Concomitant targeting of programmed death-1 (PD-1) and CD137 improves the efficacy of radiotherapy in a mouse model of human BRAFV600-mutant melanoma.

T cell checkpoint blockade with antibodies targeting programmed cell death (ligand)-1 (PD-1/PD-L1) and/or cytotoxic T lymphocyte-antigen 4 (CTLA-4) has improved therapy outcome in melanoma patients. However, a considerable proportion of patients does not benefit even from combined α-CTLA-4 and α-PD-1 therapy. We therefore examined to which extent T cell (co)stimulation and/or stereotactic body radiation therapy (SBRT) could further enhance the therapeutic efficacy of T cell checkpoint blockade in a genetically engineered mouse melanoma model that is driven by PTEN-deficiency, and BRAFV600 mutation, as in human, but lacks the sporadic UV-induced mutations. Tumor-bearing mice were treated with different combinations of immunomodulatory antibodies (α-CTLA-4, α-PD-1, α-CD137) or interleukin-2 (IL-2) alone or in combination with SBRT. None of our immunotherapeutic approaches (alone or in combination) had any anti-tumor efficacy, while SBRT alone delayed melanoma outgrowth. However, α-CD137 combined with α-PD-1 antibodies significantly enhanced the anti-tumor effect of SBRT, while the anti-tumor effect of SBRT was not enhanced by interleukin-2, or the combination of α-CTLA-4 and α-PD-1. We conclude that α-CD137 and α-PD-1 antibodies were most effective in enhancing SBRT-induced tumor growth delay in this mouse melanoma model, outperforming the ability of IL-2, or the combination of α-CTLA-4 and α-PD-1 to synergize with SBRT. Given the high mutational load and increased immunogenicity of human melanoma with the same genotype, our findings encourage testing α-CD137 and α-PD-1 alone or in combination with SBRT clinically, particularly in patients refractory to α-CTLA-4 and/or α-PD-1 therapy.

Automated determination of the ion-recombination correction factor (ksat) in ultra-high dose rate electron radiation therapy.

BACKGROUND: Plane-parallel ionization chambers are the recommended secondary standard systems for clinical reference dosimetry of electrons. Dosimetry in high dose rate and dose-per-pulse (DPP) is challenging as ionization chambers are subject to ion recombination, especially when dose rate and/or DPP is increased beyond the range of conventional radiotherapy. The lack of universally accepted models for correction of ion recombination in UDHR is still an issue as it is, especially in FLASH-RT research, which is crucial in order to be able to accurately measure the dose for a wide range of dose rates and DPPs. PURPOSE: The objective of this study was to show the feasibility of developing an Artificial Intelligence model to predict the ion-recombination factor-ksat for a plane-parallel Advanced Markus ionization chamber for conventional and ultra-high dose rate electron beams based on machine parameters. In addition, the predicted ksat of the AI model was compared with the current applied analytical models for this correction factor. METHODS: A total number of 425 measurements was collected with a balanced variety in machine parameter settings. The specific ksat values were determined by dividing the output of the reference dosimeter (optically stimulated luminescence [OSL]) by the output of the AM chamber. Subsequently, a XGBoost regression model was trained, which used the different machine parameters as input features and the corresponding ksat value as output. The prediction accuracy of this regression model was characterized by R2-coefficient of determination, mean absolute error and root mean squared error. In addition, the model was compared with the Two-Voltage (TVA) method and empirical Petersson model for 19 different dose-per-pulse values ranging from conventional to UDHR regimes. The Akiake Information criterion (AIC) was calculated for the three different models. RESULTS: The XGBoost regression model reached a R2-score of 0.94 on the independent test set with a MAE of 0.067 and RMSE of 0.106. For the additional 19 random data points, the ksat values predicted by the XGBoost model showed to be in agreement, within the uncertainties, with the ones determined by the Petersson model and better than the TVA method for doses per pulse >3.5 Gy with a maximum deviation from the ground truth of 14.2%, 16.7%, and -36.0%, respectively, for DPP >4 Gy. CONCLUSION: The proposed method of using AI for ksat determination displays efficiency. For the investigated DPPs, the ksat values obtained with the XGBoost model were in concurrence with the ones obtained with the current available analytical models within the boundaries of uncertainty, certainly for the DPP characterizing UDHR. But the overall performance of the AI model, taking the number of free parameters into account, lacked efficiency. Future research should optimize the determination of the experimental ksat, and investigate the determination the ksat for DPPs higher than the ones investigated in this study, while also evaluating the prediction of the proposed XGBoost model for UDHR machines of different centers.

