Background and Aims

DNA-damaging treatments such as radiotherapy (RT) became promising to improve the efficacy of immune checkpoint inhibitors by enhancing tumor immunogenicity. However, accompanying treatment-related detrimental events in normal tissues had been the dark side of the combination regimen, and had posed a major obstacle for patients potentially amenable for radioimmunotherapy, thus presenting new challenges to the dose delivery mode of clinical RT. In the present study, ultra-high dose rate FLASH X-ray irradiation was applied to counteract intestinal toxicity in radioimmunotherapy.

Methods

In the present study, the high-energy X-ray with ultra-high dose rate (110-120 Gy/s) was applied in the single pulse mode. Programmed cell death ligand-1 (PD-L1) knockout (KO) mice undergoing conventional dose rate (CONV) or FLASH X-ray irradiation were undertaken as animal models to simulate the concurrent administration of RT with checkpoint blockade.

Results

In the context of PD-L1 blockade, FLASH X-ray minimized mouse enteritis by alleviating CD8⁺ T cell-mediated deleterious immune response compared with CONV irradiation. Mechanistically, FLASH irradiation was less efficient than CONV X-ray in eliciting cytoplasmic double-stranded DNA (dsDNA) and in activating the cGAS-STING signaling pathway in the intestinal crypts, resulting in the suppression of the cascade feedback consisting of CD8⁺ T cell chemotaxis and gasdermin E-mediated intestinal pyroptosis in the case of PD-L1 blocking. Meanwhile, FLASH X-ray was as competent as CONV RT in boosting the anti-tumor immune response initiated by cGAS activation and achieved equal tumor control in metastasis burdens when combined with anti-PD-L1 administration.

Conclusions

Together, we proposed the ‘DNA integrity’ hypothesis to elucidate the mechanism of the FLASH sparing effect, suggesting that ‘relatively intact DNA integrity’ within intestinal cells during the ‘instantaneous’ (approximately 120 ms) IR exposure was critical for FLASH X-ray in sparing PD-L1 deficient mice from detrimental enteritis. However, the inherent genomic instability of tumor cells makes them still sensitive to FLASH radiation.

Background and Aims

Our previous studies demonstrated that single high dose FLASH total abdominal irradiation (FLASH-TAI) produced significantly reduced mortality from gastrointestinal syndrome, preserved epithelial integrity, and spared cell death in crypt base columnar cells compared to conventional dose rate total abdominal conventional irradiation (CONV-TAI) in C57BL/6J mice. Here, we compare FLASH-TAI with CONV-TAI in additional mouse strains with and without defects in DNA double strand break (DSB) repair.

Methods

FLASH-TAI and CONV-TAI were performed in S129 mice with and without a knockout in Fanca (and thus deficiency in homologous recombination DNA DSB repair) at 14 and 16 Gy; in BALB/c mice which have reduced expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (and thus deficiency in nonhomologous end joining DNA DSB repair) at 10 and 12 Gy; and in C57BL/6J mice at 12 Gy. We used a clinical linear accelerator configured to produce a ~16-17 MeV electron beam collimated to deliver a uniform dose throughout the mouse abdominal cavity in both FLASH (216 Gy/s) and conventional (0.079 Gy/s) dose rates. Regenerating crypts were counted in 3-5 hematoxylin & eosin stained mid-jejunal circumferences per mouse (4-8 mice/group) 96 hours post irradiation.

Results

While Fanca wild type mice had increased regenerating crypts compared to Fanca knockout after both FLASH-TAI and CONV-TAI, both Fanca wild type and Fanca knockout mice had increased numbers of regenerating crypts following FLASH-TAI compared to CONV-TAI. BALB/c mice had ~5-fold fewer regenerating crypts after TAI compared to C57BL/6J mice. However, BALB/c had significantly more regenerating crypts after FLASH-TAI compared to CONV-TAI.

Conclusions

These data indicate that FLASH spares crypt regeneration compared to CONV independent of FANCA-dependent and DNA-PKcs dependent DNA DSB repair processes. Future work includes an analysis of the kinetics of DNA DSB repair in these strains of mice.

Background and Aims

Radiation-induced endoplasmic reticulum stress (ERS) signaling has been described as one factor for pulmonary fibrosis. It has never been investigated in response to UHDR irradiation and this study aimed to evaluate the effect of FLASH radiotherapy on radiation-induced ERS in vitro and in vivo.

