Feasibility of BNCT radiobiological experiments at the HYTHOR facility

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NIM B Beam Interactions with Materials & Atoms

Nuclear Instruments and Methods in Physics Research B 266 (2008) 2587–2593 www.elsevier.com/locate/nimb

Feasibility of BNCT radiobiological experiments at the HYTHOR facility J. Esposito a, C. Ceballos a, M. Soncin b, C. Fabris b, E. Friso b, D. Moro a, P. Colautti a,*, G. Jori b, G. Rosi c, E. Nava d a

INFN, Laboratori Nazionali di Legnaro, Viale dell’Universita` 2, I-35020 Legnaro, PD, Italy Dipartimento di Biologia, Universita` di Padova, Via Ugo Bassi 58 B, I-35121 Padova, Italy c ENEA C.R. Casaccia, Via Anguillarese 301, I-00123 S.Maria di Galeria, Roma, Italy d ENEA C.R. Bologna, Via Martiri di Monte Sole 4, I-40129 Bologna, Italy

b

Received 25 January 2008; received in revised form 20 March 2008 Available online 12 April 2008

Abstract HYTHOR (HYbrid Thermal spectrum sHifter tapirO Reactor) is a new thermal-neutron irradiation facility, which was installed and became operative in mid 2005 at the TAPIRO (TAratura PIla Rapida potenza 0) fast reactor, in the Casaccia research centre (near Rome) of ENEA (Ente per le Nuove tecnologie Energia ed Ambiente). The facility has been designed for in vivo radiobiological studies. In HYTHOR irradiation cavity, 1–6 mice can be simultaneously irradiated to study skin melanoma treatments with the BNCT (boron neutron capture therapy). The therapeutic effects of HYTHOR radiation field on mouse melanoma has been studied as a preliminary investigation before studying the tumour local control due to boron neutron capture effect after boronated molecule injection. The method to properly irradiate small animals has been precisely defined. Results show that HYTHOR radiation field is by itself effective in reducing the tumour-growth rate. This finding has to be taken into account in studying the effectiveness of new 10B carriers. A method to properly measure the reduction of the tumour-growth rate is reported and discussed. Ó 2008 Elsevier B.V. All rights reserved. PACS: 87.50; 29.25.Dz Keywords: Neutron; BNCT; Skin melanoma

1. Introduction BNCT is a radiation therapy based on the destructive effects of helium and lithium ions emerging from the nuclear reaction 10B(n,a)7Li, which takes place when a 10 B nucleus absorbs a thermal neutron. Because of the very short ion ranges (9 and 6 lm, respectively), the destructive effect is confined inside the living cell where the 10B has been previously transported in. BNCT is therefore a cellular radiation therapy suited to treat tumours infiltrated into the surrounding healthy tissues. The therapeutic success

*

Corresponding author. Tel.: +39 0498068304; fax: +39 049641925. E-mail address: [email protected] (P. Colautti).

0168-583X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2008.03.239

depends on several factors. Namely, the capacity of concentrating a relatively high quantity of 10B inside tumour cells and contemporaneously a small quantity of 10B inside healthy cells; a high enough thermal neutron fluence rate; the minimisation of healthy tissue damage due to unwanted fast neutrons and gamma rays. BNCT is nowadays performed by using nuclear reactors. Some studies have proposed to use compact accelerators, namely radio-frequency quadrupoles (RFQ), for an accelerator-based BNCT [1,2]. At the INFN Legnaro National Laboratories (LNL), a thermal neutron source based on an intense 5 MeV proton beam is currently under construction in the framework of the radioactive-ion beam facility named SPES (Selective Production of Exotic Species). The 30 mA proton beam will be accelerated by a RFQ. The neutron

