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Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness

This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2012 Phys. Med. Biol. 57 5889 (http://iopscience.iop.org/0031-9155/57/19/5889) View the table of contents for this issue, or go to the journal homepage for more

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IOP PUBLISHING Phys. Med. Biol. 57 (2012) 58895907

PHYSICS IN MEDICINE AND BIOLOGY

doi:10.1088/0031-9155/57/19/5889

Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness


T E Schmid 1,5 , C Greubel 2 , V Hable 2 , O Zlobinskaya 1 , D Michalski 1 , S Girst 2 , C Siebenwirth 2 , E Schmid 3 , M Molls 1 , G Multhoff 1,4 and G Dollinger 2
1 2

Department of Radiation Oncology, Technische Universit t M nchen, Germany a u Institute for Applied Physics and Metrology, Universit t der Bundeswehr M nchen Neubiberg, a u Germany 3 Institute for Cell Biology, Ludwig-Maximilians Universit t M nchen, Germany a u 4 Helmholtz Zentrum M nchen, CCGInnate Immunity in Tumor Biology, Germany u E-mail: T.E.Schmid@lrz.tu-muenchen.de

Received 28 November 2011, in nal form 1 August 2012 Published 7 September 2012 Online at stacks.iop.org/PMB/57/5889 Abstract This study shows that enhanced radiobiological effectiveness (RBE) values can be generated focusing low linear energy transfer (LET) radiation and thus changing the microdose distribution. 20 MeV protons (LET = 2.65 keV m1) are focused to submicrometer diameter at the ion microprobe superconducting nanoprobe for applied nuclear (Kern) physics experiments of the Munich tandem accelerator. The RBE values, as determined by measuring micronuclei (RBEMN = 1.48 0.07) and dicentrics (RBED = 1.92 0.15), in humanhamster hybrid (AL) cells are signicantly higher when 117 protons were focused to a submicrometer irradiation eld within a 5.4 5.4 m2 matrix compared to quasi homogeneous in a 1 1 m2 matrix applied protons (RBEMN = 1.28 0.07; RBED = 1.41 0.14) at the same average dose of 1.7 Gy. The RBE values are normalized to standard 70 kV (dicentrics) or 200 kV (micronuclei) x-ray irradiation. The 117 protons applied per point deposit the same amount of energy like a 12C ion with 55 MeV total energy (4.48 MeV u1). The enhancements are about half of that obtained for 12C ions (RBEMN = 2.20 0.06 and RBED = 3.21 0.10) and they are attributed to intertrack interactions of the induced damages. The measured RBE values show differences from predictions of the local effect model (LEM III) that is used to calculate RBE values for irradiation plans to treat tumors with high LET particles.

Author to whom any correspondence should be addressed.


2012 Institute of Physics and Engineering in Medicine Printed in the UK & the USA 5889

0031-9155/12/195889+19$33.00

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1. Introduction Radiotherapy is one of the main clinical approaches to treat solid tumors. Proton and heavy ion irradiation is superior to x-ray due to enhanced linear energy transfer (LET) at the end of ion range forming the Bragg peak. Thus the dose required for tumor control can be applied at minimized energy and dose deposition into the surrounding healthy tissue. Therefore, particle tumor treatment is expected to improve therapeutic outcome by a better prevention of acute and chronic side effects and by lowering the risk for secondary tumor induction (Durante and Loefer 2010, Karger and Jakel 2007, Schulz-Ertner 2009, Newhauser and Durante 2011). In general, it is especially the low energy ions that have a high LET. The question whether high LET particles, such as high energy carbon ions, are superior to low LET particles and high energy proton beams is still a matter of debate (Durante and Loefer 2010). The optimum situation may be obtained when the differential (radiobiological effectiveness) RBE effect is optimized where the RBE inside the tumor is enhanced compared to low LET particles but keeping the RBE in the healthy tissue close to low. An optimization of this effect, however, requires an accurate knowledge of how the RBE for tumor control and also for acute as well as long term side effects depend on the details of energy deposition and thus from the LET and the specic energy of the various ions. In order to address the question which ion ts best for clinical application, a better understanding of the relation between RBE values and the ion species and their energy is necessary. Furthermore, knowledge on energy deposition and denition of biological endpoints are of major interest for an optimal tumor control when using high LET particles. High LET particles deposit therapy relevant doses of a few gray to a cell nucleus in only a few hits, whereas several hundred hits are necessary for low LET particles. As a consequence the energy deposition for high LET radiation is much more inhomogeneous in time and space than that of low LET radiation. The energy deposition of a single ion hit into a biological cell runs on the femtosecond to picoseconds time scale, while the spatial dose distribution peaks at the center of the ion track. The main question to be solved is how the RBE is affected by microdose distribution and the time course of dose deposition in order to obtain reliable knowledge on the RBE for any ion species in dependence of its energy. In earlier studies we have tested whether the RBE changes when the total dose to a cell sample is deposited by low LET particles (20 MeV protons, LET = 2.6 keV m1) that are bunched in time to one nanosecond only. We proved experimentally that with respect to micronuclei (MN) induction in HeLa cells (Schmid et al 2009) or in keratinocytes in a 3D skin model (Schmid et al 2010b) and chromosome aberration induction in monolayers of human-hamster hybrid (AL) cells (Schmid et al 2011) there is no change in RBE caused by the nanosecond time structure of dose deposition compared to continuous dose deposition within 100 ms. Obviously, the time course has no inuence on the RBE as long as no signicant overlap of the microdose distributions of the individual proton tracks occurs. These results are also most relevant for future applications of laser-based particle accelerators where the dose deposition into the tumor is expected to be performed with only a small number of pulses of 1 ns duration each. First RBE measurements with laser accelerated protons show also no enhancement (Yogo et al 2011, Doria et al 2012) while their energy spectra with a typical relative width of about 30% result in a time course of about 1020 ns duration, which is at least one order of magnitude larger than in the pulsed beam experiments mentioned above. In general, the RBE depends on the microdose distribution formed by a single ion track and the areal ion track density determining the total dose. There are approaches to model radiobiological endpoints based directly on the microdose distribution (Friedland et al 2011), the three-dimensional dose distribution with nanometer resolution deposited by a single

