Tolerance to Dose Escalation in Minibeam Radiation Therapy Applied ...

RADIATION RESEARCH

184, 314–321 (2015)

0033-7587/15 $15.00 Ó2015 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR14018.1

Tolerance to Dose Escalation in Minibeam Radiation Therapy Applied to Normal Rat Brain: Long-Term Clinical, Radiological and Histopathological Analysis Yolanda Prezado,a,1 Pierre Deman,b,c Pascale Varlet,d Gregory Jouvion,e Silvia Gil,f Ce´line Le Clec’H,b He´le`ne Bernard,b Ge´raldine Le Ducb and Sukhena Sarunb a Imagerie et Mode´lisation pour la Neurobiologie et la Cance´rologie, UMR 8165, CNRS, Baˆt. 440, Campus d’Orsay, 91405, France; b ID17 Biomedical Beamline (ESRF), 38000, Grenoble, France; c INSERM U838, E´quipe 6, Grenoble Institut de Neurosciences, Grenoble, France; d Service de Neuropathologie Hoˆpital Sainte-Anne, 75014 Paris, France; e Institut Pasteur, Histopathologie Humaine et Mode`les Animaux, 75015 Paris, France; and f Centre d’estudis de Biofı´sica, Universidad Autońoma de Barcelona, Cerdanyola del Valles, Spain

deposit a therapeutic radiation dose to the tumor while keeping the surrounding normal tissue under tolerance doses. Despite significant advances in radiation therapy, the treatment of intrinsic radioresistant tumors, such as malignant gliomas or osteosarcomas, and of tumors close to a sensitive structure, such as the spinal cord, remains unsatisfactory due to the high radiosensitivity of surrounding normal tissues. To overcome this limitation, one may exploit dose delivery methods (i.e., fractionation, dose rate, etc.) that can influence the therapeutic index of radiation therapy (1). This approach is used by two new radiotherapy techniques under development at the Biomedical Beamline of the European Synchrotron Radiation Facility (ESRF): microbeam radiation therapy (MRT) and minibeam radiation therapy (MBRT). Arrays of parallel extremely intense ( 5,500 Gy/s) submillimetric beams are used. The X-ray spectrum use ranges from 50 to 500 keV, with a mean energy of around 99 keV (2). The beam widths range from 25 to 100 lm for MRT and from 500 to 700 lm for MBRT, exploring the limits of dose–volume effect [the smaller the field size, the higher the tolerance of the normal tissues (3– 5)]. In addition, the dose is spatially fractionated by summing submillimetric beams in a comb pattern: the dose profiles then consist of peaks and valleys with high doses (. 50 Gy) in the beam paths and low doses in the spaces between them (2). Preclinical MRT studies have clearly shown a higher tolerance for the rodent brain to very high-doses of radiation (6–11). It has been hypothesized that the sparing effect in the brain is due to the repair of the microscopic lesions by the minimally irradiated cells that are contiguous to the irradiated tissue slices (8). The valley doses are believed to play an essential role in tissue sparing: to guarantee tissue sparing valley doses must be kept below the tolerance level for a seamless irradiation, around 20 Gy (8). Moreover, a remarkable preferential tumor effect of such beam arrays at high doses has been observed in MRT (12–16). As yet,

Prezado, Y., Deman, P., Varlet, P., Jouvion, G., Gil, S., Le Clec’H, C., Bernard, H., Le Duc, G. and Sarun, S. Tolerance to Dose Escalation in Minibeam Radiation Therapy Applied to Normal Rat Brain: Long-Term Clinical, Radiological and Histopathological Analysis. Radiat. Res. 184, 314–321 (2015).