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source will be mainly devoted to perform experimental medical treatments of human skin melanoma tumour by the BNCT technique. The most updated and general description of SPES facility is reported elsewhere [3]. The project foresees dosimetric and radiobiological studies for optimising BNCT irradiation protocols. Such R&D is presently performed at the 5 kW TAPIRO fast reactor at ENEA-Casaccia [4], because of the lack of an intense-enough thermal neutron source at LNL. The R&D of such a project concerns the design and construction of a tissue-equivalent proportional counter (TEPC) for a microdosimetric-based dosimetry [5,6] as well as designing, synthesizing and testing new boron carriers that display a high selectivity of accumulation in malignant melanoma lesions. The boron carriers selected are those ones already used as photodynamic therapy (PDT) agents. The ultimate aim is to explore the possible synergistic effects between the two irradiation modalities (PDT and BNCT) [7,8]. Initial irradiations performed by using an old TAPIRO thermal column and a boronated phthalocyanine pointed out that boron carriers with appreciable PDT action could perform a new positive role in BNCT [9]. HYTHOR [4] is a new thermal column designed to optimise radiobiological experimental investigations at TAPIRO.

In this paper, HYTHOR calculated dosimetry is presented as well as the experimental method to assess radiation field effectiveness in reducing the tumour-growth rate. 2. HYTHOR 2.1. Physical features HYTHOR is a thermal column that can be quickly inserted and pulled out of TAPIRO reactor experimental cave. In order to get an easy and fast access to the irradiation room, HYTHOR is made of two parts: the beam shaping assembly (BSA) and the irradiation box, which encloses the irradiation room. BSA is a hybrid Pb-CF2-RG (reactor grade)-grafite neutron spectrum shifter (see the 3D configuration in Fig. 1). When it is inserted inside TAPIRO reactor, its shaped lead layer shifts fast neutrons towards lower energies and shields against reactor gamma rays. A second teflon (CF2) layer set behind slows down neutrons further to the epithermal region (E < 10 keV). Eventually, a large RG-graphite bulk shifts neutrons towards the thermal energy range (E < 0.4 eV). In the BSA centre there is a blind tunnel of squared cross section (40  40 cm2), 108.5 cm long for the irradiation box insertion. That is

Fig. 1. Monte Carlo design of the hybrid-thermal spectrum shifter HYTHOR.

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Fig. 2. Left side: HYTHOR inserted in TAPIRO reactor. The irradiation box lid handles are visible. Right side: the irradiation box is open showing the irradiation room with the bismuth walls and the two Plexiglas planes fixed at the lid.

made of high density teflon surrounding the bismuth walls of the 14  14  24 cm3 irradiation room. Teflon provides a further neutron thermalisation without a significant increase of gamma yield. The irradiation room access is closed by a lid, extractable from the tunnel (see left side of Fig. 2). Two 4 mm thick Plexiglas planes are inserted in the lid internal surface. Up to six mice can be stretched on the two planes (Fig. 2, right side). Inside the irradiation room, radiation field parameters vary only a little transversally (2%) and 40% longitudinally. The volume-averaged neutron fluxes and their in-air tissue kerma, as well as the photon kerma and their ratios, are reported in Table 1. Further details about HYTHOR have been published elsewhere [4]. 2.2. Dosimetric features Mice exposed to HYTHOR radiation field receive an absorbed dose, the main components of which are the neutron dose Dn, the gamma dose Dk and the dose Dbnc of 10B fragments. Dn is delivered by neutrons through thermal neutron capture reactions with nitrogen nuclei 14 N(n,p)14C, proton recoils from 1H(n,n’)1H elastic reactions and heavier recoils due to elastic and anelastic fast neutron reactions. Dc is delivered by gamma rays emerging from the reactor source, by the gamma background generated by the neutron slowing down process, in which the 4.1 MeV gamma rays of 209Bi(n,n’)209Bi inelastic reaction play a relevant role, as well as by the 2.2 MeV gamma rays of capture reactions 1H(n,c)2H in mouse tissue. Dbnc is delivered by charged particles emerging from neutron-capture reactions 10B(n,a)7Li. The 0.48 MeV gamma rays from 7 Li de-excitation after neutron-capture reactions are accounted in the Dc component.