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particle. Several reports try to describe RBE dependence based on (amorphous) track structure models (Scholz and Kraft 1996, Scholz et al 2006, Els sser and Scholz 2006, 2007, Els sser a a et al 2008b, 2010, Friedrich et al 2012), which describe only the expectation value of the deposited dose depending on the distance to the ion track. The local effect model (LEM) is able to provide good estimates of clinical RBE data (Karger et al 2006) especially for the different RBE in plateau and Bragg peak region. But on the one hand there are still discrepancies between calculations and experiments: Karger et al (2006) reports a systematic underestimation of RBE in the Bragg peak of carbon ions by 25%. On the other hand, even if LEM can describe RBE of several ions, a detailed understanding of the relationship between microdose distribution and RBE is still missing since Els sser et al (2008a) demonstrated that a the impact of different track structure models, and thus different assumptions of microdose distribution, can be compensated by other parameters of the LEM models. Here, we want to elucidate how the RBE depends on the spatial distribution of dose deposition. We compare the RBE from spot like dose distributions obtained from focused low LET irradiation, where the substantial fraction of the total energy is deposited in small channels with that from statistically distributed spots (quasi homogeneous irradiation) of low LET irradiation. As low LET particles we use 20 MeV protons that can be focused to submicrometer beam size using the (superconducting nanoprobe for applied nuclear (Kern) physics experiments) SNAKE facility of the Munich tandem accelerator to severely enhance the local doses at the beam spots compared to quasi homogeneous proton irradiation. We use 117 protons applied on a single spot that deposits the same amount of energy like a single 12 C ion with 55 MeV total energy (4.48 MeV u1) and irradiate a matrix pattern to compare the effects with a 55 MeV 12C matrix irradiation and with randomly applied protons (quasi homogeneous mode) of the same average dose. The dose distribution of the focused protons changes with decreasing beam diameter until a microdose distribution similar to that of a high LET particle of the same velocity would be obtained in the limit of zero diameter focus. Beside the limited beam size that is measured to be between 0.3 and 1.0 m, the main difference to the dose deposition of a single, high LET particle is the time course of dose deposition because in our experiment the 117 protons will be deposited within about 10 ms that is many orders of magnitude slower as the femtosecond scale that is relevant for the energy transfer of a single high LET particle. It is also much slower than considered in the cluster beam effect studies (Fourkal et al 2011), and the predictions of damage effects (Kreipl et al 2009) and thus these theoretical studies cannot be compared with this actual experiment. Nevertheless, in our current study we will test if we can nd a signicant change of RBE values under our experimental conditions. 2. Materials and methods 2.1. Cell culture For the present experiments, cells of the AL cell line were used which were established in our laboratory to investigate structural or functional chromosome aberrations in monolayer after exposure to ionizing or non-ionizing radiation qualities. For example, the cells were recently used as our established model for studying the effectiveness of 20 MeV protons at nanosecond pulse lengths in producing chromosome aberrations (Schmid et al 2011). These cells contain a standard set of Chinese hamster ovary-K1 chromosomes plus a single human chromosome 11. Our previous chromosome analysis showed a modal number of 21 chromosomes (85% of analyzed cells), whereas 15% of the analyzed cells had a loss or gain of chromosomes (Schmid et al 2011). The AL cells were grown as monolayer cultures in RPMI-1640 medium,

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supplemented with 16% fetal calf serum, 100 units of penicillin and 100 g of streptomycin per ml of culture medium. The AL cell monolayers were maintained in a humidied atmosphere containing 5% CO2 and 95% air at 37 C. Under these growth conditions, the cells had a doubling time of 18 h. 2.2. Cell irradiation with 20 MeV protons and 55 MeV 12C ions For each experiment of exposure of AL cells to 20 MeV protons (LET = 2.65 keV m1) or 55 MeV 12C ions (4.48 MeV u1, LET = 310 keV m1), a frozen aliquot from the same stock culture was thawed and seeded as cultures with exponentially growing unsynchronized cells into special irradiation containers. A detailed description of the construction of these irradiation containers for cell irradiation has been given previously (Hauptner et al 2006, Schmid et al 2009). The irradiation experiments have been carried out at the ion microprobe SNAKE (Datzmann et al 2001) of the 14 MV Munich tandem accelerator which has been adopted for use in biological analysis (Hauptner et al 2004, 2006). This unique irradiation setup, which allows the microirradiation of dened cell nuclei with single or counted protons, has already been described in detail (Greubel et al 2008). In brief, irradiation containers were built by stretching and clamping a 6 m Mylar foil between two stainless steel plates. About 24 h before irradiation experiments, about 30 000 AL cells were trypsinized, carefully resuspended in fresh medium, and grown as monolayer cultures on the Mylar foil pre-treated with Cell-TAK (BD Bioscience). Shortly before irradiation, about half of the culture medium was removed and the irradiation containers were tightly closed by clamping a second 6 m Mylar foil. During irradiation at room temperature, the container with the cell monolayer, mounted directly behind the beam exit nozzle, was placed vertically in the focal plane of the ion microbeam, i.e. the cells were not covered by medium, but residual medium in a reservoir ensured a saturated atmosphere. In this irradiation geometry, the ions are focused and transported in vacuum to the beam exit nozzle covered with 7.5 m kapton foil. After 30 m of air ions transmit the 6 m thick cell carrier foil before hitting the cells. Thus the water equivalent thickness of transmitted material is 16 m. Typically, the total time required for sample preparation and irradiation, i.e. the time outside of the CO2 incubator, was less than 15 min including about 12 min in the upright position. Under these conditions, there were no differences in the number of chromosome aberrations in non-irradiated cells and cell samples remaining in the incubator (Schmid et al 2011). To demonstrate reproducibility as well as the inter-test variability, three independent experiments for MN and two independent experiments for dicentrics were carried out, mostly with up to three replicates for exposure of AL cells to 20 MeV protons or 55 MeV (4.48 MeV u1) 12C ions. Irradiation was done not targeted to single cells but in regular pattern on a central area of 3 mm diameter. Counted protons were applied in a focused mode owing 5.4 5.4 m2 matrix with 117 protons per matrix point or a quasi homogeneous mode with 4 protons per point in a 1 1 m2 matrix. For 12C ion irradiation the same 5.4 5.4 m2 matrix was irradiated but with one ion per matrix point. An upper limit of 1.0 m (FWHM; full width at half maximum) for the proton beam spot size could be determined from microscopic image of the beam spot on a scintillator, the lower limit of 0.3 m (FWHM) is given by the dimension of the object and its demagnication used for ion microbeam preparation. The shape of the beam spot is estimated to be Gaussian like that observed for heavier ions, which allow a direct measurement by nuclear track detectors (Dollinger et al 2005). Since the same experimental setup was used to prepare the microbeam for irradiation with counted protons in focused and quasi homogeneous mode as well as single 12C ions (all delivering 1.7 Gy), potential systematic errors were compensated by direct comparison of the

