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Proton minibeam radiation therapy: a promising therapeutic approach for radioresistant tumors

Proton minibeam radiation therapy (pMBRT) is a novel cancer therapy approach based on a high spatial dose modulation. pMBRT activates distinct radiobiological mechanisms and it has been shown to lead in small animal experiments to a significant increase in normal tissue dose tolerances while maintaining or enhancing tumor control effectiveness as compared with conventional radiotherapy. Although recently proposed, the biological observations collected thus far challenge the classical paradigm in RT and encourage the preparation of phase I clinical trials. Résumé. La radiothérapie par mini-faisceaux de protons (pMBRT) est une nouvelle approche de thérapie du cancer basée sur une forte modulation spatiale de la dose. La pMBRT active des mécanismes radiobiologiques distincts et il a été démontré dans des expériences sur de petits animaux qu’elle conduit à une augmentation significative des tolérances de dose pour les tissus normaux tout en maintenant ou en améliorant l’efficacité du contrôle des tumeurs par rapport à la radiothérapie conventionnelle. Bien que récemment proposées, les observations biologiques recueillies jusqu’à présent remettent en question le paradigme classique de la RT et encouragent la préparation d’essais cliniques


Introduction
Radiotherapy (RT) is one of the most important methods for cancer treatment. An estimated 2.9 million patients will be diagnosed with cancer in the EU-28 in 2025, of whom approximately 50% will receive external beam radiotherapy [1]. The technological advances in RT in recent decades have led to improved tumor dose conformation, thus reducing the dose to organs-at-risk [2]. However, adverse effects of RT are still common, and remain the main limitation of RT. Consequently, the tolerance of normal tissue to RT compromises effective treatment for radioresistant tumors.
RT is going through a paradigm shift driven by the ever-increasing evidence of the importance of the socalled "non-targeted" effects of radiation on the biological response, including cell signaling [3], and vascular [4], stromal, and immunological changes [5]. Interestingly, these effects can be activated and/or modulated by changing the physical parameters of the radiation, i.e., the dose delivery methods [6]. Proton minibeam radiation therapy (pMBRT) [7] is a novel cancer therapy that promises to overcome the limitation of RT posed by side effects. pM-BRT is based on a distinct dose delivery method using a strong spatial modulation of the dose to create alternating regions of high and low doses, in contrast to the homogeneous dose distributions used in standard RT ( Figure 1). Minibeam radiation therapy (MBRT) originated [8] at large synchrotron sources [9], facilities that provide intense kilovoltage X-ray beams with negligible beam divergence. The irradiation is performed with arrays of parallel and thin beams (0.5-1.0 mm wide). As normal tissue radiosensitivity depends on the volume irradiated [10], the use of such narrow beams may allow the use of higher and potentially curative doses in difficult-to-treat cancers. This contrasts with some other spatially fractionated RT techniques, such as GRID [11] and Lattice therapy [12], which are mainly limited to palliative treatment of bulky tumors.
Indeed, numerous experiments have shown an increase in the therapeutic index in radioresistant tumors. X-rays MBRT offers an extraordinary normal tissue sparing at very high doses (higher than 50 Gy in one fraction) [8,13,14] accompanied by a remarkable tumor control effectiveness [15,16].
In recent years, X-rays MBRT has been implemented outside large synchrotrons using low-cost equipment, such as small animal irradiators [17,18]. However, X-rays MBRT uses kV photon beams, which are inadequate for deep-seated tumor treatments, to reduce lateral scattering. The use of more energetic X-rays (megavoltage beams) would lead to reduced spatial modulation, thereby reducing the treatment benefits.
The use of protons in combination with MBRT allows to fully profit from the advantages of the spatial fractionation of the dose, which paves a direct path for optimal treatments in the future. Termed proton minibeam radiation therapy (pMBRT) [7], this method offers several advantages over X-ray-based MBRT, including negligible dose deposition in normal tissue after the Bragg peak, and the possibility of producing a uniform dose at depth, while maintaining modulation on the beam entrance side ( Figure 2). This represents an advantage over X-rays MBRT, where multiple interlacing fields are usually needed to achieve target dose homogenization, which is not only more error-prone but also technically more difficult.
The biological evaluations of pMBRT performed thus far have attested a remarkable reduction in normal tissue toxicity [19][20][21][22] and equivalent, or even superior, tumor control compared to standard proton therapy (PT) [23,24]. In the next sections the state of the art and perspectives of pMBRT will be reviewed.