The major limitation to reaching a curative radiation dose in radioresistant tumors such as malignant gliomas is the high sensitivity to radiation and subsequent damage of the surrounding normal tissues. Novel dose delivery methods such as minibeam radiation therapy (MBRT) may help to overcome this limitation. MBRT utilizes a combination of spatial fractionation of the dose and submillimetric (600 lm) field sizes with an array (‘‘comb’’) of parallel thin beams (‘‘teeth’’). The dose profiles in MBRT consist of peaks and valleys. In contrast, the seamless irradiations of the several squared centimeter field sizes employed in standard radiotherapy result in homogeneous dose distributions (and consequently, flat dose profiles). The innovative dose delivery methods employed in MBRT, unlike standard radiation therapy, have demonstrated remarkable normal tissue sparing. In this pilot work, we investigated the tolerance of the rat brain after whole-brain MBRT irradiation. A dose escalation was used to study the tissue response as a function of dose, so that a threshold could be established: doses as high as 100 Gy in one fraction were still well tolerated by the rat brain. This finding suggests that MBRT may be used to deliver higher and potentially curative radiation doses in clinical practice. Ó 2015 by Radiation Research Society

INTRODUCTION

Radiotherapy is one of the major treatment modalities for cancer (1). The main challenge in radiation therapy is to 1 Address for correspondence: CNRS, IMNC, 15 rue Georges Clemenceau, Bat 440, Orsay, Ile de France 91405, France; e-mail: [email protected].

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however, there is no comprehensive and conclusive data on the biological mechanisms underlying the differential effects of MRT on tumor versus normal tissues. Possible explanations include: 1. Induction of a denudation of tumor vessel endothelium and a decrease in tumor blood volume (16, 17) and tumor hypoxia (17). 2. A direct impact of ionizing radiation on tumor cells occurring before the vascular effect (16) and the bystander effect/cellular communication (18). Tumor cells showed extensive migration within 24 h after MRT, whereas in normal skin, peak dose irradiated cells showed minimal evidence of migration up to 3.5 days after irradiation. The rapid intermixing of lethally irradiated cells with undamaged cells within the tumor may accentuate cell-mediated communication. The induction of DNA double-strand breaks and cell migration in MRT-irradiated glioma cells appear to be mediated by bystander effects (19). 3. A significant transcriptomic modulation for 316 genes in intracranial tumor tissue after MRT, which are mainly related to the regulation of cell cycle and to immune/ inflammatory response. Among those genes, 30 are specific to brain tumors, and are undetected in normal tissue before and after MRT (20). The main drawback of MRT is that its widespread clinical implementation is currently limited due to high-dose-rate requirements. Only the high photon fluxes available at synchrotrons can enable an irradiation process with sufficient speed (in a fraction of a second) to prevent artifacts caused by cardiosynchronous pulsations (21). The thicker beams used in MBRT overcome these difficulties. Since the MBRT dose profiles are not as vulnerable as those of MRT to beam smearing from cardiac pulsations, highdose rates are not needed. Therefore, it is conceptually possible to extend this technique by using modified X-ray equipment, creating an opportunity for its implementation in clinical practice (22, 23). In addition, the use of higher beam energies is feasible in MBRT ( 200 keV), which results in lower entrance doses to deposit the same integral dose in the tumor, despite the larger penumbra doses. Penumbras are about 40 lm for a mean energy of 100 keV at 2 cm depth in a water phantom (24). Furthermore, the clinical implementation of the interlaced method producing a homogeneous dose in the tumor (while normal tissue still benefits from the spatial fractionation of the dose) is technically easier than in MRT (25). These advantages have triggered the exploration of MBRT as a new radiotherapy approach. It has been recently shown that MBRT leads to a significant increase in the mean survival time of tumorbearing rats (25, 26). Radiation doses up to 100 Gy have been used (25). Investigation of normal tissue tolerances requires a dose escalation to study the brain response to different doses and to establish an end point. Some