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results from the different irradiation modes. Using counted ions allows a very precise dose application with nearly no uctuations. The systematic dose uncertainty is only given by the uncertainty of the LET of used ions and the calibrations of the scanning unit and is estimated to be less than 3%. 2.3. X-ray irradiation for establishing reference doseresponse relationship To obtain a reference doseresponse curve for MN, irradiation of AL cells at room temperature of 2022 C was performed at the new x-ray calibration facility of our Department of Radiation Oncology in Munich. Using the same irradiation containers under identical conditions that were used for proton or carbon irradiation, cells were exposed to 200 kV x-rays (RS225, Gulmay Medical, Surrey, UK) with a dose rate of 0.88 Gy min1 (15 mA, 0.8 mm Be and 0.5 mm Cu lter) and a source-cell distance of 50 cm using a eld of 20 20 cm2. Dosimetry was performed using standard dosimetry protocols as the IAEA-TRS 398 Code of Practice and, additionally, radiochromic lm-based dosimetry. Gafchromic EBT2-lm, a self-developing radiochromic lm, was selected for dose verication after irradiation. Films were digitized with an EPSON Perfection V700 Photo scanner using the manufacturers scan software in professional mode with all image correction features turned off. Forty-eight-bit RGB tiff images with resolution of 1200 dpi were acquired from lms oriented in landscape format at the earliest 40 h after irradiation to minimize uncertainty in optical density due to post-irradiation growth. As a reference for the doseresponse relationship of dicentrics, our previously established x-ray curve for irradiation of AL cells at room temperature of 2022 C has been used. This doseresponse curve for dicentrics was determined from pooled data of two independently performed experiments (Schmid et al 2011). Cells were exposed to 70 kV x-rays at the former x-ray calibration facility of our Department of Radiation Oncology in Munich (Philips RT100; Philips Medical Systems, Eindhoven, The Netherlands) using a dose rate of approximately 1 Gy min1 (10 mA, 2 mm Al, HVL = 1.9 Al) and a source-cell distance of 30 cm to a eld of 20 20 cm2. The absorbed doses in AL cells of both experiments ranged from 0.5 to 4 Gy. 2.4. Scoring of micronuclei Immediately after irradiation, a 1 mm2 biopsy punch from the central part of the irradiated area was cut out, the irradiated AL cells were trypsinized and reseeded in fresh medium containing 10 g ml1 Cytochalasin B and the AL cells were incubated at 37 C in a humidied atmosphere of 5% CO2 in air for a further time of 24 h. Cells were xed with 2% paraformaldehyde and stained with acridine orange. Applying this cytokinesis-block (CB), the frequency of MN could precisely be detected in the rst division cycle after irradiation and easily identied from their binucleate appearance. From each sample (non-irradiated or irradiated) at least 500 binucleated CB cells with well-preserved cytoplasm were analyzed. The number of MN was counted solely if CB cells contained detached MN in the cytoplasm. 2.5. Scoring of dicentrics Immediately after irradiation, a 1 mm2 biopsy punch from the central part of the irradiated area was cut out, the irradiated AL cells were trypsinized and reseeded in 4 ml RPMI-1640 medium supplemented with 20% fetal calf serum and antibiotics (penicillin/streptomycin) and incubated at 37 C in a humidied atmosphere of 5% CO2 in air. Colcemid at the concentration of 0.1 g ml1 was present in the cultures during the last 4 h of a total incubation period of 20 h.

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By the presence of colcemid during this incubation period, a synchronization of the cycling AL cells that were at the G1-phase of the cell cycle during irradiation could be achieved, whereas AL cells irradiated at the S- and G2-phases could not be arrested by colcemid at the metaphase because these cells had already nished their rst cell cycle after irradiation. This colcemid technique has been standardized in our former experiments to identify the presence of the single human chromosome of AL cells in rst metaphases by uniformly stained sister chromatids (unpublished data), using the combined FISH (uorescence in situ hybridization) painting and harlequin staining for cell cycle-controlled chromosome analysis in human lymphocytes (Kulka et al 1995). Subsequently, the cultures were centrifuged, the supernatant removed and replaced by a hypotonic potassium chloride solution (0.075 M) at 37 C for 10 min. All subsequent steps were carried out at room temperature. The cells were xed, i.e. the culture medium was removed and the cell pellet resuspended in a mixture of three parts of methanol and one part of glacial acetic acid. Aliquots of xative were added drop by drop on individual slides. After air drying, the cells were stained with 2% acetic Orcein for 10 min. By using this technique, the cytoplasm of the cells remained well preserved and chromosome loss due to preparation procedure was avoided. Since the measured dicentric yields are generally used as the most characteristic biological endpoint of radiation-induced chromosome type aberrations, only the data for dicentrics were used in the quantitative analysis, although the yields of centric rings and supernumerary acentric fragments were also determined. The culture conditions ensured that dicentrics as structural aberrations of the chromosome-type could be analyzed exclusively in metaphases of the rst cell cycle following radiation exposure. Applying this method to analyze chromosome aberrations was necessary because former ndings in synchronized CHO cells indicated that cells irradiated in early S phase were 23 times as sensitive to damage as cells exposed in G1 (Yu and Sinclair 1967, Dewey et al 1970). Chromosome preparation was carried out according to our standardized laboratory procedure originally described for human lymphocytes (IAEA 1986, 2001). All object slides were coded. Only complete cell nuclei containing the modal number of 21 chromosomes were analyzed for dicentrics. 2.6. RBE calculation For RBE determination dose response curves of reference radiation were used. A linear quadratic model (y = c + D + D2) was tted both to MN and to dicentric data by weighted least-squares approximation. The reciprocal variances of the measured data points (1/ 2) are used as weighting factors. The RBE is dened as the ratio of the dose of the reference radiation and the applied dose of the test radiation quality necessary to induce the same effect. The necessary dose of reference radiation is determined by inverting the tted dose effect curve. As the t parameters are highly correlated for calculation of RBE errors, not only the diagonal elements of the covariance matrix, the parameter errors, are taken into account but also the non-diagonal elements, describing the correlation of parameter. 2.7. -H2AX and CENP-F staining Cells were xed with 2% paraformaldehyde 1 h after irradiation (Schmid et al 2010a). The cells were permeabilized by three washing steps in phosphate buffered saline (PBS) + 0.15% Triton X-100 (both reagents from Sigma-Aldrich, Gillingham, Dorset, UK), each for 5 min. The irradiated areas were covered with 50 l of mouse anti- -H2AX antibody (Upstate), diluted 1:200 in PBS and incubated at 4 C overnight in a humidied incubator. Unbound antibody was removed by several washing and re-blocking steps before goat-F(ab)2-anti-mouse antibody