An extensive computational investigation has been performed by using the MCNPX-2.6b Monte Carlo code [10] in order to assess the three absorbed dose components in the three main mouse compartments, namely the total body, tumour and liver, in different irradiation conditions (from 1 to 6 mice at a time). Calculations have been carried out by using a Xeon dual processor of 3.2 GHz and 64 bit. The TAPIRO-core neutron emission of 4.3  1014 s1 at the maximum power of 5 kW [11] has been taken as a reference for dose rate calculations. D_ bnc would have the highest value in the liver since all boronated drugs injected in the bloodstream are eventually accumulated in the liver. In order to minimise this unwanted dose, mouse abdomen is protected with a thermal neutron absorber layer: a kind of pant made of 0.14 g/cm2 of boric acid 98% enriched with 10 B. A simplified mouse model has been used in MCNPX absorbed dose calculations (see Fig. 3). The uniform tissue-equivalent model has the same average weight of real mice. In spite of the simple model used, the computing time to follow all the secondary charged particles set in motion by photons and neutrons was exorbitant. Therefore, calculations have been carried out assuming secondary charged particle equilibrium (CPE) and updated ICRU-63 flux-tokerma neutron conversion factors [12] and ICRU-46 fluxto-kerma photon conversion factors [13]. CPE is a good assumption for neutrons, since kerma spectrum has a mean energy of 528 eV and only 2.8% of kerma is released by fast neutrons with E > 1 MeV. The CPE is a good assumption for gamma rays, but only for depth tissues. Gamma kerma spectrum has in fact a mean energy of 1.41 MeV, being characterised by the 0.48 MeV peak (due to capture reactions of 10B in protective mouse pants), the 2.2 MeV peak of hydrogen neutron capture and the 4.1 MeV gamma rays of bismuth inelastic reactions. In order

Table 1 Volume-averaged radiation field parameters in HYTHOR irradiation room Uth(E< Irradiation Room

0.4 eV) 9

3.54  10

cm2 s1

Utotal cm2 s1 9

3.81  10

MCNPX calculated data have less than 1% statistical uncertainty.

Uth/Utotal

K_ nðE>0:4eVÞ Gy h1

K_ nðE>0:4eVÞ =K_ total

K_ c Gy h1

K_ c =K_ total

0.92

0.18

0.08

1.09

0.48

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Fig. 3. Left side: the mouse geometry model used for Monte Carlo absorbed dose calculations. Right side: irradiation room transversal cross section (MCNPX design) with 6 mice on the two Plexiglas planes.

to correct the absorbed dose data based on CPE assumption (kerma values), the mean ratio absorbed-dose/kerma has been calculated in the three mouse compartments by using just 1 mouse in the irradiation room and calculating the energy released by secondary electrons. Eventually, the corrective factors (0.41 for tumour, 0.96 for liver and 0.73 for whole body) have been used to convert kerma values in absorbed dose values. The total dose rate D_ tot , its quality (namely Dn/Dc) and D_ bnc for 1 ppm of 10B are plotted in Fig. 4 for tumour and liver against the number of mice inside the irradiation room. The figure shows that all dose parameters, do not change significantly. Such finding, which is valid as well for whole body, points out that mouse positioning as well as their number is not critical for dosimetry. The only care to have is to place the mice always with their heads towards the reactor core. Absorbed dose and radiation quality mean values are shown in Table 2 together with their standard deviations: clearly, boric acid pants are effective in shielding the mouse liver. D_ bnc for 1 ppm of 10B is 50 times lower in liver than in tumour. However, boric acid shielding increases significantly the gamma dose in the liver. Table 2 shows as well that, in contrast than in the liver and the whole body, the tumour dose has a high quality, being mainly due to neutrons. 3. Radiobiological model In order to investigate the BNCT effectiveness of new boron carriers, we used B16F1 tumour-bearing mice as

Table 2 Average (on the whole irradiation room volume) dose rates and radiation quality values in the mouse tumor, liver and whole body

Tumour bulk Liver Whole body

D_ tot [Gy/hr]

[Dn/Dc]

D_ bnc [Gy/hr. ppm 10B]

5.37 ± 0.07 11.1 ± 0.3 7.5 ± 0.1

1.3 ± 0.1 0.018 ± 0.001 0.29 ± 0.02

0.82 ± 0.05 0.028 ± 0.003 -

The uncertainties are the standard deviations of data from Fig. 4.