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remarkable insights into brain preservation using MBRT were reported by Dilmanian et al. (22) and Deman et al. (26). In the first study, a brain area of 8 3 10.2 mm2 was irradiated with a unidirectional array of minibeams. Rats, observed for 7 months, gained weight normally and did not show signs of limb weakness at any time for entrance doses up to 170 Gy. However, no in vivo imaging follow-up of the animals was performed (22). Deman et al. (26) showed that the rat brain (n ¼ 4) tolerated well doses of 120 Gy at 1 cm depth in a unidirectional irradiation covering an area of 4 3 4 mm2. Unfortunately, in these two studies, a different number of beams was used, leading to different valley doses. To the best of our knowledge, a systematic study on whole-brain dose tolerances to a dose escalation in MBRT has never been performed. In this study, 18 Fischer 344 rats received MBRT wholebrain irradiation. The same rat strain (Fischer 344) was used in previous work (22, 25, 26) and has been widely used in radiobiological studies (27). This pilot study is a first exploration of the limits of MBRT regarding rat brain tolerances, to establish the advantage with respect to conventional methods. This information is needed to proceed towards comprehensive studies of tumor control effectiveness. The data obtained would also help to refine forthcoming investigations into normal tissue response to MBRT. MATERIALS AND METHODS Radiation Source and Exposure At the ID17 Biomedical Beamline, the radiation source consists of two wigglers with 15 and 12.5 cm period, respectively. A wiggler is an insertion device in a synchrotron composed of a series of magnets designed to laterally deflect a beam of electrons periodically inside the storage ring of a synchrotron. It delivers high-intensity kilovoltage energy X-ray beams. A more detailed technical description of the beamline layout can be found in Renier et al. (28). In MBRT, the beams are filtered, and the X-ray energy spectrum after filtering ranges from about 50–600 keV, with a mean energy around 99 keV (2). The beam is then spatially fractionated by a specifically developed white-beam chopper (29). For radiation exposure, the rats were positioned on a customdesigned holder and fixed by the teeth and ears on top of a 3-axis Kappa-type goniometer (Huber SE, Berching, Germany) (see Fig. 1). The maximum achievable field dimension is 3 mm height and 36 mm width. Since the beam height is very thin (3 mm), the animals are scanned vertically through the beam. A total of 18 Fischer 344 rats (180–250 grams; Charles River Laboratories, L’Arbresle, France) were exposed to a lateral beam of radiation with horizontal minibeams of 600 lm width and 1,200 lm center-to-center distance, covering the whole brain. For each animal, a radiochromic film HD 810 was positioned in front of the rat head. Inspection of the films was performed to ensure good radiation exposure quality. Figure 2 shows a schematic representation of the irradiation configuration, and a MBRT irradiated radiochromic film is shown. The radiochromic film instantly changes color on exposure to ionizing radiation in a proportional way to the deposited dose, and can then be considered a photograph of the MBRT irradiation. A pattern of high-dose areas or peaks (dark zones) interspersed between low-dose areas or valleys (light zones) is clearly observed.

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FIG. 1. Scheme of irradiation setup. Animals were positioned on top of a high-precision goniometer allowing six degrees of freedom.

This results in strongly inhomogeneous lateral dose profiles, as shown in Fig. 2. The rats were divided into six groups of doses (n ¼ 3). The dose prescriptions (at 1 cm depth) are shown in Table 1. Peak and valley doses as well as the corresponding integral doses (the product of volume of tissue irradiated and the absorbed dose) are detailed. The doses were delivered in one session. Peak doses as high as 400 Gy were used to compare the tissue response in MBRT with the usual doses used in MRT. The dose deposited on the rat brain was assessed following the methodology previously described by Prezado et al. (30). At all stages of the experiment (implantation and irradiation), the rats were anesthetized by 2% isoflurane inhalation. All operative procedures related to animal care strictly conformed to the Guidelines of the French Government with licenses 380825 and B3818510002 and the project was reviewed by the Internal Evaluation Committee for Animal Welfare and Rights of the ESRF. Animal Follow-Up The clinical status of each rat was checked twice a week. The relative weight compared to that at day 3 (weight at day D minus the weight at day 3 after irradiation, ‘‘D3’’) was calculated. The relative average body weight was then calculated for each group. The rats were euthanized if weight loss was greater than 30%.