Proton minibeam radiation therapy: state of the art
The first worldwide technical implementation of pM-BRT at a clinical center [25] was conducted in 2014 at the Institut Curie-Orsay proton therapy center (ICPO). This first implementation was performed at one of the passive beamlines of the ICPO using mechanical collimators. In 2015, Dilmanian et al. [26] confirmed the physical feasibility of the technique with a similar implementation. Technological advances in proton minibeam generation at clinical centers include its implementation at a pencil beam scanning system with optimized multislit collimators [27], a scanning dynamic collimator for spot-scanning proton minibeam production [28], and a novel nozzle design for magnetically-focused minibeams [29].  A series of radiobiological studies have demonstrated remarkable normal tissue toxicity reduction provided by pMBRT compared to standard PT.
Initially, reduced cell viability and an increased inflammatory response were observed after homogenous broad-beam irradiation, as compared to pM-BRT irradiation, in a three-dimensional (3D) human skin model [20]. Mouse ears were irradiated either with proton minibeams or proton broad-beam irradiation. The same deposited dose (60 Gy) resulted in a significantly higher degree of edema, erythema, or desquamation in animals receiving broad-beam irradiation compared to those receiving pMBRT [19].
In another pioneering experiment, the remarkable reduction in neurotoxicity provided by pMBRT was demonstrated [22]. The whole brains (except the olfactory bulb) of two groups (n = 8/each) of normal rats (7 weeks old) were irradiated: (i) the first group received a seamless (standard) proton irradiation, with high doses (25 Gy in one session); (ii) the second group received pMBRT irradiation with peak doses of 58 Gy per fraction (corresponding to the same average dose of 25 Gy). After a 6-month follow-up, a longitudinal magnetic resonance imaging (MRI) study using a 7-T small-animal MRI scanner, together with histological and immunochemical analyses, was conducted. Rats treated with conventional proton irradiation presented grade 3 skin damage (moist desquamation) and extensive brain damage, including necrosis, demyelination, astrogliosis, and microglial activation. On the contrary, pMBRT treatment resulted in no significant damage [22] ( Figure 3, left). It has also been demonstrated that pMBRT has no significant impact on cognitive, motor, or emotional processes after whole rat brain irradiations [21].
This reduction in neurotoxicity observed with pM-BRT compared to standard RT may improve the therapeutic index in cases that have good rates of tumor control but are accompanied with substantial side effects, such as pediatric astrocytoma. It also opens the door for a more efficient treatment of radioresistant tumors, such as high-grade gliomas (GBM), which remain one of the most challenging diagnoses in clinical oncology.
Indeed, in a previous study in glioma-bearing rats, pMBRT showed a tumor-control efficacy equal or superior to that of standard RT, and without the side effects observed in different rat glioma models, thus opening up the possibility of more aggressive irradiation schemes [23,24,30]. Besides, in some configurations, a 70% long-term survival rate (>6 months, free of tumor) was obtained in the pM-BRT group [24]. See Figure 3, right. The mean survival time was significantly higher than that in the standard PT group, despite the heterogeneous target dose coverage. Moreover, tumor sterilization was even achieved in cases where the beam spacing was large (3.5 mm) and large areas of the tumor received low (non-tumoricidal) doses [30]. This contradicts the classical paradigm of RT requiring the deposition of a lethal dose in every tumor cell for tumor ablation. Moreover, the fact that large areas (or even the whole brain) could be irradiated without significant side effects would allow the infiltrative nature of the tumor to be overcome, one of the major difficulties to be faced in glioma treatment.
The studies conducted to date have used a singlefraction irradiation scheme. Standard RT usually employs temporal fractionation of the dose to profit from both the reoxygenation of the tumor and redistribution of cells to more sensitive phases of the cell cycle (late G2 and M phase) [31]. Local tissue toxicity is also reduced due to the increased time available for the repair of sublethal damage, in addition to the repopulation of cell fractions [31]. Thus, temporal fractionation could also help and sum up to the inherent benefit of the spatial fractionation of the dose for normal tissue preservation. A recent study reported, for the first time, the response of glioma-bearing rats to a temporal fractionation in pMBRT delivered under a crossfire geometry. A significant increase in the median survival time was obtained when the dose was delivered over more than two sessions as opposed to in a single fraction. Moreover, 83% of longterm survivors had a total lack of tumoral cells, which is 3.3 times higher than that observed with conventional broad-beam irradiation, without excess of toxicity. This result could facilitate the path toward pM-BRT treatments.
Compared to direct damage by ionizing radiation, pMBRT seems to activate different biological mechanisms; with the immune system already observed to play an important role [32]. Other factors may include cell signaling effects, i.e., bystander-like effects [33], and the preferential effect on the tumoral versus normal vasculature [4]. The participation of these phenomena needs to be further investigated  [22]. Center: Dose distributions received by the tumor-bearing rats. Right: Tumor control. Survival curves of tumor-bearing rats: controls (black line); standard PT (green); pMBRT with quasi-homogeneous dose in the target (blue). An enhanced lifespan is observed. Taken from [24].
with a comprehensive approach. Additionally, it remains unclear how these effects are translated when the spatial dose distribution is modified. Indeed, no systematic evaluation of the influence of the beam width and spacing on the tissue response to spatially fractionated RT has been performed.