experimental model. The cell line B16F1, used in our studies is a pigmented variant of murine melanoma B16 [14], which differs because of its highly metastatic potential. The cell line, cultured as a monolayer at 37 °C in a humidified atmosphere with 5% CO2, was grown in Dulbecco’s modified minimal essential medium containing 10% heatinactivated foetal calf serum and supplemented with 100 units/ml penicillin, 100 lg/ml streptomycin, 0.25 lg/ ml amphotericin and 2 mM glutamine. The six-week-old female C57BL/6 mice (20–22 g body weight) were supplied from Charles River Laboratories (Como, Italy) and kept in standard cages with free access to tap water and standard dietary chow. Animal care was performed according to the guidelines established by the Italian Committee for experimental Animals. B16F1 pigmented melanoma was subcutaneously transplanted into the upper flank of the mice by injecting 20 ll (106 cells) of a sterile cell suspension in phosphate-buffered saline (PBS).

Fig. 4. Total absorbed-dose rate D_ tot , nuclear-fragment dose rate due to 1 ppm of liver (right side) against the number of mice in the irradiation room.

10

B D_ bnc and radiation quality [Dn/Dc] in tumour bulk (left side) and

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The R&D on boron carriers foresees to inject the drug intravenously in B16F1 tumour-bearing mice at 8 days after transplantation, when the major axis of the lentilshaped tumour is about 0.6 cm. Mice will be then irradiated in HYTHOR, with the desired radiation dose, 24 h later. After irradiation the tumour size will be measured daily by means of a calliper. Individual tumour volumes (V) will be calculated by assuming a hemiellipsoidal structure for the tumour nodule and measuring the two perpendicular axes (a and b) and the height (c). Application of the relationship V = 2/3p (a/2 b/2 c) will provide the tumour volume. The number of days for the tumour volume to grow from its initial value of 0.02 ± 0.005 cm3 to a value of 0.8–1 cm3 will be calculated for the individual neoplastic lesions. Mice will be treated according to the rules established by the University of Padova ethical committee for humane treatment of experimental animals. They will be sacrificed by euthanasia before the tumour development becomes too large in order to minimize the risk of undue suffering and spreading of the malignancy across the body through lung or liver metastases. A preliminary investigation and aim of this paper, was to study the radiation field efficacy in reducing by itself the skin melanoma growth. Mice not injected with the boron compound have been therefore irradiated according to the upper-mentioned protocol. HYTHOR radiation field effect has been studied by comparing the tumour growth in irradiated mice with that one in control mice. To obtain accurate average values, several irradiations have been performed in different experimental shifts. To properly assess the radiation field effect, tumour-growth fluctuations in different mice populations have to be taken into account. In Fig. 5 the tumour growth in control mice of 12 different experimental shifts is plotted. Time 0 is the time of irradiation (8 days after tumour transplantation). Fig. 5 shows that the tumour volume is rather different in different experimental shifts already at 0 time. The average tumour-growth is exponential, however the relative standard deviation of the 12 experimental shifts is as large as about 40% (see right side of Fig. 5).

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In order to minimise the tumour-growth biological variance, we have defined the quantity relative tumour-growth at the ith day or RTGi: RTGi ¼