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(Alexa488 labeled, 1:500, Invitrogen) was applied as secondary antibody. Finally, DNA was stained with DAPI and cells were mounted with vectashield and a cover slip. Microscopic -H2AX foci were immunolocalized and images were acquired using epiuorescence sectioning microscopy (Zeiss Axiovert 200 M), a LCI Plan Neouar 63 objective with 1.3 numerical aperture and a Zeiss AxioCam MRm resulting in a pixel size of 0.10 0.10 m2. The Alexa488 and DAPI digital images were captured serially. Z-stacks of at least 15 different positions were taken from each sample with at least 15 slices per stack (2025 cells per stack) at different positions. 2.8. Statistics A null hypothesis that the observed differences between the mean frequencies of MN or dicentrics from up to three replicates ( SE) obtained from replicated experiments for exposure of AL cells to 20 MeV protons at different exposure conditions or to 55 MeV (4.48 MeV u1) 12 C ions are not different was tested by using the z-test statistics. Since the conditions for the applications of the normal probability distribution hold for these data (i.e. both sample sizes are much larger than 100 cells and the products of sample sizes and proportions are greater than ve), this statistical procedure could be applied. A difference at the two-sided p-value <0.05 was considered statistically signicant. 2.9. Calculation of RBE by LEM III The local effect model as described by Els sser et al (2008b) is used to predict the RBE for a MN and dicentrics of focused protons. According to this model the local dose distribution is calculated for all three irradiation qualitiescarbon ions, focused protons applied matrix wise and quasi homogeneous proton irradiationusing the modied radial dose prole of the work of Els sser which takes into account the diffusion of radicals (Els sser et al 2008b, Els sser a a a and Scholz 2007). This dose prole is given by a two dimensional Gaussian with a width of = 4 nm which is convoluted with the following initial prole: 2 B/rmin r < rmin rmin < r < rmax (1) D0 (r) = B/r 0 rmax < r rmin = v/c 40 nm, where v is the speed of the particle and c the speed of light in vacuum, rmax = 0.062 m (EA/1 MeV)1.7, EA is the energy per nucleon and B is constant to ensure energy conservation. rmax is approximately 10.1 m for 20 MeV protons and 0.825 m for 55 MeV 12C ions (EA = 4.48 MeV u1). For the local dose distribution on a rectangular area of interest of 2.7 2.7 m2 with 10800 10800 pixels the contributions of ion hits are summed up. The length of this area of interest is half the point distance in matrix irradiation mode. In this mode one matrix point is located in the corner of this area. Thus due to the symmetry of the irradiation pattern this dose distribution represents the dose distribution of the total irradiated area. For focused protons applied matrix wise the hit positions of 117 protons at each matrix point are generated randomly following a Gaussian distribution of the areal distribution of ion hits. The FWHM of the Gaussian distribution is varied between 1 nm and 10 m. To cover all contributing ion hits all matrix points with a distance smaller than 18.9 m to any pixel of the area of interest are included into the calculation. The dose distribution for carbon irradiation is generated similarly assuming only one particle in the corner of the area of interest. Due to the small rmax of carbons ions ion hits at all other matrix point give no dose contribution to the area of interest. For quasi homogeneous proton irradiation 29 protons are distributed randomly inside

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(a)

(b)

(c)

Figure 1. The appearance of AL cells with immunouorescence of -H2AX foci obtained 1 h post irradiation with an average dose of 1.7 Gy (scale bar: 10 m): (a) foci pattern from 55 MeV 12C ions applied in a 5.4 5.4 m2 matrix pattern, (b) foci pattern from 20 MeV protons applied quasi homogeneously, (c) foci pattern from 117 protons (20 MeV) focused to 650 350 nm and applied in the same 5.4 5.4 m2 matrix pattern as for 12C ions.

the area of interest and 2337 protons outside within a circle with radius of 13.7 m. Thus all proton hits with a distance of less than 11 m to any pixel are included and the proton density, the number of protons per area, is the same as in matrix mode. The dose effect curve, in our case the effect is not a lethal event but the induction of MN or dicentrics, is parameterized by Nx (D) = (D) c + D + D2 c + Dt + Dt2 + (D Dt ) ( + 2Dt ) for D < Dt for D > Dt . (2)

For doses smaller the threshold dose, Dt, the dose effect is linear quadratic (parameterized as usual by constants and ) with an offset c accounting for number of MN or dicentrics in non-irradiated cells. For doses above Dt the dependence is linear and the curve and its slope are continuous as previously described (Scholz and Kraft 1996, Els sser et al 2008b). (D) a is the damage enhancement factor describing the number of additional double strand breaks (DSBs) formed by two single strand breaks separated by less than 25 base pairs (Els sser and a Scholz 2007). The parameter , and c were extracted from the measured dose effect curve (gure 3) for induction of MN or dicentrics by x-ray irradiation, respectively. For both endpoint Dt is adjusted to Dt = 6 Gy to reproduce the measured RBE of homogeneous proton irradiation. The areal effect density, v(D) = Nx(D)/A, is calculated for each pixel. A is the pixel area. The effect density is summed up over the area of interest to calculate the total effect of the given irradiation quality. The photon dose Dx-ray that is necessary to induce the same effect is calculated by inverting the dose effect curves of equation (2) and the RBE = Dx-ray/D is calculated. 3. Results 3.1. Spatial distribution of -H2AX foci In gure 1, the appearance of AL cells with immunouorescence of the -H2AX foci pattern obtained 1 h after irradiation by 1.7 Gy average dose is shown. The cells of gure 1(a) were irradiated by 55 MeV 12C ions in a 5.4 5.4 m2 matrix that can be well recognized by the -H2AX foci pattern in a major fraction of cells. In contrast to this irradiation condition, all cells of the quasi homogeneously applied 20 MeV proton irradiation show a random

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Table 1. Results of experiments I, II and III: frequency ( SE) and intercellular distribution of micronuclei (MN) in cytokinesis-blocked (CB) binucleate AL cells following exposure to 1.7 Gy of 55 MeV 12C ions (C ions) or 20 MeV protons either at quasi homogeneous exposure condition (Phom) or in a focused mode owing 5.4 5.4 m2 matrix (P5.4 5.4); 2/y, dispersion ratio; u-value, test quantity.

Intercellular distribution of MN Radiation quality Control C ions C ions C ions Phom Phom Phom P5.4 5.4 P5.4 5.4 P5.4 5.4 C ions C ions C ions Phom Phom Phom P5.4 5.4 P5.4 5.4 P5.4 5.4 C ions C ions C ions Phom P5.4 5.4 Cells scored 1000 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 MN per CB cell 0.022 0.005 0.484 0.488 0.528 0.260 0.222 0.296 0.328 0.274 0.342 0.536 0.488 0.524 0.208 0.260 0.280 0.236 0.314 0.298 0.496 0.486 0.502 0.250 0.316 0 980 1 18 155 171 166 89 81 93 92 85 94 176 156 169 72 90 96 75 101 88 153 159 168 85 94 2 2 39 27 43 16 12 20 24 20 25 34 41 39 10 14 19 17 13 23 40 36 31 17 23 3 0 3 6 4 3 2 5 8 4 9 8 2 5 4 4 2 3 10 5 5 4 7 2 6 2/y 1.161 0.915 0.879 0.890 1.127 1.105 1.179 1.260 1.196 1.269 0.899 0.899 0.890 1.218 1.142 1.079 1.207 1.236 1.215 0.950 0.911 0.914 1.120 1.205 u-value 3.60 1.35 1.92 1.74 2.01 1.66 2.84 4.12 3.11 4.26 1.60 1.60 1.76 3.45 2.26 1.26 3.29 3.74 3.40 0.80 1.41 1.36 1.91 3.26