IL-15-secreting CAR natural killer cells directed toward the pan-cancer target CD70 eliminate both cancer cells and cancer-associated fibroblasts.

BACKGROUND: It remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma. METHODS: RNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70+ tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs. RESULTS: In this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70+ tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70+ tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner. CONCLUSIONS: We disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70+ cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.

Optically stimulated luminescence system as an alternative for radiochromic film for 2D reference dosimetry in UHDR electron beams.

Radiotherapy is part of the treatment of over 50% of cancer patients. Its efficacy is limited by the radiotoxicity to the healthy tissue. FLASH-RT is based on the biological effect that ultra-high dose rates (UHDR) and very short treatment times strongly reduce normal tissue toxicity, while preserving the anti-tumoral effect. Despite many positive preclinical results, the translation of FLASH-RT to the clinic is hampered by the lack of accurate dosimetry for UHDR beams. To date radiochromic film is commonly used for dose assessment but has the drawback of lengthy and cumbersome read out procedures. In this work, we investigate the equivalence of a 2D OSL system to radiochromic film dosimetry in terms of dose rate independency. The comparison of both systems was done using the ElectronFlash linac. We investigated the dose rate dependence by variation of the (1) modality, (2) pulse repetition frequency, (3) pulse length and (4) source to surface distance. Additionally, we compared the 2D characteristics by field size measurements. The OSL calibration showed transferable between conventional and UHDR modality. Both systems are equally independent of average dose rate, pulse length and instantaneous dose rate. The OSL system showed equivalent in field size determination within 3 sigma. We show the promising nature of the 2D OSL system to serve as alternative for radiochromic film in UHDR electron beams. However, more in depth characterization is needed to assess its full potential.

Application of a novel diamond detector for commissioning of FLASH radiotherapy electron beams.

PURPOSE: A diamond detector prototype was recently proposed by Marinelli et al. (Medical Physics 2022, https://doi.org/10.1002/mp.15473) for applications in ultrahigh-dose-per-pulse (UH-DPP) and ultrahigh-dose-rate (UH-DR) beams, as used in FLASH radiotherapy (FLASH-RT). In the present study, such so-called flashDiamond (fD) was investigated from the dosimetric point of view, under pulsed electron beam irradiation. It was then used for the commissioning of an ElectronFlash linac (SIT S.p.A., Italy) both in conventional and UH-DPP modalities. METHODS: Detector calibration was performed in reference conditions, under 60 Co and electron beam irradiation. Its response linearity was investigated in UH-DPP conditions. For this purpose, the DPP was varied in the 1.2-11.9 Gy range, by changing either the beam applicator or the pulse duration from 1 to 4 µs. Dosimetric validation of the fD detector prototype was then performed in conventional modality, by measuring percentage depth dose (PDD) curves, beam profiles, and output factors (OFs). All such measurements were carried out in a motorized water phantom. The obtained results were compared with the ones from commercially available dosimeters, namely, a microDiamond, an Advanced Markus ionization chamber, a silicon diode detector, and EBT-XD GAFchromic films. Finally, the fD detector was used to fully characterize the 7 and 9 MeV UH-DPP electron beams delivered by the ElectronFlash linac. In particular, PDDs, beam profiles, and OFs were measured, for both energies and all the applicators, and compared with the ones from EBT-XD films irradiated in the same experimental conditions. RESULTS: The fD calibration coefficient resulted to be independent from the investigated beam qualities. The detector response was found to be linear in the whole investigated DPP range. A very good agreement was observed among PDDs, beam profiles, and OFs measured by the fD prototype and reference detectors, both in conventional and UH-DPP irradiation modalities. CONCLUSIONS: The fD detector prototype was validated from the dosimetric point of view against several commercial dosimeters in conventional beams. It was proved to be suitable in UH-DPP and UH-DR conditions, for which no other commercial real-time active detector is available to date. It was shown to be a very useful tool to perform fast and reproducible beam characterizations in standard clinical motorized water phantom setups. All of the previously mentioned demonstrate the suitability of the proposed detector for the commissioning of UH-DR linac beams for preclinical FLASH-RT applications.

Development of an ultra-thin parallel plate ionization chamber for dosimetry in FLASH radiotherapy.