DV i  DV control i DV control i

ð1Þ

where DVi is the tumour volume under investigation at the ith day minus the tumour volume at the 0 day and DV control i is the same difference for un-irradiated control mice. The 0 day is that one of the irradiation in HYTHOR. DVi is averaged for all the irradiated mice at a given dose. DV control is averaged for the 12 control mice. When the irrai diated tumour grows as well as the un-irradiated tumour in control mice, RTGi  0. If the irradiated tumour grows slower than the control tumour, RTGi is negative. If the irradiated tumour stops growing, RTGi = 1. Eventually, if the irradiated tumour decreases its volume, RTGi < 1. 4. Results and discussion After several irradiations to set up the experimental procedures, 66 mice have been irradiated in three different experimental shifts for times varying from 10 to 40 min at a reactor power of 5 kW. All the animals had a B16F1 pigmented melanoma subcutaneously transplanted on the left shoulder 8 days before. In order to obtain data for significant comparisons with future irradiations in the presence of boronated molecules, all irradiated animals were protected with 0.14 g/cm2 of boric acid pants (see HYTHOR dosimetric feature paragraph). Before irradiation animals were anaesthetized by intraperitoneal injection of 20 mg/kg of ZoletilÒ. Control mice underwent identical experimental procedures (including anaesthesia, transport to the nuclear reactor and back) as the irradiated mice, but were not exposed to irradiation. Irradiation dosimetry has been monitored by using one of the ionisation chambers inserted inside the TAPIRO reactor. The ionisation chamber current was measured with a Keithley 614 electrometer interfaced with a personal

Fig. 5. Control mice tumour-growth in 12 different experiments (left side). The average growth and the relative standard deviation is in the right side of figure. The reference time 0 is the irradiation day.

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computer. The ionisation chamber current (I) has been measured to be linearly correlated with TAPIRO power (P) as read by power level monitors, being the linear relationship: I [lA] = 6.49  104 + 5.41  105 P [W]. Therefore, TAPIRO-core neutron emission per charge unity has been measured to be 1.59  1015 lC1 at 5 KW. The ionisation chamber current has been monitored and integrated over all the irradiation time by using PC-resi-

dent LabView software. An irradiation time of 30 min at 5 kW corresponds to about 500 lC of ionisation chamber charge. The absorbed dose in tumour bulk was calculated by using HYTHOR dosimetric data (see HYTHOR dosimetric feature paragraph) and the above-mentioned linear relationship. At 5 kW, the absorbed dose D in tumour bulk is proportional to the ionisation chamber charge: D [Gy] = 0.0053 I [lC]. In Fig. 6, RTGi is plotted against

Fig. 6. Relative tumour-growth (see text) after 4 tumour absorbed doses. Error bars (shown only for 3.6 Gy data) are RTGi relative errors for 6 mice.

Fig. 7. Relative, with respect unirradiated mice, tumour-growth against the total absorbed dose in tumour. The tumour total absorbed dose (upper X axis) scales with the reactor ionisation-chamber charge (lower X axis), see text. The thick line is a by-eye fit of experimental data. Dashed lines are drawn by hands for including experimental uncertainties.

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the post-irradiation time for four different total absorbed doses in the tumour. After some fluctuation during the first days, mainly due to the uncertainty in measuring small tumour volumes, RTGi reaches an almost constant value, which undergoes no further changes. When the tumour volume reached 1 cm3 the mice were sacrificed by prolonged exposure to CO2 vapours. Averaging RTGi values over the plateau region (after the 5th day), we obtain the mean relative tumour-growth (RTG), the value of which can be related to the absorbed dose. In Fig. 7, RTG is plotted against the ionisation chamber charge and the total tumour dose. Error bars in Fig. 7 represent standard deviations of RTGi values measured after the 5th day (i > 5), for a given absorbed dose. At the beginning, the mean tumour-growth rate decreases quite quickly with the dose. Such a decrease slows down at higher doses. The dashed lines in Fig. 7 define an envelope including all RTGi standard deviations. The band in between the two dashed lines is an assessment of RTG uncertainty. The band width of 0.15 seems to be rather constant at different doses. Fig. 7 shows that BNCT therapeutic effects can be observed in HYTHOR radiation field when they reduce RTG by 0.15 or more. In other words, 10B effects can be detected, when they reduce by more than 15% the tumour-growth rate, with respect to the tumour-growth rate in control mice (see Eq. 1). 5. Conclusion The fast nuclear reactor TAPIRO has been recently equipped with a new thermal column, called HYTHOR, designed for radiobiological studies with mice. The main aim of HYTHOR will be to perform studies on new boron carriers for treating skin melanoma tumours with the BNCT technique. In this paper, dosimetric features of HYTHOR irradiation room have been presented. The tumour absorbed-dose is almost independent on the number of mice inside the irradiation room. Such a finding increases the flexibility in using the irradiation facility. Moreover, the thermal fluence rate in HYTHOR is so high that 1 ppm of 10B in the tumour is enough to increase the local total dose by15%. Because of the high biological variance of the mouse melanoma growth, an experimental method has been developed based on the definition of RTGi (relative tumour-growth at the ith day). Results show that RTGi values become rather constant after the 5th post-irradiation day. Such a constant value can be used as a significant marker of the radiation-field biological effect. At the highest mouse-tolerable dose, the HYTHOR radiation field is just able of reducing by itself of about 70% the tumourgrowth rate. In spite of that, the relatively small variance