Experiment I 0.030 303 0.029 296 0.031 287 0.024 392 0.022 405 0.026 382 0.029 376 0.026 391 0.029 372 Experiment II 0.031 282 0.030 301 0.031 287 0.023 414 0.024 392 0.025 383 0.024 405 0.028 376 0.027 384 Experiment III 0.031 302 0.030 301 0.030 294 0.024 396 0.028 377

distribution of -H2AX foci (gure 1(b)). The 5.4 5.4 m2 matrix is also visible in many cells that were irradiated with this matrix applying 117 protons per spot. The localized spots (gure 1(c)) being slightly larger than in the carbon matrix irradiation case (gure 1(a)) reect the well-focused proton irradiation with a spot size between 0.3 and 1.0 m. 3.2. Micronuclei and dicentrics data Table 1 summarizes the results of the MN test in AL cells after quasi homogeneous and a 5.4 5.4 m2 matrix spot exposure to 1.7 Gy of 20 MeV protons as well as to 55 MeV 12C ions derived from three independent experiments. The corresponding intercellular distributions of MN for all three radiation qualities are given together with the results of the tests for Poisson distributions. The corresponding yields of dicentrics and their intercellular distribution in AL cells exposed to the three radiation qualities are presented in table 2, which have been determined in two independent experiments. The relative variance, 2/mean y, approximates unity, if the intercellular distributions of the MN or dicentric data follow a Poisson distribution. The signicance is assessed in terms of the test quantity u which

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T E Schmid et al Table 2. Results of experiment I and II: frequency ( SE) of dicentrics and their intercellular distribution in AL cells following exposure to 1.7 Gy of 55 MeV 12C ions (C ions) or 20 MeV protons either at quasi homogeneous exposure condition (Phom) or in a focused mode owing 5.4 5.4 m2 matrix (P5.4 5.4). Control, background frequency (Schmid et al 2011); 2/y, dispersion ratio; u-value, test quantity.

Intercellular distribution of dicentrics Radiation quality Control Control Control Control C ions C ions C ions Phom Phom Phom P5.4 5.4 P5.4 5.4 P5.4 5.4 Control C ions Phom P5.4 5.4 Cells scored 3000 500 500 500 310 325 338 285 302 381 321 325 310 812 721 578 684 Dicentrics per cell 0.0007 0.0005 Experiment I 0 0.002 0 0.207 0.212 0.192 0.060 0.043 0.053 0.081 0.077 0.087 0.002 0.030 0.028 0.028 0.015 0.013 0.012 0.015 0.015 0.017 Experiment II 500 499 500 260 267 287 269 290 362 295 301 284 812 608 552 630 1 40 49 41 15 11 18 26 23 25 96 24 51 6 7 6 1 1 1 1 1 12 2 2 5 1 4 2 4 1.0 1.36 1.17 1.37 1.06 1.12 1.05 0.92 1.01 0.99 1.22 1.10 1.09 0 4.47 2.15 4.79 0.68 1.48 0.71 1.01 0.08 0.13 4.10 1.68 1.61 0 2998 1 2 2 3 2/y 1.0 u-value 0.02

0 0.187 0.018 0.048 0.010 0.085 0.012

approximates to a unit normal deviation; values of u > 1.96 indicate an over- or underdispersion at the 5% level of signicance (Schmid et al 1995). For the nine exposures of AL cells to carbon ions, there is a trend of under-dispersion in the MN data because all u values are lower than unity. However, these intercellular distributions of the MN data show no signicant deviation from the Poisson distribution (u values between 0.80 and 1.92). In contrast, at 11 out of 14 proton-exposed cell samples, the intercellular distribution of MN is signicantly over-dispersed compared to Poisson (u values between 2.01 and 4.26), whereas there is a trend of over-dispersion in the MN data of the residual three proton-exposed cell samples in the 5.4 5.4 m2 matrix exposure condition (u values between 1.26 and 1.91). Since for dicentrics, similar to the MN data, no substantial differences between the results of the three replicates of experiment I could be observed, experiment II was carried out only with single exposure samples. However, the numbers of cells that have been analyzed in experiment II are nearly doubled compared to those of experiment I. In contrast to the MN data, the intercellular distribution of dicentrics is signicantly over-dispersed (u values between 2.15 and 4.79) for carbon ion exposure, whereas for proton-induced dicentrics at both application modes (quasi homogeneous and 5.4 5.4 m2 matrix exposure conditions) the intercellular distribution of data showed no deviation from a Poisson distribution (u values between 1.01 and 1.68). Table 3 demonstrates the results of the z-test statistics between the mean MN data obtained from experiment I and II, or experiment III as well as between the mean dicentric yields obtained from experiment I and II. No signicant differences were determined for these comparisons, either for exposure to 55 MeV 12C ions (p-values from 0.492 to 0.872 for MN data and p-value of 0.480 for dicentric yields) or to 20 MeV protons both at quasi homogeneous

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Table 3. The results of the z-test statistics for the differences between the MN data obtained from experiment I (Exp. I) and experiment II (Exp. II) or experiment III (Exp. III) as well as between the dicentric data obtained from Exp. I and Exp. II. Data are given for exposure of AL cells to 20 MeV protons at homogeneous (Phom) or 5.4 5.4 m2 matrix conditions (P5.4 5.4) and to 55 MeV 12C ions (C ions). Signicance is considered at p-values less than 0.05.

Micronuclei Comparison of experiments Exp. I Exp. II Exp. I Exp. II Exp. I Exp. II Exp. I Exp. III Exp. I Exp. III Exp. I Exp. III Radiation qualities C ions C ions Phom Phom P5.4 5.4 P5.4 5.4 C ions C ions Phom Phom P5.4 5.4 P5.4 5.4 z-value 0.69 1.16 1.46 0.16 0.32 0.03 p-value 0.492 0.245 0.145 0.872 0.746 0.975

Dicentrics z-value 0.71 0.31 0.20 p-value 0.480 0.755 0.841

Table 4. RBE ( SE) of 20 MeV protons at quasi homogeneous (Phom) or 5.4 5.4 m2 matrix conditions (P5.4 5.4) as well as 55 MeV 12C ions (C ions) relative to x-rays derived for the induction of MN or dicentrics in AL cells applied to obtain an average dose of 1.7 Gy. MN and dicentric data are given either as mean values ( SE) from two or three replicates of each single experiment or as mean values of the single experiments. An extrapolation of the dose effect curve was used for calculation of the carbon RBE for dicentrics. A possible undetected deviation from the assumed linear quadratic model above 4 Gy may introduce additional uncertainties.