In addition, anatomical magnetic resonance imaging (MRI) followup was performed in two animals per group at day 1, 30, 150 and 365 after irradiation. MRI was performed on a 7T preclinical magnet (Avance III system; Bruker Inc., Billerica, MA) using a volume/ surface cross coil configuration. The rats were anesthetized using 2% isoflurane. Two series were acquired: 1. Morphological T2-weighted (T2w) images [repetition time (TR): 4,000 ms; echo time (TE): 11 ms; resolution: 118 3 118 lm, 1 mm thick; field of view: 3 3 3 cm2]. 2. T1-weighted (T1w) turbo RARE SE sequences [TR ¼ 800 ms; TE ¼ 4.85 ms; resolution: 234 3 234 lm, 1 mm thick; field of view: 3 3 3 cm2] were acquired before and 5 min after injection of a bolus of Gd-DOTA (200 M/kg; Guerbet SA, Villepinte, France). Histological Analysis After euthanasia (intracardiac injection of 1 ml of Dolethal), the brain was quickly removed, snap frozen in liquid nitrogen-cooled isopentane and stored at 808C. Ten-micron brain frontal sections were cut using a cryostat (Microme HM560, Thermo Fisher Scientific, Inc., Waltham, MA), stained in hematoxylin and eosin (H&E) and examined under a microscope (Axioplan, Carl Zeiss Microscopy, Jena, Germany) jointly by a senior neuropathologist (PV) and a senior veterinary pathologist (GJ).

FIG. 2. Panel A: Representation of a MBRT rat irradiation. A radiochromic image shows the aspect of the radiation field at the entrance of the rat’s head. Panel B: Radiochromic film irradiated with MBRT. High-dose regions (dark zones) interspersed with low-dose regions (light zones) are observed. Panel C shows the resulting lateral dose profile, consisting in peaks and valleys.

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TABLE 1 Peak, Valley and Integral Doses Deposited at 1 Cm Depth in Whole-Rat Brain Irradiations Group 1 2 3 4 5 6

Peak doses (Gy) 50 100 150 200 250 400

6 6 6 6 6 6

4 8 12 16 20 32

Valley doses (Gy) 3.3 6.6 10.0 13.3 16.6 26.0

6 6 6 6 6 6

0.3 0.5 0.8 1.0 1.3 2.0

Integral dose (Gy L) (4.3 (8.2 (1.2 (1.6 (2.0 (3.3

6 6 6 6 6 6

0.4) 0.8) 0.1) 0.2) 0.2) 0.3)

3 3 3 3 3 3

105 104 104 104 104 104

Note. Doses were delivered in one fraction.

RESULTS

Survival Curves and Follow-Up

Survival curves are shown in Fig. 3. All of the rats that received peak doses higher than 200 Gy were euthanized due to visible symptoms of radiation dose intolerance such as apathy and inability to feed. Only one of the three rats that received a 200 Gy dose survived more than a few days (193 days), presenting occasional signs of hyperactivity. All rats that received 150 Gy peak radiation dose or less were alive 560 days after irradiation, behaving normally. They were euthanized once the study was determined to be finished. Figure 4 shows the average weight for the different groups versus time. The weight gain is less for the longterm survivor that received a 200 Gy dose than for the groups that received radiation doses equal to or lower than 150 Gy. The long-term survivor that received a 200 Gy dose had a lower mean weight than the others. Indeed, during the first 20 days after irradiation, the rat was losing weight, and then started to gain weight again. Comparative MRI and Follow-Up

