CHUV
Radio-Oncology

Moderator Of 1 Session

Session Type
Live E-Poster Discussions
Date
Wed, 01.12.2021
Session Time
11:30 - 12:30
Room
Station 01

Presenter Of 1 Presentation

A PURSUIT FOR A HIGH-THROUGHPUT INDICATOR OF THE FLASH EFFECT

Session Type
FLASH Modalities Track (Oral Presentations)
Date
Wed, 01.12.2021
Session Time
10:20 - 11:30
Room
Room 2.31
Lecture Time
11:00 - 11:10

Abstract

Background and Aims

Despite an immense research interest in FLASH-RT, the precise beam requirements for obtaining the FLASH effect have still not been elucidated. Yet, there is an increasing number of reports assigning the name FLASH to beams and irradiators without any supportive biological data.

Methods

Currently, the FLASH effect can be confirmed only in vivo, which requires time-consuming animal studies and corresponding ethical approvals. The FLASH community would therefore greatly benefit from a high-throughput FLASH beam indicator that can validate an UHDR beam for FLASH-RT. Such indicator has to generate an observable that follows the same dependency on temporal beam characteristics as the FLASH effect.

Results

We used our published data on sparing of the normal mouse brain and killing of GBM to monitor the impact of gradually changing electron beam parameters (dose rate, dose per pulse) on the occurrence of the FLASH effect. These data were considered as a template to investigate responses of various high-throughput assays over the same range of beam parameters. In particular, we studied assays that previously showed differential response to our FLASH and Conv beams: H2O2 yield, O2 depletion, plasmid DSB, lipid peroxidation and zebrafish embryo. Only the length of zebrafishes grown from irradiated embryos showed the dependency on beam parameters mimicking the cognitive protection in vivo with low energy electron beam (Oriatron).

Conclusions

Since mechanistic differences between different types of beams (protons, X-ray) are possible, further investigations are mandatory to confirm universal validity of this model as a general FLASH indicator.
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Author Of 6 Presentations

A PURSUIT FOR A HIGH-THROUGHPUT INDICATOR OF THE FLASH EFFECT

Session Type
FLASH Modalities Track (Oral Presentations)
Date
Wed, 01.12.2021
Session Time
10:20 - 11:30
Room
Room 2.31
Lecture Time
11:00 - 11:10

Abstract

Background and Aims

Despite an immense research interest in FLASH-RT, the precise beam requirements for obtaining the FLASH effect have still not been elucidated. Yet, there is an increasing number of reports assigning the name FLASH to beams and irradiators without any supportive biological data.

Methods

Currently, the FLASH effect can be confirmed only in vivo, which requires time-consuming animal studies and corresponding ethical approvals. The FLASH community would therefore greatly benefit from a high-throughput FLASH beam indicator that can validate an UHDR beam for FLASH-RT. Such indicator has to generate an observable that follows the same dependency on temporal beam characteristics as the FLASH effect.

Results

We used our published data on sparing of the normal mouse brain and killing of GBM to monitor the impact of gradually changing electron beam parameters (dose rate, dose per pulse) on the occurrence of the FLASH effect. These data were considered as a template to investigate responses of various high-throughput assays over the same range of beam parameters. In particular, we studied assays that previously showed differential response to our FLASH and Conv beams: H2O2 yield, O2 depletion, plasmid DSB, lipid peroxidation and zebrafish embryo. Only the length of zebrafishes grown from irradiated embryos showed the dependency on beam parameters mimicking the cognitive protection in vivo with low energy electron beam (Oriatron).

Conclusions

Since mechanistic differences between different types of beams (protons, X-ray) are possible, further investigations are mandatory to confirm universal validity of this model as a general FLASH indicator.
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CHARACTERIZATION OF DAMAGE ASSOCIATED MOLECULAR PATTERNS AFTER FLASH RADIOTHERAPY TO AMPLIFY ANTI-TUMOR IMMUNE RESPONSE

Session Type
FLASH Mechanisms Track (Oral Presentations)
Date
Wed, 01.12.2021
Session Time
10:20 - 11:30
Room
Room 2.15
Lecture Time
10:30 - 10:40

Abstract

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.

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COMPARISON OF H2O2 AND HO· PRIMARY YIELDS AND O2 DEPELTION AFTER IRRADIATION AT UHDR AND CONVENTIOANL-DOSE RATE WITH 6MEV ERT6/ORIATRON

Session Type
FLASH Mechanisms Track (Oral Presentations)
Date
Wed, 01.12.2021
Session Time
18:00 - 19:00
Room
Room 2.15
Lecture Time
18:00 - 18:10

Abstract

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)

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PLASMID DNA DAMAGES AFTER FLASH VS CONVENTIONAL DOSE RATE IRRADIATIONS IN VARIOUS OXYGEN CONDITIONS

Session Type
FLASH Mechanisms Track (Oral Presentations)
Date
Wed, 01.12.2021
Session Time
18:00 - 19:00
Room
Room 2.15
Lecture Time
18:50 - 19:00

Abstract

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).

layout 3_redim.jpg

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.

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MODELLING OF WATER RADIOLYSIS FOR ULTRA-HIGH DOSE RATE (FLASH) ELECTRON BEAMS IN GEANT4-DNA

Session Name
Session Type
FLASH Mechanisms Track (Oral Presentations)
Date
Thu, 02.12.2021
Session Time
11:00 - 12:00
Room
Room 2.15
Lecture Time
11:50 - 12:00

Abstract

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 (eaq) 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.

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NOT JUST HEALTHY TISSUE SPARING: HYPOXIA DOES NOT IMPACT FLASH-RT ANTI-TUMOR EFFICACY

Session Type
FLASH Mechanisms Track (Oral Presentations)
Date
Thu, 02.12.2021
Session Time
15:10 - 16:10
Room
Room 2.15
Lecture Time
15:30 - 15:40

Abstract

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

all data oxygen.jpg

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)

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