MN data Exp. I II III I II III I II III Radiation quality C ions C ions C ions (Phom) (Phom) (Phom) (P5.4 5.4) (P5.4 5.4) (P5.4 5.4) C ions (Phom) (P5.4 5.4) mean value ( SE) 0.499 0.516 0.495 0.259 0.236 0.250 0.315 0.283 0.316 0.017 0.018 0.017 0.014 0.014 0.024 0.016 0.015 0.028 x-ray dose RBE ( SE) mean value ( SE) 0.204 0.187 0.052 0.048 0.082 0.085

Dicentric data x-ray dose 5.58 5.30 2.42 2.30 3.24 3.31 RBE ( SE) 3.28 3.12 1.43 1.35 1.90 1.95 0.29 0.29 0.16 0.21 0.18 0.21

Single experiments 3.71 2.18 0.11 3.80 2.24 0.11 3.69 2.17 0.11 2.22 1.31 0.09 2.05 1.21 0.09 2.15 1.27 0.12 2.61 1.53 0.09 2.39 1.40 0.09 2.61 1.54 0.13

0.015 0.018 0.007 0.010 0.009 0.012

Mean values of the above experiments 0.503 0.010 3.74 2.20 0.09 0.197 0.012 0.254 0.009 2.18 1.28 0.07 0.051 0.006 0.301 0.010 2.51 1.48 0.07 0.083 0.007

5.47 2.39 3.26

3.21 0.27 1.41 0.14 1.92 0.15

and 5.4 5.4 m2 matrix conditions (p-values from 0.145 to 0.975 for MN data and p-values from 0.755 to 0.841 for dicentric yields). 3.3. RBE determination for micronuclei and dicentrics induction Since there were no signicant differences between the MN data derived in three experiments as well as between the dicentric data obtained in two experiments, it was justied to calculate the RBE of 20 MeV protons (quasi homogeneous and 5.4 5.4 m2 matrix irradiation) or 55 MeV 12C ions using the pooled MN data and the pooled dicentric data from the different experiments (table 4). For the reference radiation 200 kV x-rays, the MN data determined in the dose range up to 4 Gy are given in table 5. Only at the highest dose, the intercellular

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T E Schmid et al Table 5. Frequency ( SE) and distribution of micronuclei (MN) in cytokinesis-blocked (CB) binucleate AL cells induced by 200 kV x-rays used as reference radiation ( 2/y, dispersion ratio; u-value, test quantity).

Intercellular distribution of MN Dose (Gy) 0 0.5 1 2 4 Cells scored 1000 1000 1000 1000 1000 MN per CB cell 0.022 0.053 0.099 0.265 0.541 0.005 0.008 0.011 0.017 0.022 0 980 951 910 780 558 1 18 45 82 180 352 2 2 4 7 35 81 3 0 0 1 5 9 2/y 1.161 1.099 1.104 1.114 0.859 u-value 3.60 2.23 2.34 2.54 3.15

Table 6. Frequency ( SE) of dicentrics and their intercellular distribution in AL cells following exposure to 70 kV x-rays. Data are pooled from our two independently performed experiments obtained previously (Schmid et al 2011). ( 2/y, dispersion ratio; u-value, test quantity).

Intercellular distribution of dicentrics Dose (Gy) 0 0.5 1 2 4 Cells scored 3000 1900 1400 1100 800 Dicentrics per cell 0.0007 0.0047 0.0164 0.0490 0.1088 0.0005 0.0016 0.0034 0.0067 0.0117 0 2998 1891 1378 1050 720 1 2 9 21 46 74 2 3 2/y 1.00 1.00 1.07 1.10 1.08 u-value 0.02 0.13 1.92 2.37 1.54

1 4 5

distribution of MN is under-dispersed but over-dispersed at the other doses. For the reference radiation 70 kV x-rays, pooled dicentric data from two independently performed experiments (table 6) have been used which were recently obtained for exposure of AL cells to 70 kV x-rays (Schmid et al 2011). Only at the dose of 2 Gy, the intercellular distribution is over-dispersed but there is a regular distribution at the other doses. A least-squares approximation was used to t the MN data with a linear-quadratic model y = c + D + D2. The resulting coefcients ( SE) of the doseresponse curve are c = (0.019 0.005), = (0.077 0.012) Gy1 and = (0.014 0.004) Gy2 and the nondiagonal elements of the correlation matrix are corr(, ) = 0.918, corr caused by the time structure of dose deposition: (, c) = 0.419 and corr(c, ) = 0.560. The corresponding weighted least-squares regression curve is shown in gure 3 (upper panel) as a solid line with the gray areas representing the standard error bands. Applying the weighted least-squares approximation to t the pooled dicentric data also with the linear-quadratic model results in c = (0.0006 0.0005), = (0.010 0.003) Gy1 and = (0.0048 0.0012) Gy2. The elements of the correlation matrix are: corr(, ) = 0.822, corr(, c) = 0.179 and corr(c, ) = 0.297. The corresponding reference curve is shown in gure 3 (lower panel). The results of the RBE evaluations relative to x-rays as reference are summarized in table 4. The RBE of 20 MeV protons (quasi homogeneous and 5.4 5.4 m2 matrix) and 55 MeV 12C ions was calculated in each experiment or the pooled experiments as the ratio between the dose, Dx-rays, of the reference radiation and the dose, Dprotons = 1.7 Gy or Dcarbon ions = 1.7 Gy, which produced equal response, i.e. RBE = Dx-rays/Dprotons or Dx-rays/Dcarbon ions. To calculate Dx-rays, the linear-quadratic doseresponse curves for MN or dicentric induction were used. In the proton experiments IIII for MN induction, RBE values of 1.31 0.09, 1.21 0.09 and 1.27 0.12 resulted for the quasi homogeneous exposure as well as 1.53 0.09, 1.40 0.09 and 1.53 0.13 for the 5.4 5.4 m2 matrix exposure,