The rats were followed up with a MRI on day 1, 30, 150 and 365 after irradiation. MRI performed one day after irradiation showed no signs of damage, even for the highest dose group (400 Gy). Edema and vascular leakage were visible 30 days after irradiation (Fig. 5A and B) in the cortex of one of the three rats receiving 150 Gy peak dose. Some signs of hemorrhage along the path of one of the minibeams (see Fig. 5C and D) were visible in the rats that received 100 Gy peak dose. MRI performed 150 days after irradiation showed some signs of hemorrhage on the beam paths and cerebrospinal fluid accumulation in the ventricles of the rats receiving 150 Gy (Fig. 6A and B). In the rats that received 100 Gy peak dose, only a hyposignal in the T2w image (Fig. 6C) was observed, which could correspond to a former partially repaired hemorrhage according to the low leakage to GdDOTA observed at this time (Fig. 6D). MRI was also performed 365 days after irradiation for long-term analysis. Histopathology studies were performed

FIG. 3. Survival curves corresponding to the six irradiated groups.

on the brain tissue extracted from the same rats sacrificed at the end of the study. The histopathological analysis revealed no abnormalities in the rats that received 50 Gy peak dose, except for the presence of a well-defined nodule of 1 mm diameter in the leptomeningeal frontal location in one of the rats from this group. This might correspond to a spontaneous neoplasm typical of Fischer rats (31). As for the rats that received 100 Gy peak dose, the MRI images at 365 days after irradiation indicated that the small damages observed in previous acquisitions appeared to have been repaired. A light hyposignal in T2w images (see Fig. 7A) was detected in only one rat, probably corresponding to healed hemorrhages, as in the subtraction of the T1w images with and without Gd contrast where no signs of leakage were observed (Fig. 7B). These results are consistent with the histopathological examination. At the microscopic level, an extensive unilateral plus bilateral calcification of cortical layer of the posterior part of primary motor cortex was indeed detected, associated with calcifications of the anterior part of the primary and secondary visual cortex and the main zone of the primary somatosensory cortex (Fig. 7C). In addition, a bilateral laminar calcification of the pyramidal cell layer of hippocampus from CA1 to CA3 and lateral and symmetric nodular calcification of the ventromedial part of the thalami were observed. MRI images 365 days after irradiation revealed important hemorrhages in the rats that received a 150 Gy dose (Fig. 8A and B). The histopathological analysis testifies to the presence of hematomas, as shown in Fig. 8C. In the worst case, a well-demarcated radionecrosis situated in the central medium paraventricular dorsal area of the thalami nuclei was observed. Table 2 summarizes the temporal evolution of MRI observations, as well as the histological findings for the rats receiving 100 and 150 Gy peak doses. DISCUSSION

To our knowledge, this is the first systematic and longterm study of the response for normal rat whole brain to a dose escalation in MBRT (peak doses from 50 to 400 Gy).

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FIG. 4. Long-term weight evolution of MBRT whole-brain-irradiated rats.

The status of the rats was checked regularly by observing external symptoms along with the acquisition of four series of MRI images, although comparative behavioral experiments were not done, this allowed us to assess the evolution of the animals with time. Radiation doses higher or equal to 200 Gy were not well tolerated by the animals, as shown by their early deaths or abnormal behavior. All 150 Gy irradiated rats survived long-term and behaved normally, but important and persistent brain damage was observed at MRI and histopathological

analyses. In particular, radionecrosis, considered the most serious side effect of radiation exposure to the brain (5), was observed in one of the rats from this group. Previous studies where the rat brain was exposed to a single dose of unsegmented X-ray beams using a semicircular lead aperture of 10 mm radius (half the rat brain) found end point (50%), ED50, values for necrosis in white matter at 39 weeks after 23 Gy irradiation and at 52 weeks after 21 Gy irradiation (32, 33).

FIG. 5. MRI images taken 30 days after irradiation with 150 Gy (panels A and B) and 100 Gy (panels C and D) peak radiation doses. The worst cases of each group are shown. Panels A and B show the presence of edema in the T2w image (panel A) and vascular leakage (B) in group 3 (150 Gy). Panels C and D show T2w (panel C) and T1w (panel D) images of one of the rats from group 2 (100 Gy), showing some evidence of hemorrhage.