BACKGROUND: Conventional air ionization chambers (ICs) exhibit ion recombination correction factors that deviate substantially from unity when irradiated with dose per pulse magnitudes higher than those used in conventional radiotherapy. This fact makes these devices unsuitable for the dosimetric characterization of beams in ultra-high dose per pulse as used for FLASH radiotherapy. PURPOSE: We present the design, development, and characterization of an ultra-thin parallel plate IC that can be used in ultra-high dose rate (UHDR) deliveries with minimal recombination. METHODS: The charge collection efficiency (CCE) of parallel plate ICs was modeled through a numerical solution of the coupled differential equations governing the transport of charged carriers produced by ionizing radiation. It was used to find out the optimal parameters for the purpose of designing an IC capable of exhibiting a linear response with dose (deviation less than 1%) up to 10 Gy per pulse at 4 µ $\umu$ s pulse duration. As a proof of concept, two vented parallel plate IC prototypes have been built and tested in different ultra-high pulse dose rate electron beams. RESULTS: It has been found that by reducing the distance between electrodes to a value of 0.25 mm it is possible to extend the dose rate operating range of parallel plate ICs to ultra-high dose per pulse range, at standard voltage of clinical grade electrometers, well into several Gy per pulse. The two IC prototypes exhibit behavior as predicted by the numerical simulation. One of the so-called ultra-thin parallel plate ionization chamber (UTIC) prototypes was able to measure up to 10 Gy per pulse, 4 µ $\umu$ s pulse duration, operated at 300 V with no significant deviation from linearity within the uncertainties (ElectronFlash Linac, SIT). The other prototype was tested up to 5.4 Gy per pulse, 2.5 µ $\umu$ s pulse duration, operated at 250 V with CCE higher than 98.6% (Metrological Electron Accelerator Facility, MELAF at Physikalisch-Technische Bundesanstalt, PTB). CONCLUSIONS: This work demonstrates the ability to extend the dose rate operating range of ICs to ultra-high dose per pulse range by reducing the spacing between electrodes. The results show that UTICs are suitable for measurement in UHDR electron beams.

Design, realization, and characterization of a novel diamond detector prototype for FLASH radiotherapy dosimetry.

PURPOSE: FLASH radiotherapy (RT) is an emerging technique in which beams with ultra-high dose rates (UH-DR) and dose per pulse (UH-DPP) are used. Commercially available active real-time dosimeters have been shown to be unsuitable in such conditions, due to severe response nonlinearities. In the present study, a novel diamond-based Schottky diode detector was specifically designed and realized to match the stringent requirements of FLASH-RT. METHODS: A systematic investigation of the main features affecting the diamond response in UH-DPP conditions was carried out. Several diamond Schottky diode detector prototypes with different layouts were produced at Rome Tor Vergata University in cooperation with PTW-Freiburg. Such devices were tested under electron UH-DPP beams. The linearity of the prototypes was investigated up to DPPs of about 26 Gy/pulse and dose rates of approximately 1 kGy/s. In addition, percentage depth dose (PDD) measurements were performed in different irradiation conditions. Radiochromic films were used for reference dosimetry. RESULTS: The response linearity of the diamond prototypes was shown to be strongly affected by the size of their active volume as well as by their series resistance. By properly tuning the design layout, the detector response was found to be linear up to at least 20 Gy/pulse, well into the UH-DPP range conditions. PDD measurements were performed by three different linac applicators, characterized by DPP values at the point of maximum dose of 3.5, 17.2, and 20.6 Gy/pulse, respectively. The very good superimposition of three curves confirmed the diamond response linearity. It is worth mentioning that UH-DPP irradiation conditions may lead to instantaneous detector currents as high as several mA, thus possibly exceeding the electrometer specifications. This issue was properly addressed in the case of the PTW UNIDOS electrometers. CONCLUSIONS: The results of the present study clearly demonstrate the feasibility of a diamond detector for FLASH-RT applications.

A new calculation method for the free electron fraction of an ionization chamber in the ultra-high-dose-per-pulse regimen.

The free electron fraction is the fraction of electrons, produced inside the cavity of an ionization chamber after irradiation, which does not bind to gas molecules and thereby reaches the electrode as free electrons. It is a fundamental quantity to describe the recombination processes of an ionization chamber, as it generates a gap of positive charges compared to negative ones, which certainly will not undergo recombination. The free electron fraction depends on the specific chamber geometry, the polarizing applied voltage and the gas thermodynamic properties. Therefore, it is necessary to evaluate such fraction in an accurate and easy way for any measurement condition. In this paper, a simple and direct method for evaluating the free electron fraction of ionization chambers is proposed. We first model the capture process of the electrons produced inside an ionization chamber after the beam pulse; then we present a method to evaluate the free electron fraction based on simple measurements of collected charge, by varying the applied voltage. Finally, the results obtained using an Advanced Markus chamber irradiated with a Flash Radiotherapy dedicated research Linac (ElectronFlash) to estimate the free electron fraction are presented. The proposed method allows the use of a conventional ionization chamber for measurements in ultra-high-dose-per-pulse (UHDP) conditions, up to values of dose-per-pulse at which the perturbation of the electric field due to the generated charge can be considered negligible.