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whereas RBE values of 2.18 0.11, 2.24 0.11 and 2.17 0.11 resulted in the carbon ion experiments IIII, respectively. In the proton experiments I and II for dicentric induction RBE values of 1.43 0.16 and 1.35 0.21 for the quasi homogeneous exposure as well as 1.90 0.18 and 1.95 0.21 for the 5.4 5.4 m2 matrix exposure were determined, whereas RBE values of 3.28 0.29 and 3.12 0.29 resulted in the carbon ion experiments. The RBE value obtained for the pooled MN data from the different proton experiments are 1.28 0.07 for the quasi homogeneous exposure and 1.48 0.07 for the 5.4 5.4 m2 matrix exposure as well as 2.20 0.09 for exposure to carbon ions. The corresponding RBE values obtained for dicentric induction by protons are 1.41 0.14 for the quasi homogeneous exposure, 1.92 0.15 for the 5.4 5.4 m2 matrix exposure to protons as well as 3.21 0.27 for exposure to carbon ions (table 3). It should be mentioned that calculation of carbon RBE for dicentrics requires an extrapolation of the x-ray dose curve up to about 5.5 Gy. Possible undetected deviations of the dose effect curve from the assumed linear-quadratic model above 4 Gy may introduce additional uncertainties. It is remarkable that the RBE values determined for proton exposure in the 5.4 5.4 m2 matrix condition are signicantly higher (z-value = 2.31; p = 0.0211 using MN data and z-value = 2.66; p < 0.01 using dicentric data) compared to those after quasi homogeneous exposure. 4. Discussion The main physical difference between low LET (LET <10 keV m1, RBE 1) and high LET (LET > 10 keV m1, RBE > 1) irradiation is the temporal and spatial dose deposition. In case of x-ray, electron and high energy proton irradiation, a great number of particles have to penetrate a cell within timespans of milliseconds to minutes to deposit relevant doses of several Gy. As a consequence, a close to homogeneous microdose distribution is obtained from the random particle penetration generating only small dose spikes where low energy secondary electrons are stopped. In case of high LET ions the structure of the microdose distribution differs signicantly. In order to exemplarily show the differences, the microdose distributions along a line through a cell nucleus of typical size (10 m diameter) are calculated applying the same average dose of 1.7 Gy by randomly distributed 20 MeV protons (quasi homogeneous mode) and by a matrix of 55 MeV (total energy) 12C ions (gure 2). The dose distributions are calculated according to equation (1). In case of the quasi homogeneous proton irradiation, 320 protons are randomly distributed within a single cell nucleus that is equivalent to a uence of 4 108 cm2. The dose is quite homogeneously distributed but several dose spikes can be recognized that are attributed to the dose deposition from the core of the proton tracks. In contrast, only a small number of 55 MeV 12C ions are necessary to deposit the same average dose because the LET of the 55 MeV 12C ions in water (310 keV m1) is 117 times that of 20 MeV protons (2.65 keV m1). Thus, a matrix irradiation of single carbon ions having a point to point distance of 5.4 m (areal density of 3.4 106 ions cm2) results in the same average dose. Each carbon ion creates ultra high doses that are mainly concentrated within a nanometer sized track (gure 2) surrounded by a radial dose fall off up to a micrometer distance. The dose spikes are deposited within femtoseconds resulting in ultra high dose rates while the total dose delivered by the whole set of particles takes milliseconds to seconds. The microdose distribution of 117 protons (20 MeV) focused to a beam spot size of 300 nm, the lower limit of assumed beam focus and applied by/in the same matrix as in the carbon case is additionally shown in gure 2. The 117 protons deposit the same amount of energy like a single 55 MeV 12C ion because the LET of carbon ions is 117 times that of protons. The dose distribution of the focused protons differs much from that of a quasi homogeneous proton irradiation (see gure 2) and its gross structure looks similar to the dose distribution

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Figure 2. Simulation of dose distribution of 5.4 5.4 m2 matrix irradiation of 55 MeV carbon ions with one ion per point (dotted line), 20 MeV protons with 117 protons per point and a beam spot size of 300 nm FWHM (solid line) and quasi homogeneous irradiation with randomly distributed 20 MeV protons (dashed line). All irradiation modes results in a mean dose of 1.7 Gy. For the simulation the radial dose distribution of (equation (1) was used and at each point along the line (x = 5 m, . . . , + 5 m; y = 0 m) the dose contributions of all ions were summed up. For matrix irradiation mode the line hit two matrix points, at x = 2.7 m and x = +2.7 m. The highest local dose is deposited by carbon ions, but only on a very thin core. By focusing 117 protons the radial dose distributions of single protons overlap and result in an increased local dose approaching that of a single carbon ion.

of the carbon matrix. The maximum doses achieved are an order of magnitude larger than the maximum of the dose spikes in case of the random proton irradiation. However, the ultra high microdoses in the core of the carbon tracks are still several orders of magnitude larger than in the case of the focused protons. Besides this inner part the microdose distributions are roughly the same within a radius of about 1 m. 4.1. RBE values The aim of the present study was to study the effect of the different spatial distributions of low LET radiation on the RBE in view of genetic damage. The experiments show that the microdose distribution inuences the dose effect relationships and that high LET effects and thus enhanced RBE can be induced by submicrometer focused low LET proton irradiation. Obviously, single proton tracks cluster in space and lead to an enhanced yield of MN and dicentrics by inter-track interaction in a similar way as by intra-track effects of high-LET radiation. The density of DSBs is enhanced by the high ionization load of the bunch of protons leading to a greater chance of DSBs lying close together and to form clusters of DSBs where the ends of different chromosome strands can intermix. Thus the probability is enhanced to form chromosome aberrations. In detail, RBE is enhanced for MN induction from RBE = 1.28 0.07 for quasi homogeneous proton irradiation to RBE = 1.48 0.07 for the 117 protons focused to submicrometer diameter. The RBE enhancement is even larger for the induction of dicentrics, from RBE = 1.41 0.14 for homogeneous proton irradiation to RBE = 1.92 0.15. Since the present comparison was performed on the same batch of cell cultures thawed from frozen aliquots of the same stock culture, the different RBE enhancements have to be attributed to a different sensitivity of the investigated biological endpoints. The RBE enhancements for focused protons are statistically signicant and the experiments show for the rst time that higher RBE values can be produced by a focused application of low LET radiation although

Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness


0,8
C ions Phom

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micronuclei / CB cell

0,6

P5.4x5.4

0,4

0,2

0,0

2 3 absorbed dose / Gy

0,3
C ions Phom

dicentrics / cell

0,2

P5.4x5.4

0,1

0,0

2 3 absorbed dose / Gy

Figure 3. Linear-quadratic doseresponse curve for induction of MN by 200 kV x-rays in cytokinesis-blocked (CB) binucleated AL cells (upper panel) and for induction of dicentrics by 70 kV x-rays in AL cells (lower panel). The dots and the associated binomial standard error bars correspond to the data from the two different proton irradiation modes and from carbon irradiation (tables 3 and 4). The solid lines are the weighted least-squares ts to the data. The gray areas represent the standard error bands. Additionally, the corresponding mean frequencies of MN or dicentrics each pooled from different experiments of exposure to 1.7 Gy of carbon ions as well as 20 MeV protons at quasi homogeneous (Phom) or 5.4 5.4 m2 matrix (P5.4 5.4) exposure conditions are demonstrated.

the high RBE values of carbon ions representing ions of very large LET is not yet obtained. The previous work of Durante et al (2010) and Hill et al (2011) have also shown that biological response is not solely dependent on the mean absorbed dose and radiation quality but also on the pattern of energy deposition within the cell and cell population. 4.2. Comparison with LEM III The measured RBE enhancements can be compared with predictions of the local effect model (LEM III; Els sser et al 2008b) that is used to predict RBE from the local dose distributions a of low and high LET ionizing particles. The model is used in therapy planning to calculate the required equivalent doses when using high LET particles. The basis is to calculate a local effect probability according to x-ray data. To calculate RBE enhancement of focused proton irradiation in dependence of the beam diameter rst the relevant parameters , , c

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Figure 4. Calculated RBE (diamonds) in dependence of the diameter of the focused beam of 117 protons using LEM III as described in (Els sser et al 2008b). Due to the increasing overlap of the a radial dose distribution of single protons with decreasing beam diameter the RBE increases from approximately 1.3 and 1.4 for induction of MN and dicentrics to 1.9 and 2.1, respectively. The stars show the measured RBE values for single carbon ions and the triangles for 117 protons focused to a diameter 650 350 nm. For induction of dicentrics by carbon ions the LEM calculation is in good agreement with the measurement, for induction of micronuclei the RBE is overestimated by LEM III. In contrast for focused protons the calculation underestimates the RBE for dicentrics whereas the RBE for micronuclei induction is in good agreement with the measured data.

