming advancements in FLASH-RT remains to be seen, but this intriguing technology has certainly captured the imagination of the radiation sciences and holds appeal at several levels, except perhaps for clinical profit margins.

Background and Aims

Methods

Results

Conclusions

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

At the Photo Injector Test facility at DESY in Zeuthen (PITZ, near Berlin, Germany), the realization of an R&D platform for electron FLASH radiation therapy, VHEE radiation therapy and radiation biology is under preparation. The name of the new platform is HP²eFLASH-RT@PITZ, which stands for high power, high performance electron FLASH radiation therapy at PITZ.

Methods

The beam parameters that PITZ can provide are unique in the world: ps scale electron bunches with up to 5nC bunch charge at MHz repetition rate in bunch trains of up to 1 ms in length.

Results

The individual bunches can produce dose rates up to 10¹⁴ Gy/s and dose deposition up to 1000 Gy. Upon demand, each bunch of the bunch train can be guided to a different transverse location on the tumor, so that either a “painting” with micro beams, or a cumulative increase of absorbed dose using a wide beam distribution can be realized at the tumor. Full tumor treatment can hence be finished in a time interval of 1 ms, mitigating organ movement issues. Together with 20 years of operational experience at PITZ, and availability of detailed beam characterization and extremely flexible beam manipulation capabilities, this R&D platform will cover current parameter range of successfully demonstrated FLASH effects and extend the parameter range towards yet unexplored and unexploited short treatment times and high dose rates.

Conclusions

A summary of the plans for HP²eFLASH-RT@PITZ and the status of the preparations will be presented, with as goal to stimulate interest and broaden out our cooperation.

Background and Aims

Future RT devices using very-high energy electrons (VHEE) (50-250MeV) may produce suitable beams to treat deep-seated tumours conformally and at ultra-high dose rates (UHDR) capable of triggering the FLASH effect. The FLASH effect has been observed for large doses delivered with overall treatment times less than 200ms. Such treatment durations do not allow the use of a movable gantry and multiple fixed beam lines (FBL) become mandatory. This treatment planning study evaluates VHEE dose distributions in patients using a varying number of FBL with different energies and source-axis-distances (SAD). The minimum requirements for delivering conformal VHEE RT comparable to conventional VMAT plans and trade-offs between plan quality and number of beam lines are assessed.

Methods

We performed VHEE and VMAT treatment planning for multiple indications (glioblastoma, mediastinum, lung, prostate) using RayStation (research version) and compared the dosimetric quality of VHEE plans to VMAT while assessing the impact of arrangement and number of FBL, beam energies, and SAD.

Results

Most substantial coverage and conformity improvement is achieved when increasing the beam energy from 50 to 100MeV. Further improvement is obtained specifically for deep-seated targets (>10-15cm) when increasing energies further to 200MeV. While VHEE plans using 16 coplanar beams outperform VMAT plans, we found that VHEE plans using only 3-5 beams have DVH metrics that are comparable to VMAT plans. Beams with SAD>1m are preferable for treatments using few beams.

Conclusions

UHDR VHEE devices with only a few FBL may provide competitive dosimetric conformity that may be additionally enhanced by the FLASH effect.

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)

Background and Aims

Synchrotron Microbeam Radiation Therapy (S-MRT) consists of Synchrotron X-rays fractionated into an array of quasi-parallel beamlets delivered in FLASH mode. S-MRT achieves excellent tumour control and normal tissue sparing. This study aimed to evaluate S-MRT efficacy in a preclinical mouse lung carcinoma model.

Methods

Lewis-lung carcinoma implanted C57BL/6J mice were treated with two cross-fired arrays of S-MRT or Synchrotron-Broad Beam (S-BB) at 11 days after implantation. An array composed of seventeen microbeams 50 µm wide, spaced 400 µm apart was employed. S-MRT peak-dose was 400 Gy with a valley-dose of 4.76 Gy (delivery 361 ms, dose-rate 991.7 Gy/s). S-BB delivered a homogeneous dose of 5.16 Gy (delivery 129 ms, dose-rate 37.0 Gy/s). In addition, mouse lungs without tumours were irradiated with S-MRT, and radiation-related effects were assessed up to 6 months post-treatment.

Results

Mice in the S-MRT group had notably smaller tumour volumes compared to the S-BB group however, there was no difference in animal survival. This was attributed to pulmonary oedema found around the S-MRT-treated tumours. A mild transient form of fluid effusion was also observed in the S-MRT-treated normal lungs. Six months after S-MRT, the lungs of healthy mice were completely absent of radiation-induced pulmonary fibrosis.

Conclusions

Our study indicates that FLASH S-MRT is a promising tool for treating mouse lung carcinoma, i.e. reducing tumour size compared to mice treated with FLASH S-BB and sparing healthy lung from pulmonary fibrosis. Future experiments should focus on optimizing S-MRT parameters to minimize pulmonary oedema and maximize its therapeutic ratio.