a ENEA Radiation Sources Laboratory, Research Center Frascati, Rome, Italy b Istituto Superiore di Sanità - Technology and Health Department, Viale Regina Elena 299, Roma, Italy c ENEA C.R. Casaccia, UT. BIORAD Radiobiology, Via Anguillarese, 301, 00123, Roma, Italy d INFN-Sezione di Roma1, Gr.coll.Sanità M. Vadrucci a, A. Ampollini a, P. Anello b, M. Balduzzi c, G. Bazzano a, G. Esposito b,d, C. Maricno c, C. Patrono c, L. Picardi a, C. Ronsivalle a, C. Snels c, M. A. Tabocchini b,d, A. Testa c Radiobiology Experiments for Characterization of the low-energy TOP- IMPLART Proton Beam ABSTARCT: The TOP (Oncological Therapy with Protons)-IMPLART (Intensity Modulated Proton Linear Accelerator for RadioTherapy) [1] accelerator is a facility for proton-therapy under construction in ENEA C. R. Frascati Radiation Sources Laboratory in collaboration with ISS and IFO. It is a completely linear pulsed accelerator consisting of a 7 MeV injector followed by a high frequency booster up to 150 MeV. A vertical beam extraction-line at the exit of injector has been devoted to in-vitro radiobiology experiments for the characterization of the low-energy proton beam. Irradiation experiments were carried out on V79 and CHO Chinese hamster cells. Both cell lines have been widely used in radiobiology studies with different radiation qualities – including by the authors [2, 3] - thus allowing the evaluation and the comparison of results. In order to carry out the biological characterization of the facility, the radiation responses of V79 and CHO cells have been evaluated in terms of clonogenic survival. Moreover, a protocol of Micronuclei Assay for the in situ evaluation of radiation induced DNA damage has been adapted to the specific exposure conditions of the vertical proton beam. Cells were plated in stainless steel Petri dishes, especially designed for charged particle irradiation [1], and grown attached to a 60 μm mylar foil representing the base of the dish. The irradiations have been performed with protons extracted in air and impinging on the cells with energy of 5 MeV (incident LET=7.7 keV/μm in MS20); the clonogenic survival was evaluated in the dose range Gy. The dose-response curves - characterized by an initial shoulder followed by a straight portion - were well fitted by a linear-quadratic function. The obtained results were found in good agreement with literature data [3, 4]. [1] C. Ronsivalle et al., The TOP IMPLART Project, Eur. Phys. J Plus (2011) 126, number 7, 68; [2] Belli et al, NIM 1987; [3] Belli et al, IJRB 1998; [4] JT Tang'et al, BJC ABSTARCT: The TOP (Oncological Therapy with Protons)-IMPLART (Intensity Modulated Proton Linear Accelerator for RadioTherapy) [1] accelerator is a facility for proton-therapy under construction in ENEA C. R. Frascati Radiation Sources Laboratory in collaboration with ISS and IFO. It is a completely linear pulsed accelerator consisting of a 7 MeV injector followed by a high frequency booster up to 150 MeV. A vertical beam extraction-line at the exit of injector has been devoted to in-vitro radiobiology experiments for the characterization of the low-energy proton beam. Irradiation experiments were carried out on V79 and CHO Chinese hamster cells. Both cell lines have been widely used in radiobiology studies with different radiation qualities – including by the authors [2, 3] - thus allowing the evaluation and the comparison of results. In order to carry out the biological characterization of the facility, the radiation responses of V79 and CHO cells have been evaluated in terms of clonogenic survival. Moreover, a protocol of Micronuclei Assay for the in situ evaluation of radiation induced DNA damage has been adapted to the specific exposure conditions of the vertical proton beam. Cells were plated in stainless steel Petri dishes, especially designed for charged particle irradiation [1], and grown attached to a 60 μm mylar foil representing the base of the dish. The irradiations have been performed with protons extracted in air and impinging on the cells with energy of 5 MeV (incident LET=7.7 keV/μm in MS20); the clonogenic survival was evaluated in the dose range Gy. The dose-response curves - characterized by an initial shoulder followed by a straight portion - were well fitted by a linear-quadratic function. The obtained results were found in good agreement with literature data [3, 4]. [1] C. Ronsivalle