Point scintillator dosimetry in ultra-high dose rate electron "FLASH" radiation therapy: A first characterization.

FLASH radiation therapy is a novel technique combining ultra-high dose rates (UHDR) with very short treatment times to strongly decrease normal tissue toxicity while preserving the anti-tumoral effect. However, the radiobiological mechanisms and exact conditions for obtaining the FLASH-effect are still under investigation. There are strong indications that parameters defining the beam structure, such as dose per pulse, instantaneous dose rate and pulse repetition frequency (PRF) are of importance. UHDR irradiations therefore come with dosimetric challenges, including both dose assessment and temporal ones. In this work, a first characterization of 6 real-time point scintillating dosimeters with 5 phosphors (Al2O3:C,Mg; Y2O3:Eu; Al2O3:C; (C38H34P2)MnBr4 and (C38H34P2)MnCl4, was performed in an UHDR pulsed electron beam. The dose rate independence of the calibration was tested by calibrating the detector at conventional and UHDR. Dose rate dependence was observed, however, further investigation, including intermediate dose rates, is needed. Linearity of the response with dose was tested by varying the number of pulses and a linearity with R2> 0.9989 was observed up to at least 200 Gy. Dose per pulse linearity was investigated by variation of the pulse length and SSD. All point scintillators showed saturation effects up to some extent and the instantaneous dose rate dependence was confirmed. A PRF dependence was observed for the Al2O3:C,Mg and Al2O3:C- based point scintillators. This was expected as the luminescence decay time of these materials exceeds the inter-pulse time.

Radiosensitivity Is an Acquired Vulnerability of PARPi-Resistant BRCA1-Deficient Tumors.

The defect in homologous recombination (HR) found in BRCA1-associated cancers can be therapeutically exploited by treatment with DNA-damaging agents and PARP inhibitors. We and others previously reported that BRCA1-deficient tumors are initially hypersensitive to the inhibition of topoisomerase I/II and PARP, but acquire drug resistance through restoration of HR activity by the loss of end-resection antagonists of the 53BP1/RIF1/REV7/Shieldin/CST pathway. Here, we identify radiotherapy as an acquired vulnerability of 53BP1;BRCA1-deficient cells in vitro and in vivo. In contrast to the radioresistance caused by HR restoration through BRCA1 reconstitution, HR restoration by 53BP1 pathway inactivation further increases radiosensitivity. This highlights the relevance of this pathway for the repair of radiotherapy-induced damage. Moreover, our data show that BRCA1-mutated tumors that acquire drug resistance due to BRCA1-independent HR restoration can be targeted by radiotherapy. SIGNIFICANCE: These findings uncover radiosensitivity as a novel, therapeutically viable vulnerability of BRCA1-deficient mouse mammary cells that have acquired drug resistance due to the loss of the 53BP1 pathway.

The curative outcome of radioimmunotherapy in a mouse breast cancer model relies on mTOR signaling.

Radiotherapy is a successful treatment modality for localized cancer. Our group has been exploring radiotherapy in combination with immunotherapy (radioimmunotherapy) to enhance systemic antitumor responses. Previously, we have shown that when local radiotherapy was combined with monoclonal antibodies (mAbs) (that enable T-cell responses by engaging costimulation [anti (α)-CD137] and blocking coinhibition [α-PD-1] [corrected], up to 100% of mice bearing established syngeneic AT-3 mammary tumors were cured, but single modality treatments were not curative. Here, we investigated the molecular mechanisms underlying responses to this radioimmunotherapy approach. We observed that inhibition of signaling through the mammalian target of rapamycin (mTOR) pathway during the first 10 days of treatment severely impaired the curative effect of radioimmunotherapy, at least in part by reducing MHC class I expression on tumor cells, reducing dendritic cell (DC) activation status and CD8+ T-cell function. This data indicates that the efficacy of this type of radioimmunotherapy approach involves mTOR signaling and therefore, mTOR inhibitory drugs may impede the efficacy of similar radioimmunotherapy approaches in humans.