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FIG. 6. MRI images taken 150 days after irradiation. The worst cases of each group are shown. Panel A and B show T2w (panel A) and T1w (panel B) images of one of the rats from group 3 (150 Gy peak dose), where hemorrhage in the trajectory of one of the beams and accumulation of cerebrospinal fluid in the ventricles are identified. Panels C and D show the presence of a hyposignal area in the T2w image (panel C) and vascular leakage (panel D) in the worst case of group 2 (100 Gy peak dose).

In contrast, the analysis of group 2 (100 Gy) showed transitory and less important radiation-induced effects. Few microcalcifications were observed in the long term (365 days), without any external symptoms. Microcalcifications are the most commonly seen neuroradiologic abnormality in cancer patients receiving whole-brain radiation therapy at

doses greater than 20 Gy in one fraction. It is uncertain whether these lesions produce any abnormal clinical manifestations in patients (34). No significant deleterious effects were observed in the group treated with peak radiation doses of 50 Gy, with the exception of a well-defined nodule, probably corresponding

FIG. 7. Analysis of group 2 (100 Gy peak doses) 365 days after irradiation. The worst case is shown. Panels A and B show T2w (panel A) and T1w (panel B) images. A light hyposignal is visible in the T2w image, while no leakage is observed in subtraction T1w images with and without Gd contrast. In panel C, calcifications in different parts of the cortex are visible.

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FIG. 8. T2w (panel A) and subtraction T1w images with and without Gd contrast 5 (panel B) images of one of the rats from group 3 (150 Gy peak dose) 365 days after irradiation, indicating the presence of hemorrhage. A large hematoma from CA2 to CA3 areas of the hypocampus (panel C) and a radionecrosis in the central medium paraventricular dorsal area of the thalami nuclei (panel D) are observed in the histopathological study.

to a spontaneous neoplasm. This dose (50 Gy) is already a very high dose for a single-dose fractionation scheme. The most common dose/fractionation schedule used in wholebrain irradiation is 30 Gy delivered in 10 fractions over the course of 2 weeks (35). TABLE 2 Temporal Evolution of MRI and Histological Findings MRI follow-up (n ¼ 2) Time 1 day 30 days

150 days

365 days

Group 2 (100 Gy) No apparent changes (n ¼ 2). Some signs of hemorrhage along some beam paths (n ¼ 2). Small vascular damage (n ¼ 2).

No apparent changes (n ¼ 2).

Group 3 (150 Gy) No apparent changes (n ¼ 2). Edema, vascular damage in cortex (n ¼ 2). Hemorrhage along some beam paths, cerebrospinal fluid accumulation in ventricles (n ¼ 2). Important hemorrhage (n ¼ 2).

Despite being a pilot study, this work provides, clear evidence of the shift of the normal tissue complication probability curve in MBRT (whole-brain irradiation). These results also provide the basis for continued study of this technique. In addition, an upper threshold (150 Gy peak radiation dose) was established. This information will serve to guide forthcoming biological experiments. The high tissue resistance observed with respect to conventional seamless irradiation opens the door for a more effective treatment of intrinsic radioresistant tumors such as malignant gliomas (26) or osteosarcomas (36). It might also allow retreatment of the brain months or years after the initial radiation treatment(s), as well as a lower risk treatment for pediatric brain tumors. Because this technique could be extended outside large synchrotron sources, such as ESRF, it is clearly an asset. MBRT could be implemented in hospitals with costeffective X-ray equipment such as modified X-ray tube, or with a compact synchrotron. Received: January 7, 2015; accepted: July 2, 2015; published online: August 18, 2015

REFERENCES Histology examination (n ¼ 2) Microcalcifications (n ¼ 2) in hippocampi.

Hematomes (n ¼ 2); calcifications in hippocampi (n ¼ 2); radionecrosis (n ¼ 1).

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