et al., The TOP IMPLART Project, Eur. Phys. J Plus (2011) 126, number 7, 68; [2] Belli et al, NIM 1987; [3] Belli et al, IJRB 1998; [4] JT Tang'et al, BJC TOP – IMPLART PROJECT The TOP-IMPLART project, developed by ENEA, the Italian National Institute of Health (ISS) and Regina Elena National Cancer Institute IFO, Rome, is devoted to the realization of a new proton therapy center to be sited in Rome (Italy). The project is based on a 230 MEV proton accelerator consisting of a sequence of linear accelerators. The low-energy segment (up to 35 MeV) is composed by a commercial 7MeV proton Linac (sequence of a source, a RFQ and a DTL operating at the frequency of 425 MHz) produced by AccSys-Hitachi, followed by 4 SCDTL-modules added to leading the proton energy up to 11.6, 18, 27 and 35MeV. The TOP-IMPLART facility is designed for fully active scanning 4D (intensity, energy and transversal position x-y) IMPT protocols. Peculiar characteristics of the system are: modularity, pulsed operation naturally suited to IMPT, fast energy variation, high quality beam. TOP – IMPLART PROJECT The TOP-IMPLART project, developed by ENEA, the Italian National Institute of Health (ISS) and Regina Elena National Cancer Institute IFO, Rome, is devoted to the realization of a new proton therapy center to be sited in Rome (Italy). The project is based on a 230 MEV proton accelerator consisting of a sequence of linear accelerators. The low-energy segment (up to 35 MeV) is composed by a commercial 7MeV proton Linac (sequence of a source, a RFQ and a DTL operating at the frequency of 425 MHz) produced by AccSys-Hitachi, followed by 4 SCDTL-modules added to leading the proton energy up to 11.6, 18, 27 and 35MeV. The TOP-IMPLART facility is designed for fully active scanning 4D (intensity, energy and transversal position x-y) IMPT protocols. Peculiar characteristics of the system are: modularity, pulsed operation naturally suited to IMPT, fast energy variation, high quality beam. Vertical Beam Line 7 MeV Protons Dedicated to In vitro Radiobiology 70 cm long - 2 mm aluminium collimator - 2  m gold scatterer spreading uniformly the beam on an area of 13 mm diameter – 50  m Kapton vacuum/air window degrading the beam energy to 6.2 MeV SCDTL-1 RADIOBIOLOGICAL EXPERIMENTS The first radiobiological experimental campaign was carried out to characterize the proton beam. To this purpose, cell killing experiments and micronuclei (MN) assay were conducted at the vertical transport line on V79 and CHO cells. Irradiations have been performed with protons extracted in air and impinging on the cells with energy of 5 MeV (incident LET=7.7 keV/μm in MS20); the clonogenic survival was evaluated in the dose range Gy. RADIOBIOLOGICAL EXPERIMENTS The first radiobiological experimental campaign was carried out to characterize the proton beam. To this purpose, cell killing experiments and micronuclei (MN) assay were conducted at the vertical transport line on V79 and CHO cells. Irradiations have been performed with protons extracted in air and impinging on the cells with energy of 5 MeV (incident LET=7.7 keV/μm in MS20); the clonogenic survival was evaluated in the dose range Gy. STAINLESS STEEL CELLS SAMPLE HOLDER ASSEMBLAGE mylar foil protons cell monolayer beam Mylar Cells Dose-response curves for clonogenic survival in V79 and CHO cells The dose-response curves for clonogenic survival were found to be characterized by an initial shoulder (more pronunced in V79 than in CHO cells) followed by a straight portion, that can be well fitted by a linear-quadratic function of the dose.  = 0.208±0.016  = 0.020±0.003  = 0.186±0.019  = 0.090±0.003 In situ MN assay ~ 10 4 CHO cells seeded on the irradiation dish (60 μm mylar) fresh medium containing Cytochalasin-b (1.5 μg/ml) added h incubation (37°C, 5% CO2) Hypotonic shock and fixing Staining with 5% Giemsa 24h 5 MeV proton irradiation All the steps of experimental protocol used for MN assay have been adapted to the specific exposure conditions of the TOP-IMPLART vertical proton beam. MN 40 X After the staining the mylar membrane is mounted on a glass slide for microscope analysis Up to now layout of the TOP – IMPLART facility The results on V79 cells, widely used in hadrontherapy experiments, were found in good agreement with the previous data obtained at LNL [3]. Also the data obtained using CHO cells were consistent with literature results [4].