are tted to the x-ray data of gure 3. The parameter Dt, above which the dose effect curve is assumed to be linear, according to LEM, is adjusted to reproduce data of quasi homogeneous proton irradiation. The resulting function of RBE versus beam diameter is plotted in gure 4 for induction of both MN and dicentrics. It shows an enhancement of RBE at small beam diameters and an RBE close to the RBE of quasi homogeneous distributed protons for beam diameters larger than 3 m. For induction of MN, the calculation is in good agreement with measured focused proton data, but overestimates the RBE for carbon. Discrepancies in describing carbon and protons simultaneously with LEM III (and below) are well known but normally in the opposite direction. Els sser et al (2010) report overestimation of RBE of lighter ions and in the a entrance channel, Karger et al (2006) reports systematic underestimation of Bragg peak RBE for carbon ions. In contrast, for induction of dicentrics, the calculation can nearly reproduce the RBE for carbon ions and due to the choice of Dt the RBE in the limit of very large focus sizes is in agreement with quasi homogeneous proton data. However, the calculation underestimates the RBE for focused protons, which dose distribution is between the highly concentrated dose deposition of single carbon ions and the quasi homogeneous distribution of applied protons. We assume that the discrepancies are caused because of LEM III using only the distribution of dose levels but neglecting the spatial correlation of dose distribution and thus the spatial DSB distribution. However, since structural chromosome aberrations require at least two DSB in proximity the spatial correlation is essential. Taking into account this correlation a better prediction of the newest LEM IV model (Els sser et al 2010, Friedrich et al 2012) is expected. a 4.3. Micronuclei and dicentric distributions The intra-laboratory comparison of scoring MN and dicentrics in the same cell type indicated that reasonable consistency was obtained for analyzing the dependency of the RBE of 20 MeV protons on quasi homogeneous and spot application modes. In general, it can be

Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness

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stated that scoring of MN or dicentrics appeared not to produce notable differences. As already mentioned in our previous investigation on evaluating the RBE of pulsed and continuous 20 MeV protons using MN analysis in HeLa cells (Schmid et al 2009), it should be considered that in contrast to radiation-induced structural chromosome aberrations, the relevance of radiation-induced MN for quantifying the chromosome damage may be limited under the following circumstances. The slope of a reference doseresponse curve for MN can be inuenced by the mechanisms of MN formation and MN content, because with increasing number of acentric fragments the relative proportion of acentric fragments formed in independent MN may decrease. Consequently, individual MN may contain several acentric fragments at higher doses resulting in a signicant reduction of proton-induced MN yields. In fact, if such saturation effects occur, the doseresponse for MN induction by x-rays tends to turn down with increasing dose. Therefore, to minimize this problem in the present investigation, a dose of 1.7 Gy has been applied for exposure of AL cells to 20 MeV protons at both spatial application modes, whereas a dose of 3.4 Gy of 20 MeV protons was applied in our previous investigation in AL cells analyzing differently the temporal application modes (Schmid et al 2011). However, as shown in tables 1 and 2 for exposure of AL cells to 1.7 Gy of 55 MeV 12C ions, there is no agreement in the observed patterns of the cellular distribution of MN and dicentrics, respectively. Whereas there is no deviation from regular dispersion in the MN data (u values from 0.80 to 1.92, table 1), signicant over-dispersion could be observed in the dicentric data (u values from 2.15 to 4.79, table 2). Thus 55 MeV 12C ions have produced an excess of cells with more than one dicentrics which can be suspected for a high-LET radiation quality. As already mentioned, the decreased dispersion ratio observed in the MN data relative to the dicentric data may reect a saturation effect even at the lower dose of 1.7 Gy used in the present investigation. Contrary to these ndings for a high-LET radiation quality it is more difcult to interpret in detail the present results for the observed patterns of the cellular distribution dicentrics and MN induced by 20 MeV protons with respect to the differently applied exposure conditions. For induction of dicentrics (table 2), a regular distribution was observed in all three replicates of experiment I as well as in the single sample of experiment II, independently of the exposure conditions. For induction of MN (table 1), a regular distribution could only be observed in 3 of 7 cell samples exposed to 20 MeV protons at homogeneous irradiation conditions, whereas the intercellular distribution of MN in all other cell samples, which were exposed at a homogeneous mode or in a 5.4 5.4 m2 matrix condition, was signicantly over-dispersed compared to Poisson. Such an effect could be explained by a combination of different phenomena. As a rst explanation, one may assume a depression of cell kinetics of highly damaged cells. This error can arise from the fact that some cells, especially the damaged ones with more MN, may be sufciently delayed and may not even have reached the binucleate CB cell stage of the division cycle. Although a sufcient number of binucleate cells has been scored one could argue as a second explanation that particularly cells containing several acentric fragments in one or more MN might have already died during the rst karyokinesis and thus prior to the end of cytokinesis. Therefore, such badly damaged cells would escape scoring as micronucleated CB cells. Thus the present investigation was carried out in parallel on the production of MN as well as chromosome aberrations to avoid such potential saturation effects in data. 5. Conclusions Our experiments demonstrate evidence that low LET radiation focused to submicrometer diameters but depositing the same average dose as quasi homogeneous low LET irradiation

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results in an increase in RBE for the induction of MN and dicentrics similar to the increase observed with high-LET radiation. The small focus size of the protons is not meant to enhance the efciency in creating DSB but affects the distribution of the DSB inside the cell nuclei. The local density of DSB is increased at the irradiated spots enhancing also the probability for the interaction of the DSB and thus rising the probability of connecting the wrong ends. We hypothesize that a tighter beam spot of protons might further enhance the RBE value. It is even possible that for instance, at beam diameters in the 10100 nm range, the RBE might be larger than that of a broad beam of heavy ions since a higher density of DSB and even a larger number of severe DNA lesions through inter track interactions may be created but avoiding an overkill situation as it may occur within a single heavy ion track core. In general, our results provide evidence that the increased RBE of heavy ions can be approached by spot application of a bunch of protons. These results strongly support the assumption of LEM that the spatial dose distribution is the source of relative biological effectiveness. In addition, a test eld is opened to understand the creation of higher RBE values through high LET radiation by modeling these effects by focused low LET radiation. Reducing the proton beam diameter and/or additionally focusing in time to apply the radiation in nanoseconds provide a parameter eld for further investigations of high LET effects. It should be mentioned that the presented microbeam approach to enhance RBE is limited to thin targets like conuent growing cells in culture only. It will not be possible to keep a submicrometer beam spot size for tumor irradiation inside the body of a patient due to lateral straggling of the particles inside the tissue.

Acknowledgments Supported by the DFG-Cluster of Excellence Munich-Centre for Advanced Photonics, by the DFG INST 95/980-1 FUGG and by the Maier Leibnitz Laboratory Munich. We thank the MLL staff for operation of the accelerator and Fridtjof N sslin for his valuable comments on u the manuscript.

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Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness

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