Methods

Using eRT6/Oriatron Linac (5.5 MeV), SV2 lung adenocarcinoma cells were irradiated at 10Gy using CONV (0.1 Gy/s, 10Hz) or FLASH irradiation (>3x10⁶ Gy/s, 100Hz). Protein lysates were collected 24-72h post-RT. SV2 cells were also orthotopically engrafted in the lung of C57BL6/J male mice (n=7/group) and irradiated with hypo-fractionated 2x6Gy CONV or FLASH-RT on two consecutive days. Tumor growth delay was evaluated by mCT. Whole lung protein lysates (tumor + normal parenchyma) were collected 3 and 10 days post-RT. Biomarkers of ERS pathway (p-PERK/p-eIF2α/CHOP/IRE1α/XBP1s) were analyzed by Western-blot.

Results

In vitro experiments showed that both CONV and FLASH-RT activated ERS with a similar pattern. A dynamic change in the expression of the anti-survival pathway dependent of p-PERK-p-eIF2α-CHOP was observed. In vivo, tumor growth was delayed by 7 days both after CONV and FLASH-RT vs. control. At 3 day post-RT p-PERK-p-eIF2α-CHOP were activated in the CONV group vs. control but not in the FLASH group. Levels returned to normal level at 10 days.

Conclusions

These experiments suggest that in vitro experiments do not recapitulate in vivo regulations. In vivo studies suggest a possible role for p-PERK-p-eIF2α-CHOP pathway in the differential response to CONV vs. FLASH in the lung. More investigations are needed to determine the respective changes occurred in the tumor microenvironment vs. normal lung.

Background and Aims

Post-lumpectomy radiotherapy (RT) reduces in-breast tumor recurrence by eradicating residual, occult breast cancer (BC) that may be in the mm size scale. The ability of FLASH-RT to eradicate BC relative to conventional dose-rate (CONV) RT is unknown. ~ 20Gy RT is currently used clinically for single-fraction breast IORT. Determine the effectiveness of FLASH compared to CONV in eradicating small tumors in an orthotopic, syngeneic model of BC using single-fraction 20 or 30Gy RT.

Methods

Radiation sensitive, mammary tumor cell-line Py117 from the transgenic model of the mouse mammary tumor virus promoter driving the polyoma middle T antigen (MMTV- PyMT) efficiently forms non-metastatic, orthotopic tumors in C57BL/6 mice. 10⁶ Py117 cells were injected orthotopically into the left 4^th mammary fat pad of C57Bl/6J mice. Radiotherapy was performed with a custom jig that allows for fixed positioning of the target volume (2x2cm radiation field) with 5mm of margin into surrounding tissue. Tumors were irradiated at ~30mm³ volume or, for comparison, at a range of greater volumes (200-800mm³) with 20 or 30Gy FLASH or CONV with 16-17 MeV electrons.

Results

Small 30mm³ tumors regressed until ~day 15 after 20Gy single-fraction RT then regrew for both FLASH and CONV. 30mm³ tumors were eradicated with both FLASH and CONV at 30Gy with no regrowth up to day 35 post-RT. Larger tumors irradiated with 30Gy regressed until ~day 12 post-RT then regrew for both FLASH and CONV. There was no significant difference in growth delay or tumor eradication between FLASH and CONV groups.

Conclusions

FLASH was as effective as CONV in controlling growth and eradicating murine BC. Based on other preclinical studies, single-fraction doses between 20 and 30Gy, as well as hypofractioned RT schedules, may identify FLASH doses that achieve comparable tumor control with less toxicity than CONV. Such findings would encourage clinical trials of FLASH in human BC.

Background and Aims

Background: The beam stability and the precision of the dose delivery are essential to guarantee the safety and quality of proton FLASH therapy in ultra-high dose rates (UHDRs).

Aim: A proton pencil-beam scanning pattern was designed and irradiated on a high spatiotemporal resolution 2D strip ionization chamber array (SICA) with an in-house designed phantom as an all-in-one pre-treatment QA for proton FLASH therapy.

Methods

Methods: A PBS plan with a single field was designed with a 5x5 cm² uniform field at the center for dose and dose rate consistency. Asymmetrical lines on the perimeter are used to test positioning accuracy in discrete spot mode, varied speeds in continuous scanning mode, and range verification. The 250 MeV proton beam was irradiated on the SICA with the Varian ProBeam system at 100nA and 215nA nozzle currents. A reference Advanced Markus ionization chamber was placed downstream to the SCIA. The QA items, including the spot positioning, spot scanning speed, absolute dose, and average dose rates were recorded for trend analysis.