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of RTG will allow for detecting a BNCT effectiveness in decreasing the tumour-growth rate as small as the 15%. Acknowledgments The physics activities of that research have been supported by the Italian Institute of Nuclear Physics in the frame of the SPES project. The radiobiological activities have been supported by the University of Padova project No. CPDA 048247 on ‘‘Combined PDT/BNCT treatment of melanotic melanoma”. References [1] Y.D. Harker, J.F. Harmon, G.W. Irwine, Inel and ISU BNCT research using a 2 MeV RFQ-based neutron source, Nucl. Instr. and Meth. B 99 (1995) 843. [2] R. Terlizzi, N. Colonna, E. Bisceglie, P. Colangelo, S. Marrone, A. Raino, G. Tagliente, V. Variale, Feasibility of an epithermal neutron source for BNCT based on RFQ accelerator, Nucl. Instr. and Meth. B 213 (2004) 210. [3] G. Prete, SPES Advanced Exotic Ion Beam facility at LNL, Technical Design Report, INFN-LNL-220 (2007), 2007. [4] J. Esposito, G. Rosi, S. Agosteo, The new hybrid thermal neutron facility at Tapiro reactor for BNCT radiobiological experiments. Radiat. Prot. Dosim. Advance Access published on May 15, 2007; NEUDOS-10 SPECIAL ISSUE, doi:10.1093/rpd/ncm015. [5] L. De Nardo, E. Seravalli, G. Rosi, J. Esposito, P. Colautti, V. Conte, G. Tornielli, BNCT microdosimetry at the Tapiro Reactor thermal column, Radiat. Prot. Dosim. 110 (2004) 579. [6] L. De Nardo, V. Cesari, G. Dona’, P. Magrin, P. Colautti, V. Conte, G. Tornielli, Mini-TEPCs for radiation therapy, Radiat. Prot. Dosim. 108 (2004) 345. [7] A.Z. Diaz, J.A. Coderre, A.D. Chanana, R. Ma, Boron neutron capture therapy for malignant gliomas, Ann. Med. 32 (2000) 81. [8] R. Ackroyd, C. Kelty, N. Brown, M. Reed, The history of photodetection and photodynamic therapy, Photochem. Photobiol. 74 (2001) 656. [9] E. Friso et al., A novel10 B-enriched carboranyl-containing phthalocyanine as a radio- and photo-sensitising agent for Boron Neutron Capture Therapy and Photodynamic Therapy of tumours: in vitro and in vivo studies, Photochem. Photobiol. Sci. 5 (1) (2006) 39. [10] J.S. Hendricks, et al. MCNPX, version 2.6b Los Alamos National Laboratory Report. LA-UR-06-3248, June 2006. [11] K.W. Burn, L. Casalini, D. Mondini, E. Nava, G. Rosi, R.L. Tinti, The epithermal neutron beam for BNCT under construction at TAPIRO: physics, Journal of Physics: Conference Series 41 (2006) 187. [12] International Commission on Radiation Units and Measurements, Nuclear Data for Neutron and Proton Radiotherapy and for Radiation Protection, 2000, ICRU-63. [13] International Commission on Radiation units and Measurements, Photon, Electron, Proton, and Neutron Interaction Data for Body Tissues, 1992, ICRU-46. [14] L.H. Graf, P. Kaplan, S. Silagi, Efficient DNA-mediated transfer of selectable genes and unselected sequences into differentiated and undifferentiated mouse melanoma clones, Somatic Cell and Molecular Genetics 10 (1984) 139.