Perspectives: toward clinical trials
The ultimate goal is to translate and reproduce the findings of pMBRT from small animal experiments into the clinic.
Indeed, ten patients with large tumors have already been treated with proton GRID therapy (1-cm wide beams) [34] with palliative intent. These patients had tumors that could not be treated with photons, standard broad-beam irradiation, or photon GRID due to the tolerance of normal tissues. The improvement rate in terms of symptom relief and partial tumor regression was 80% for all patients, while acute side effects (grade 1/2) appeared in 50% of the patients, all of which were well-tolerated.
The experimental data gathered so far indicate that pMBRT can increase the therapeutic index, allowing the move from palliative to curative treatments. Patients thought to benefit the most from the advantages of pMBRT include those suffering from lymphomas, low-grade gliomas, or benign lesions, such as meningioma; these patients may benefit from the reduction in side effects while maintaining a comparable local control in diseased tissue. Moreover, in patients who are affected by radiationresistant tumors, such as GBM, osteosarcoma, chordoma, or chondrosarcoma, pMBRT may facilitate higher potentially curative doses in the tumor. The first therapeutic trials could include primitive brain tumors with a poor prognosis, such as recurrent gliomas.
The first theoretical investigation was conducted on possible treatment plans for two brain tumors, glioma and meningioma, in virtual patients derived from anonymized human patient imaging records. The pMBRT treatment plans were compared to standard (seamless) proton therapy plans, and they met the criteria recommended for clinically acceptable dose-volume histograms in organs-at-risk. Satisfactory homogeneity was achieved in the target, while a similar level of spatial dose modulation was obtained in normal tissues as that observed in preclinical studies [35]. Future research will be focused on unraveling the underlying mechanisms, how these effects are translated when the spatial distribution is modified, and linked to that, determining the best way to prescribe the dose in pMBRT, i.e., in terms of peak, valley, average dose, or potentially, a novel metric.

Conclusion
pMBRT is a novel cancer therapy approach that promises a high therapeutic index in difficult-to-treat cases, such as GBM. pMBRT is considered to be particularly appealing for use in pediatric oncology due to its remarkably low neurotoxicity. pMBRT offers the best balance between the broad beams employed in GRID and lattice therapy, both of which are currently limited to palliative treatments, and the extreme conditions of microbeam radiation therapy which difficult the passage of this latter technique to patients' treatments. The alliance of protons and MBRT allows the physical advantages of protons for therapy to be combined with the biological advantages of MBRT, promising an optimal treatment. Indeed, strong spatial dose modulation in pMBRT was shown to lead to a significant reduction in side effects while providing equivalent or superior tumor (high-grade glioma) control than standard homogeneous irradiation in small animal experiments. Despite its infancy, the biological data gathered thus far challenge the classical paradigm in RT and encourage the preparation of phase I clinical trials. This preparation is ongoing at Institut Curie.

Conflicts of interest
Authors have no conflict of interest to declare.

Acknowledgments
Y. Prezado warmly thanks the work of the current and former members of her team "New Approaches in Radiotherapy", the personnel of the Orsay proton therapy centre and the Experimental Radiotherapy platform of Institut Curie. Y. Prezado acknowledges the funding from European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 817908).

Perspectives : vers les essais cliniques
L'objectif ultime est de traduire et de reproduire en clinique les résultats de la pMBRT issus des expériences sur les petits animaux.