Results

Results: The overall delivered spot positionings are within 0.2mm compared with the planned positions. The low and high spot scanning speeds are within 1 mm/ms deviation to the requested specification of the beam control system. The output variation of the central field was within ±5% compared with the average and ±2.5% after normalized with the reference. More than 79.6% and 97.8% of the measurement points reached the FLASH threshold (40 Gy/s) at 100 nA and 215 nA in the 5x5 cm²region defined by a dose threshold of 80% of the center dose, respectively.

Conclusions

Conclusions: The pre-treatment QA program for FLASH therapy in UHDR with the designed pattern irradiated on a SICA can be performed in single irradiation. The SICA is a reliable tool to fulfill a QA solution for proton PBS FLASH radiotherapy.

Background and Aims

There is a need for beam monitoring and feedback control systems for FLASH that are dose-rate independent and capable of controlling the beam at the level of individual pulses. We aim to use a beam current monitor (BCT) to measure dose in real-time, validate time structure of the beam, and build a feedback control loop.

Methods

We modified a 3D printed collimator for pre-clinical anatomy-specific irradiation of mice to accommodate a 55 mm diameter BCT positioned surrounding the beam path. The signal from the BCT was digitized on a fast oscilloscope. We determined the effect of the modified collimator with BCT on the entrance beam profile. The linearity of the BCT was established by varying dose per pulse in the range 0.2 to 2.3 Gy/pulse. The pulse width on the clinical linac was varied, and this change was measured using the BCT. Finally, the ability of the BCT to measure the variability of dose per pulse and pulse width due to a mistuned automatic frequency control (AFC) system was demonstrated.

Results

The BCT had minimal effect on the entrance profile and exhibited excellent linearity with dose per pulse up to 2.5 Gy/Pulse (R² =0.99). The peak beam current for the 17 MeV FLASH beam was measured to be ~10 mA for a 2Gy/pulse output. Manual tuning of AFC showed the presence of a ramp-up in dose per pulse and pulse width. During the ramp-up period, the pulse width was observed to be as small as 0.5 us and the pulse output was 1/4^th of the stable output.

Conclusions

We demonstrated real-time per pulse readout of electron beam charge, correlated with entrance dose. Going forward, we will perform a failure modes and safety analysis of the control system and assess long-term stability with the goal of supporting a human clinical trial.

Background and Aims

Despite the remarkable developments in radiation oncology a significant patient population remains hopeless and predestined to palliative or best supportive care. These are patients affected by unresectable bulky tumors, whose volume and close relationship with neighboring organs do not allow the ablative radiation dose to be delivered through conventional radiotherapy. We assessed the impact of a novel partial bulky-tumor irradiation using particles on tumor control and survival among patients with unresectable bulky tumors unsuitable for conventional radio-chemotherapy who failed previous state-of-the-art treatments. The hypothesis is that for effective immunomodulation resulting in bystander and abscopal effects, the entire tumor volume may not need to be irradiated but only a partial volume to initiate the immune cycle in radiation-spared peri-tumoral-immune microenvironment.

Methods

11 consecutive patients were treated from March 2020 until December 2021 (Fig.1). The targeted Bystander Tumor Volume (BTV) was created by subtracting 1 cm from GTV surface corresponding to approximately 30% of its central volume and was irradiated with 30–45 Gy RBE in three consecutive fractions (Fig.2). The Peritumoral Immune Microenvironment (PIM)surrounding the GTV, containing nearby tissues, blood-lymphatic vessels and lymph nodes, was considered an OAR and protected by highly conservative constraints.

Results

With the median follow up of 6.3 months (range: 3-16), overall survival was 64% with a median survival of 6.7 months; 46% of patients were progression-free. The average tumor volume regression was 61%. The symptom control rate was 91%, with an average increase of the Karnofsky Index of 20%. No side effects other than fatigue G1 were reported. The abscopal effect has been observed in 60% of patients (Fig.3).

Conclusions

Partial bulky-tumor irradiation is an effective, safe and well tolerated treatment for patients with unresectable recurrent bulky disease. Abscopal effects elucidate an immunogenic pathway contribution. Extensive tumor shrinkage in some patients might permit definitive treatment—otherwise previously impossible.